Refine Search

New Search

Results: 305

(searched for: 10.29328/journal.aac.1001009)
Save to Scifeed
Page of 7
Articles per Page
by
Show export options
  Select all
Mohei Menul Islam, Muhammad Harunur Rashid, Aqib Muntasir
Journal of Engineering Science, Volume 12, pp 11-17; https://doi.org/10.3329/jes.v12i3.57475

Abstract:
Autoclaved aerated concrete (AAC) prepared by the mixing of ordinary Portland cement, lime powder, sand, aluminium powder and water. This study covers the variation of physical, mechanical and functional properties of autoclaved aerated concrete with autoclaving temperature and aluminium content and compared with that of normal weight cement mortar sample. In this work, two dosage of aluminium content of 0.4% and 0.8% of the dry weight of ordinary Portland cement and three different autoclaving temperature of 160oC, 180oC and 200oC were used. AAC sample with 0.8% aluminium and 160oC temperature had unit weight of 1490kg/m3 which was lowest among all samples including the control or normal weight cement blocks. Weight reduction of AAC sample was 31.53%. AAC sample with 0.4% aluminium and 200oC autoclaving temperature gave maximum compressive and tensile strength of 19.4MPa and 1.81MPa respectively which were close to that of normal weight concrete and strength of AAC increased with autoclaving temperature and decreased with aluminium content. In this research, the functional propertiesof AAC, absorption capacity was much higher than normal weight concrete and this capacity was increased with aluminium content and with decreasing autoclaving temperature and unit weight of AAC. For AAC with 0.8% aluminium and 160oC temperature gave maximum water absorption capacity (=9.93%). Again, surface absorption rate was higher for first 12hours and with time it would be constant because of its saturated position. Journal of Engineering Science 12(3), 2021, 11-17
Sina Jasim
Published: 1 January 2022
AACE Clinical Case Reports, Volume 8; https://doi.org/10.1016/j.aace.2021.12.004

Abstract:
Happy New Year and welcome to another issue of AACE Clinical Case Reports (ACCR). We are excited that it has been a year since we fully transitioned to Elsevier and the new online AACE Journals platform. ACCR continues to grow in both manuscript submissions and readership with the number of yearly article downloads has increased from 142,122 in 2020 to over 278,698 in 2021. This growth would not have been possible without the dedication of our associate editors, editorial board members, and editorial/publication staff. Special thank you to our excellent reviewers that provided meaningful and constructive reviews to help the educational value of the published cases.
Gabriela N Tenea, Daniela Olmedo, Pamela Ascanta, Gabriela Gonzalez
Proceedings of MOL2NET'21, Conference on Molecular, Biomedical & Computational Sciences and Engineering, 7th ed.; https://doi.org/10.3390/mol2net-07-10913

Abstract:
Abstract: The contamination of food by microorganisms, their persistence, growth, multiplication, and/or toxin production has emerged as an important public health concern . The demand for consuming fresh and low-processed foods free of chemicals and pathogens is increasing. Despite advances in food safety, annually, more than 9 million persons developed illnesses caused by food contamination (Scallan et al., 2011). In Ecuador, the risk of diseases associated with food contaminations is increasing due to incorrect food manipulation, hygiene, and inappropriate storage conditions (Garzon et al., 2017). Although the vendors are continuously capacitated, no improvement on selling sites was made. The food is continuously sold on streets, near parks, transportation terminals, as a common habit. Along with the excessive use of chemicals for the purpose of preservation, food safety is of concern. To overcome this problem, the application of natural methods for preservation might be a suitable solution. Lactic acid bacteria are producing peptides or small proteins namely bacteriocins which could be the next generation of antimicrobials. Thus, their incorporation in food to prevent poisoning or spoilage has been an area of dynamic research in the last decade (Backialakshmi et al., 2015). Previously, we identified two native bacteriocinogenic strains, Lactobacillus plantarum UTNGt2 and L. plantarum UTNCys5-4, producing peptides with a broad spectrum of antibacterial activity against several foodborne pathogens in vitro (Tenea and Pozo, 2019; Tenea and Guana, 2019). Moreover, the addition of those peptide extracts at the exponential phase of growth of the target bacteria (Staphylococcus aureus ATCC1026) results in a decrease of total cell viability with about 3.2-fold (log CFU/ml) order of magnitude at 6 h of incubation, indicating their bactericidal mode of action. In this study, the possible mechanism of action against Staphylococcus aureus was investigated through a series of cell biology analyses such as membrane permeabilization, cell integrity, and structural changes of the target cells. Altogether, the results demonstrated the effectiveness of peptides produced by native lactic acid bacteria to kill Staphylococcus and further investigation is need it to prove the effect in a food matrix. Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M. A., Roy, S. L., Jones, J. L., & Griffin, P. M. (2011). Foodborne illness acquired in the United States--major pathogens.Emerging infectious diseases,17(1), 7–15. https://doi.org/10.3201/eid1701.p11101.Garzón, K., Ortega, C., & Tenea, G. N. (2017). Characterization of Bacteriocin-Producing Lactic Acid Bacteria Isolated from Native Fruits of Ecuadorian Amazon.Polish journal of microbiology,66(4), 473–481. https://doi.org/10.5604/01.3001.0010.7037Backialakshmi, S., Rn, M., Saranya, A., Ms, J.T., Ar, K., Js, K., & Ramasamy, S. (2015). Biopreservation of Fresh Orange Juice Using Antilisterial Bacteriocins101 and Antilisterial Bacteriocin103 Purified from Leuconostoc mesenteroides.Journal of Food Processing and Technology, 6, 1-5.Tenea, G. N., & Delgado Pozo, T. (2019). Antimicrobial Peptides fromLactobacillus plantarumUTNGt2 Prevent Harmful Bacteria Growth on Fresh Tomatoes.Journal of microbiology and biotechnology,29(10), 1553–1560. https://doi.org/10.4014/jmb.1904.04063Tenea, G. N., & Guana, J. M. (2019). Inhibitory substances produced by native Lactobacillus plantarum UTNCys5-4 control microbial population growth in meat. Journal of Food Quality, a9516981. https://doi.org/10.1155/2019/9516981.Wang, X., Teng, D., Mao, R., Yang, N., Hao, Y., & Wang, J. (2016). Combined Systems Approaches Reveal a Multistage Mode of Action of a Marine Antimicrobial Peptide against Pathogenic Escherichia coli and Its Protective Effect against Bacterial Peritonitis and Endotoxemia.Antimicrobial agents and chemotherapy,61(1), e01056-16. https://doi.org/10.1128/AAC.01056-16.
Corrigendum
, Nicholas J. Scott, Rachel A. Matson, Mark E. Everett, Elise Furlan, Crystal L. Gnilka, David R. Ciardi, Kathryn V. Lester
Frontiers in Astronomy and Space Sciences, Volume 8; https://doi.org/10.3389/fspas.2021.696011

Abstract:
A Corrigendum on The NASA High-Resolution Speckle Interferometric Imaging Program: Validation and Characterization of Exoplanets and Their Stellar Hosts by Steve B. Howell, Nicholas J. Scott, Rachel A. Matson, Mark E. Everett, Elise Furlan, Crystal L. Gnilka, David R. Ciardi, Kathryn V. Lester. (2021). Front. Astron. Space Sci. 10:635864. doi: 10.3389/fspas.2021.635864 In the original article, there were incorrect parameters listed in the last paragraph of Section 2. A correction has been made to that paragraph as follows: “As an aside, Robo-AO is another high-resolution imaging technique used in the optical wavelength range. Ziegler et al. (2017) discuss their results using this method for exoplanet host stars. Unlike speckle imaging, Robo-AO uses the mechanical deformable mirror techniques of IR/AO and applies them to optical light. See Ziegler et al. (2018) for details. Ref is: @ARTICLE2018AJ....156...83Z, author = Ziegler, Carl and Law, Nicholas M. and Baranec, Christoph and Howard, Ward and Morton, Tim and Riddle, Reed and Duev, Dmitry A. and Salama, Ma¨ıssa and Jensen-Clem, Rebecca and Kulkarni, S. R., title = ”Robo-AO Kepler Survey. V. The Effect of Physically Associated Stellar Companions on Planetary Systems”, journal =, keywords = binaries: close, instrumentation: adaptive optics, methods: data analysis, methods: observational, planets and satellites: fundamental parameters, techniques: high angular resolution, Astrophysics - Earth and Planetary Astrophysics, year = 2018, month = aug, volume = 156, number = 2, eid = 83, pages = 83, doi = 10.3847/1538-3881/aace59, archivePrefix = arXiv, eprint = 1804.10208, primaryClass = astro-ph.EP, adsurl = https://ui.adsabs.harvard.edu/abs/2018AJ....156...83Z, adsnote = Provided by the SAO/NASA Astrophysics Data System.” The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Ziegler, C., Law, N. M., Baranec, C., Howard, W., Morton, T., Riddle, R., et al. (2018). Robo-AO Kepler Survey. V. The Effect of Physically Associated Stellar Companions on Planetary Systems. AJ 156 (2), 83. doi:10.3847/1538-3881/aace59 CrossRef Full Text | Google Scholar Keywords: exoplanets, exoplanetary systems, binary host stars, speckle interferometry, high-resolution imaging Citation: Howell SB, Scott NJ, Matson RA, Everett ME, Furlan E, Gnilka CL, Ciardi DR and Lester KV (2021) Corrigendum: The NASA High-Resolution Speckle Interferometric Imaging Program: Validation and Characterization of Exoplanets and Their Stellar Hosts. Front. Astron. Space Sci. 8:696011. doi: 10.3389/fspas.2021.696011 Received: 30 April 2021; Accepted: 07 May 2021;Published: 30 September 2021. Edited and reviewed by: Copyright © 2021 Howell, Scott, Matson, Everett, Furlan, Gnilka, Ciardi and Lester. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Steve B. Howell, [email protected]
Sina Jasim
Published: 28 August 2021
AACE Clinical Case Reports, Volume 7; https://doi.org/10.1016/j.aace.2021.08.003

Abstract:
Thank you for being part of our journal and welcome to another issue of AACE Clinical Case Reports. The current issue includes many interesting and educational case reports to share. We will provide a summary of those cases. For more details, please access ACCR online journal available at https://www.aaceclinicalcasereports.com/
Published: 27 August 2021
by 10.5281
Abstract:
These are the results files for the following peer-reviewed article: Price, M.H., J.M. Capriles, J. Hoggarth, R.K. Bocinsky, C.E. Ebert, and J.H. Jones, (2021). End-to-end Bayesian analysis for summarizing sets of radiocarbon dates. Journal of Archaeological Science. They were generated inside a Docker container as outlined in the README of this github repository: https://github.com/MichaelHoltonPrice/price_et_al_tikal_rc The analyses rely on an R package located in this github repository: https://github.com/eehh-stanford/baydem For the results archived here, the commits for each repository are: price_et_al_tikal_rc 3ac1e35f4277ef878f8e3aac3d05159928a09a2b baydem 1220a60a860633b51f9f07cbff3eb78f458efc1a
Published: 27 August 2021
by 10.5281
Abstract:
These are the results files for the following peer-reviewed article: Price, M.H., J.M. Capriles, J. Hoggarth, R.K. Bocinsky, C.E. Ebert, and J.H. Jones, (2021). End-to-end Bayesian analysis for summarizing sets of radiocarbon dates. Journal of Archaeological Science. They were generated inside a Docker container as outlined in the README of this github repository: https://github.com/MichaelHoltonPrice/price_et_al_tikal_rc The analyses rely on an R package located in this github repository: https://github.com/eehh-stanford/baydem For the results archived here, the commits for each repository are: price_et_al_tikal_rc 3ac1e35f4277ef878f8e3aac3d05159928a09a2b baydem 1220a60a860633b51f9f07cbff3eb78f458efc1a
Imaekhai Lawrence
Published: 16 August 2021
by 10.5281
Abstract:
{"references": ["Arditi, D., Akan, G. T., & Gurdamar, S. (1985). Cost overruns in public projects. International Journal of Project Management, 3(4), 218-224.", "Dlakwa MM, Culpin MF (1990) Reasons for overrun in public sector construction projects in Nigeria. International Journal of Project Management 1990;8(4):237\u201341.", "Frimpong, Y. (2000). Project management in developing countries: causes of delay and cost overruns in construction of groundwater projects. Unpublished Masters Research Project, University of Technology, Sydney, Australia.", "Giridhar P, Ramesh K. (1998) Effective management of Turnkey projects. Aace Transactions, PM7- PM11 1998.", "Khalil ALMI, AL-Ghafly MA. (1999) Delay in public Utility projects in Saudi Arabia. International Journal of Project Management,17(2):101\u20136.", "Mansfield NR, Ugwu OO, Doran T. (1994) Causes of delay and cost overruns in Nigeria construction projects. International Journal of Project Management,12(4):254\u201360.", "Ogunlana SO, Promkuntong K, Vithool J. (1996) Construction delays in a fast-growing growing economy: comparing Thailand with other economies. International Journal of Project Management; 14(1):37\u201345.", "Ogunlana SO, Olomolaiye PO. (1989) A survey of site management practice on some selected sites in Nigeria. Building Environ, 24(2):191\u20136.", "Oglesby C, Parker H, Howell G. (1989) Productivity improvement in construction. New York: McGraw-Hill; 1989.", "Okpala DC, Aniekwu AC. (1988) Causes of high costs of construction in Nigeria. Journal of Management in Engineering, ASCE, 114:233\u201344."]}
Imaekhai Lawrence
Published: 16 August 2021
by 10.5281
Abstract:
{"references": ["Arditi, D., Akan, G. T., & Gurdamar, S. (1985). Cost overruns in public projects. International Journal of Project Management, 3(4), 218-224.", "Dlakwa MM, Culpin MF (1990) Reasons for overrun in public sector construction projects in Nigeria. International Journal of Project Management 1990;8(4):237\u201341.", "Frimpong, Y. (2000). Project management in developing countries: causes of delay and cost overruns in construction of groundwater projects. Unpublished Masters Research Project, University of Technology, Sydney, Australia.", "Giridhar P, Ramesh K. (1998) Effective management of Turnkey projects. Aace Transactions, PM7- PM11 1998.", "Khalil ALMI, AL-Ghafly MA. (1999) Delay in public Utility projects in Saudi Arabia. International Journal of Project Management,17(2):101\u20136.", "Mansfield NR, Ugwu OO, Doran T. (1994) Causes of delay and cost overruns in Nigeria construction projects. International Journal of Project Management,12(4):254\u201360.", "Ogunlana SO, Promkuntong K, Vithool J. (1996) Construction delays in a fast-growing growing economy: comparing Thailand with other economies. International Journal of Project Management; 14(1):37\u201345.", "Ogunlana SO, Olomolaiye PO. (1989) A survey of site management practice on some selected sites in Nigeria. Building Environ, 24(2):191\u20136.", "Oglesby C, Parker H, Howell G. (1989) Productivity improvement in construction. New York: McGraw-Hill; 1989.", "Okpala DC, Aniekwu AC. (1988) Causes of high costs of construction in Nigeria. Journal of Management in Engineering, ASCE, 114:233\u201344."]}
Charneal L. Dixon,
Published: 19 July 2021
Journal of Lipid Research, Volume 62; https://doi.org/10.1016/j.jlr.2021.100097

Abstract:
NOD2 (nucleotide-binding and oligomerization domain containing protein 2) is a cytosolic pattern recognition receptor that detects intracellular peptidoglycan (muramyl dipeptide) from bacteria. Membrane association of NOD2 is essential for its ability to activate nuclear factor κB and mitogen-activated protein kinase signaling pathways via the kinase, receptor-interacting serine/threonine-protein kinase 2. The post-translational addition of palmitate to NOD2 results in an acylated protein with increased affinity for membrane bilayers (1Lu Y. Zheng Y. Coyaud É. Zhang C. Selvabaskaran A. Yu Y. Xu Z. Weng X. Chen J.S. Meng Y. Warner N. Cheng X. Liu Y. Yao B. Hu H. Xia Z. Muise A.M. Klip A. Brumell J.H. Girardin S.E. Ying S. Fairn G.D. Raught B. Sun Q. Neculai D. Palmitoylation of NOD1 and NOD2 is required for bacterial sensing.Science. 2019; 366: 7Crossref Scopus (26) Google Scholar). Palmitoylation of cysteine residues (shown in orange) at positions C395 and C1033 en face of the structural model (A) based on the crystal structure of rabbit NOD2 (PDB 5IRL) (2Maekawa S. Ohto U. Shibata T. Miyake K. Shimizu T. Crystal structure of NOD2 and its implications in human disease.Nature Communications. 2016; 7: 11813Crossref PubMed Scopus (74) Google Scholar) is mediated by the protein acyltransferase enzyme zDHHC5 (1Lu Y. Zheng Y. Coyaud É. Zhang C. Selvabaskaran A. Yu Y. Xu Z. Weng X. Chen J.S. Meng Y. Warner N. Cheng X. Liu Y. Yao B. Hu H. Xia Z. Muise A.M. Klip A. Brumell J.H. Girardin S.E. Ying S. Fairn G.D. Raught B. Sun Q. Neculai D. Palmitoylation of NOD1 and NOD2 is required for bacterial sensing.Science. 2019; 366: 7Crossref Scopus (26) Google Scholar). The model suggests that this face of the protein is juxtaposed to the plasmalemmal surface. Palmitoylation of the expressed GFP-NOD2WT and GFP-NOD2C395,1033S in HCT116 cells was further characterized using a biorthogonal chemical reporter assay (B) (3Yap M.C. Kostiuk M.A. Martin D.D.O. Perinpanayagam M.A. Hak P.G. Siddam A. Majjigapu J.R. Rajaiah G. Keller B.O. Prescher J.A. Wu P. Bertozzi C.R. Falck J.R. Berthiaume L.G. Rapid and selective detection of fatty acylated proteins using ω-alkynyl-fatty acids and click chemistry.Journal of Lipid Research. 2010; 51: 1566-1580Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Here,15-hexadecynoic acid (15-HDYA), also referred to as alkynyl palmitic acid, was metabolically incorporated into cells. The 15-HDYA covalently attached to the immunocaptured GFP-NOD2 proteins was reacted with azide-PEG3-FLAG via a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction. The proteins were resolved by SDS-PAGE and subsequently detected by an anti-FLAG antibody. The metabolic label, which was incorporated into NOD2WT and was notably absent in the double cysteine mutant, was removed by treatment with 2.5% hydroxylamine (HAM) that hydrolyzes the 15-HDYA – protein thioester linkage. Finally, the palmitoylation-deficient GFP-NOD2C395,1033S does not localize to the plasma membrane in HCT116 cells (C). Mutations that disrupt S-palmitoylation are associated with severe immunologic and inflammatory diseases such as Crohn’s disease whereas mutations associated with Blau syndrome demonstrate increased S-palmitoylation and membrane localization (1Lu Y. Zheng Y. Coyaud É. Zhang C. Selvabaskaran A. Yu Y. Xu Z. Weng X. Chen J.S. Meng Y. Warner N. Cheng X. Liu Y. Yao B. Hu H. Xia Z. Muise A.M. Klip A. Brumell J.H. Girardin S.E. Ying S. Fairn G.D. Raught B. Sun Q. Neculai D. Palmitoylation of NOD1 and NOD2 is required for bacterial sensing.Science. 2019; 366: 7Crossref Scopus (26) Google Scholar).
Thomas Kovacs
American Journal of Speech-Language Pathology, Volume 30, pp 1038-1048; https://doi.org/10.1044/2020_ajslp-20-00224

Abstract:
Purpose The aim of the study was to collect information about American speech-language pathologists' preprofessional training, practice, self-perceived competence, adequacy of resources, and interest in continuing education related to augmentative and alternative communication (AAC) assessment and intervention strategies addressing each of the five language domains: semantics, pragmatics, phonology, morphology, and syntax. Method An anonymous online survey of American speech-language pathologists was conducted. Results A majority of participants rated their preprofessional training for assessing semantic and pragmatic skills positively. Otherwise, a majority of participants rated preprofessional training for assessment and intervention negatively across language domains. High interest in continuing education opportunities addressing assessment and intervention was found across language domains. A discrepancy between responses to questions addressing semantic and pragmatic skills and responses to questions addressing phonological, morphological, and syntactic skills was consistently found for ratings of preprofessional training, practice, perceived competence, and adequacy of resources. In all cases, higher frequencies of positive ratings were found for questions addressing semantic and pragmatic skills. Conclusions Improved preprofessional training and continuing education opportunities are needed to support AAC assessment and intervention across language domains. Perspectives and practice patterns reflect a historical emphasis on semantic and pragmatic skills in the external evidence base, even though there are several recent journal articles addressing morphology and syntax in clients who use AAC.
Frontiers in Environmental Science, Volume 9; https://doi.org/10.3389/fenvs.2021.664313

Abstract:
The year 2021 offers a critical opportunity for concerted action to influence the future of freshwater biodiversity, ecosystem services and human well-being. The United Nations Decade on Biodiversity 2011–2020 has ended, and governments around the world are reviewing major international agreements relevant to biodiversity conservation, including the Convention on Biological Diversity (CBD)1, the Sustainable Development Goals (SDGs)2, and the UN Framework Convention on Climate Change (UNFCCC)3. A Post-2020 Global Biodiversity Framework4 is under development, with the grand mission to “Halt the loss of species, ecosystems and genetic diversity by 2030; restore and recover biodiversity to ensure a world of people “living in harmony with nature' by 2050”. Freshwater ecologists have acted quickly to draw attention to the global dimensions of the freshwater biodiversity crisis and address the lack of a comprehensive framework to guide policy responses (Bunn, 2016; Darwall et al., 2018). An Emergency Recovery Plan for freshwater biodiversity, published by 25 authors from 14 organizations (Tickner et al., 2020), sets out six major priorities for global action and policy development to “bend the curve of freshwater biodiversity loss.” It has been submitted to the working committees of the Post-2020 Global Biodiversity Framework, and further promoted as a dramatic OUPblog “Bring living waters back to our planet5” Comprehensive reviews have since enumerated many research questions, actions and policy refinements needed to “bend the curve” and protect the world's freshwater ecosystems (van Rees et al., 2020; Buxton et al., 2021; Harper et al., 2021; Maasri et al., 2021). Each review cuts across important scientific, societal, management and policy issues. The purpose of this brief challenge paper is, likewise, to strengthen and support the Emergency Recovery Plan, but in a different way, by advocating a broader package of strategic activities that too often operate in silos, with patchy coverage of the world's freshwater ecosystem types and biogeographic diversity. This package presents traditional areas of scientific and societal activity that require more strategic, integrated and collaborative global effort to deliver evidence-based freshwater conservation outcomes, conjoined with terrestrial and estuarine/marine conservation, depending on context: (i) inventory, evaluation and research; (ii) restoration and rehabilitation; (iii) protected area design and management; and (iv) socio-ecological science and governance. The paper is intended to motivate greater interest, commitment and collaboration of all stakeholders in the most urgent and ambitious conservation enterprise of the next decade—to protect and sustain freshwater biodiversity in the socio-ecological systems of the Anthropocene. Evidence-based ecosystem restoration and biodiversity protection depend upon a credible foundation of scientific and sociological data, process understanding and a capacity to model, predict and evaluate ecological/societal outcomes from natural processes, pressures and management actions. Notwithstanding a huge body of erudite freshwater research, there remains an ongoing need to increase understanding of the biodiversity, biophysical processes and ecosystem services of the world's freshwater and connected terrestrial and estuarine/marine ecosystems. The IUCN Commission on Ecosystem Management has developed a globally consistent, spatially explicit Ecosystem Typology for conservation purposes (Keith et al., 2021). It is designed to help identify the ecosystems most critical to conservation of biodiversity and supply of ecosystem services, as well as structuring global risk assessments for the Red List of Ecosystems and reporting against CBD and SDG targets and other framings. The typology distinguishes 28 natural freshwater ecosystem types within subterranean systems, palustrine wetlands, streams, rivers, freshwater and saline lakes, artesian springs, oases, and transitional waters (fjords, estuaries, intermittently closed and open lakes and lagoons–ICOLLS). Depending on ecosystem type, geography and knowledge gaps, freshwater inventory and research is traditionally integrated around taxonomy, genetics and organismal biology, population and community ecology, and ecosystem functions, the latter including the processes that link landscapes, connected boundary systems (riparian areas, floodplains, wetlands/lakes, and groundwater systems) and freshwater ecosystems (Geist, 2011; Reis et al., 2017; Flitcroft et al., 2019). Likewise, the pathways and processes that connect rivers and estuaries via surface flows and submarine groundwater discharges are vital dimensions of interconnected freshwater and coastal ecosystems. The IUCN Ecosystem Typology provides a geographic framing and scientific resource to help guide priorities for basic inventory and ecological research on understudied ecosystem types and biogeographic regions. For example, groundwater-dependent ecosystems such as artesian springs and oases are relatively poorly studied but coming to attention globally (Cantonati et al., 2020). Intermittent rivers and ephemeral streams (IRES) and episodic arid-zone floodplains are of growing interest because even when dry they perform multiple ecosystem services that complement those of nearby perennial rivers (Datry et al., 2018). Given the exceptional biodiversity of the Amazon Basin and poor knowledge of many aquatic taxa (e.g., migratory fishes), there is an outstanding need for inventory, knowledge synthesis and risk assessment to guide recovery and conservation (Duponchelle et al., 2021). Innovative biodiversity assessment techniques (remote sensing, GIS, environmental DNA, camera traps, sound recordings, radiotelemetry) can be integrated with established field methods to document biodiversity patterns and hotspots, and track flagship, umbrella and endangered species of high conservation value (Harper et al., 2021). Systematic reviews, meta-analysis, natural and laboratory experiments and modeling offer scope to relate biodiversity patterns and processes with dominant environmental drivers (climate, hydrological regime and water quality, etc). Broad stakeholder engagement is essential across the spectrum of biodiversity inventories, identification of knowledge gaps and research priorities, evaluation of ecosystem services and formulation of targets for restoration and protection of species, ecosystem processes and valued services. The major threats to freshwater ecosystems have been comprehensively synthesized in six main categories: hydrological alterations, habitat degradation and loss, pollution, overexploitation, invasive species, and climate change (Dudgeon et al., 2006). These have been mapped at global scale (Vörösmarty et al., 2010; Reis et al., 2017; Grill et al., 2019), elaborated as new pollutants and configurations of stress emerge (Reid et al., 2019) and widely publicized (Bunn, 2016; Flitcroft et al., 2019). Yet despite prodigious management efforts, biodiversity loss and ecosystem degradation continue, creating huge deprivation for millions of people whose diets and livelihoods depend directly on freshwater biota (Lynch et al., 2016). Biodiversity decline has significant implications for ecosystem resilience, recovery potential and adaptation to climate change. The Emergency Recovery Plan offers a blueprint focused on reducing biodiversity decline and recovering from these major threats, a well as a new threat category on connectivity to highlight the implications of habitat fragmentation for freshwater biota and ecosystems (Grill et al., 2019). Numerous methods and sound protocols already enable mitigation of these major threats, as demonstrated in successful ecological restoration projects around the world (Palmer et al., 2005). For example, the restoration of connectivity patterns and processes has contributed to recovery of biodiversity and ecosystem processes in many regulated rivers (Horne et al., 2017; Opperman et al., 2019). The bolder objective of the Emergency Recovery Plan is to transition from local freshwater restoration successes to a strategic approach that achieves biodiversity and ecological recovery at larger spatial scales. The European Water Framework Directive6 offers one well-established jurisdictional framing for freshwater ecosystem recovery to good ecological status. Building on European case studies, challenges and successes under this and other directives, van Rees et al. (2020) extend the ideas of the freshwater Emergency Recovery Plan into 15 special recommendations with potential to protect freshwater life globally. Beyond the main categories of threat to freshwater biodiversity and ecosystems lie new kinds of stress and new configurations of familiar stressors (Reid et al., 2019). Many, if not most, freshwater ecosystems are affected by several types of stress that interact, often with effects greater than (synergism), less than (antagonism) or equal to the sum of their individual effects (Sabater et al., 2018). The daunting scientific challenge is to identify the most significant causes of stress and define the most beneficial blend, geographic placement and timing of management actions (Omerod et al., 2010; Craig et al., 2017). This approach has worked reasonably well for the urban stream “syndrome” (Sheldon et al., 2012; Booth et al., 2016). Other multiple-stressor syndromes that threaten freshwater ecosystems include irrigated agriculture, forestry, mining, energy production, transport systems and the recreation and tourism sectors. Climate change, itself a complex mix of stressors, already compounds multiple stressor syndromes (Sabater et al., 2018), by altering river flow and flooding regimes, while rising temperatures are driving higher evaporation rates, water scarcity, and aquatic habitat loss. Shifting climatic regimes intensify the urgency of multiple stressor research and adaptive management solutions. In multiple-stressor contexts, Tickner et al. (2020) recommend the assembly of “strategic portfolios of measures” rather than relying on interventions that address individual stressors, although these will always be necessary in particular contexts. Methods for mapping individual and cumulative stressors are well-developed (e.g., Vörösmarty et al., 2010), and analytical tools for prioritizing ecological restoration among sites in multi-stressor landscapes are emerging (Hermoso et al., 2015; Neeson et al., 2016). Strategic portfolios of restoration measures require development of cause-and-effect relationships to understand and predict the responses of species and communities to individual and multiple-stressor configurations. Maasri et al. (2021) recommend assessment of restoration outcomes using large-scale replication of before-after-control-impact (BACI) designs, and long-term post-monitoring phases. Relatively few restoration projects meet these stringent design and monitoring requirements (Palmer et al., 2005; Geist and Hawkins, 2016). Meta-analyses of results from post-monitoring can help to identify restoration failures (often under-reported, Geist, 2011) as well as successes, extract learnings and guide adaptation toward more effective strategies. In many situations with a long history of anthropogenic stress it is important to be realistic about the potential for restoration of near-natural ecological systems (Geist and Hawkins, 2016). Rehabilitation or remediation to recover and sustain selected ecosystem values and species may be the only feasible approach, especially where novel ecosystems with well-established alien species have replaced natural system structures, biodiversity and processes, as in many impounded rivers and degraded floodplain wetlands (Acreman et al., 2014; Poff et al., 2017). These novel circumstances require careful development of explicit and realistic targets for the recovery of the system at project onset (Geist, 2011, 2015; Geist and Hawkins, 2016). A framing termed Strategic Adaptive Management (SAM) offers a structured step-wise process from development of a shared vision and hierarchy of objectives linked to management actions, monitoring, evaluation and publication of outcomes (Kingsford et al., 2021). It amply meets the criteria for measuring restoration and management success from an ecological perspective (Palmer et al., 2005) and provides a powerful model of effective stakeholder collaboration. Broad stakeholder engagement throughout project design, implementation and monitoring strengthens comprehension of the multiple challenges of ecosystem restoration, and encourages appreciation of what can be achieved and is worthy of investment. Freshwater ecosystem restoration, rewilding, rehabilitation and remediation are technically feasible with existing and emerging technologies, collaborative human commitment and adequate resourcing. The IUCN ecosystem typology provides a template for identification of risks and restoration priorities at global scale. As an example, severe threats to freshwater biodiversity in the Amazon Basin (overexploitation, deforestation, extensive hydroelectric dam development and climate change) demand a portfolio of recovery actions (Duponchelle et al., 2021) and spatially explicit prioritization of future hydropower developments to minimize loss of aquatic connectivity and biodiversity (Winemiller et al., 2016). Ecosystem restoration is challenging, expensive and may require decades of sustained effort to maintain the desired outcomes. Prevention of biodiversity loss is a far better option than struggling for cures. Perfectly located, designed and managed freshwater protected areas (PAs) represent the pinnacle of global conservation policy. Many categories of area-based protected ecosystems (IUCN I–VI PAs, Ramsar list of Wetlands of International Importance, private protected areas, landholder covenants, indigenous stewardship) play significant roles in freshwater biodiversity conservation. In 2010, the Convention on Biological Diversity (CBD) included an area target of 17% protection for inland waters. However, 70% of river reaches (by length) have no protected areas in their upstream catchments, and only 11.1% (by length) achieve full integrated protection (Abell et al., 2017). Seasonal inland wetlands represent ~6% of the world's land surface, yet around 89% are unprotected by IUCN PAs and Ramsar sites (Reis et al., 2017). Urgent calls for increased protection of freshwater ecosystems and biodiversity include free-flowing rivers (Perry et al., 2021), river-wetland mosaics (Reis et al., 2017), springs (Cantonati et al., 2020) and other groundwater-dependent ecosystems, as well as integrated terrestrial-freshwater-estuary/marine protection coordinated across spatial scales, jurisdictions and sectors (Abell et al., 2017; Leal et al., 2020; Buxton et al., 2021). Systematic conservation planning offers data-driven methods for prioritizing restoration and protected area strategies (Abell et al., 2017; Linke et al., 2019). Applications of these approaches have addressed vital issues for freshwater conservation planning (source catchment condition, dimensions of river connectivity, integrated river, wetland and aquifer protection, threatening processes, species distribution shifts under climate change, and trade-offs between freshwater biodiversity conservation and human water requirements). Other tools that can aid similar spatial analysis, provide insights into trade-offs, and inform strategic multi-objective decision-making include pareto-optimal assessments (Hurford and Harou, 2014), Strategic Environmental Assessment (Lazarus et al., 2018) and system-scale infrastructure planning (Winemiller et al., 2016; Opperman et al., 2019). Significant improvements in the placement, spatial configuration and connectivity of protected areas are feasible using these techniques. Recent studies have sought to evaluate the benefits of freshwater protected areas for conservation of freshwater biodiversity. A systematic review found that only 51% of 75 case studies demonstrated beneficial outcomes relative to comparable unprotected areas (Acreman et al., 2020). Activities within and external to protected areas were held responsible, including landscape modifications, riparian loss, alterations to hydrological regimes, loss of floodplain connectivity, habitat alterations, chemical contamination, fishing, harvesting (e.g., turtle eggs) and the presence of non-native species. Over-exploited and degraded protected areas add to the burden of ecosystem restoration and recovery facing many societies. Ecological principles and guidelines for improved use, management and monitoring of freshwater protected areas and their surrounding landscapes warrant far wider appreciation and application (Finlayson, 2018; Acreman et al., 2020). Strengthening the conservation benefits of freshwater protected areas requires engagement and collaboration among scientists, management agencies and the people who visit, know and use these areas. Increased public engagement, citizen science and participatory monitoring of trends in condition or species abundance by committed stakeholders can raise the profile of freshwater biodiversity and help to change behaviors that might otherwise lead to ecosystem damage. Positive socio-economic outcomes as well as biodiversity conservation are important, and more likely to occur when PAs adopt co-management regimes (e.g., fisheries), empower local people, reduce economic inequalities, and maintain cultural and livelihood benefits (Oldekop et al., 2016). Freshwater ecosystems and their catchments are increasingly viewed as coupled human and natural systems, wherein setting objectives and devising management solutions, require engagement and collaboration among engineers and hydrologists, ecologists, social scientists and citizens (Bunn, 2016). This has been advocated and implemented in the field of environmental water management for decades (Poff et al., 2003, 2017) and is a strong element of The Brisbane Declaration and Global Action Agenda on Environmental Flows (Arthington et al., 2018; Anderson et al., 2019). Ecosystem-based Management (EBM), also referred to as the ‘Ecosystem Approach', jointly considers societal and ecological goals and scenarios in an impressive modeling framework (Langhans et al., 2019). The EBM and similar framings (e.g., SAM) recognize the need for coupling of social and ecological systems, and engagement of all stakeholders. The concept of “stakeholders” has often meant token representation of indigenous, marginalized or poorly recognized societal groups. Yet increasingly, solving complex conflicts about water use and management, especially in times of scarcity and uncertainty, requires collaboration and enduring partnerships among all stakeholders with indigenous, societal and scientific knowledge, technical expertise, and credentials at all levels of governance. Recent reviews consistently call for improved practices to enhance communication, understanding and respect for different “ways of knowing,” and methods for blending of stakeholder knowledge (especially indigenous knowledge) with conventional science (Anderson et al., 2019; Buxton et al., 2021; Maasri et al., 2021; Perry et al., 2021). Others call for evidence-based and targeted guidance to facilitate working with the complex dynamic interactions of ecological and societal systems (Harper et al., 2021). The framing termed Coupled Human and Natural Systems (CHANS) is especially relevant. It proposes strategic integration of patterns and processes that connect human and natural systems, as well as within-scale and cross-scale interactions and feedbacks between human and natural components of such systems (Liu et al., 2021). Interesting applications to freshwater systems include evaluation of water availability, use, quality, management and governance in Canadian agricultural watersheds (Liu et al., 2019) and fisheries management (Lynch and Liu, 2014). CHANS, SAM and EBM embrace important principles of socio-ecological collaboration and governance, including building trust, maintaining respectful interactions, upholding rights, embracing mutual understanding, and development of enduring partnerships. These integrated socio-ecological frameworks and partnership models offer fundamental tools to guide understanding and management of increasingly degraded Anthropocene ecosystems, in which societal and ecological processes are deeply entwined and interact. Socio-ecological systems in turn require participatory management and governance regimes that can foster biodiversity conservation alongside societal benefits and social justice. For example, a “Just Aquatic Governance” framework has been proposed for the Amazon Basin, based on three pillars of social justice: recognitional, procedural and distributional (Lopes et al., 2021). The need for inclusive socio-ecological freshwater science and governance is particularly acute in the biodiverse, multicultural Amazon Basin (Castello, 2021). The Post-2020 Global Biodiversity Framework is visionary and compelling, and especially relevant to the recovery of freshwater biodiversity—the most overlooked and urgent conservation challenge of the next decade. The IUCN has distinguished 28 global freshwater ecosystem types, a powerful framing for activities to promote the recovery and conservation of freshwater biodiversity. This challenge paper supports the freshwater Emergency Recovery Plan by promoting a broader package of strategic activities that too often operate in silos, with patchy coverage of the world's freshwater ecosystem types and biogeographic diversity and cultural heritage. This portfolio urges integration of biodiversity inventory and basic ecosystem science, stressor assessment and mapping with systematic restoration and protected area management in a strategic global freshwater conservation strategy, with links to terrestrial and estuarine/marine realms as required. An overarching and integrative theme is the coupling of ecological and human systems, and the importance of collaboration among all stakeholders with indigenous, societal and scientific knowledge, technical expertise, and experience with governance models and policy development. There is an urgent need to build shared knowledge, trust, mutual understanding and enduring respectful partnerships in coupled human-ecological systems if we want a world of people “living in harmony with nature.” The author confirms being the sole contributor of this work and has approved it for publication. The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 1. ^ https://www.cbd.int/convention/guide/?id=web4 2. ^ https://sustainabledevelopment.un.org/sdgs 3. ^ https://www.iucn.org/theme/global-policy/our-work/united-nations-framework-convention-climate-change-unfccc 4. ^ http://www.fao.org/forestry/48209-0cb7240cc9f200dcf507a40e71c39a591.pdfs 5. ^ https://blog.oup.com/2020/09/bring-living-waters-back-to-our-planet/ 6. ^ https://ec.europa.eu/environment/pubs/pdf/factsheets/wfd/en.pdf Abell, R., Lehner, B., Thieme, M., and Linke, S. (2017). Looking beyond the fence line: assessing protection gaps for the world's rivers. Conserv. Lett. 10, 384–394. doi: 10.1111/conl.12312 CrossRef Full Text | Google Scholar Acreman, M., Arthington, A. H., Colloff, M. J., Couch, C., Crossman, N. D., Dyer, F., et al. (2014). Environmental flows for natural, hybrid, and novel riverine ecosystems in a changing world. Frontiers Ecol. Env. 12, 466–473. doi: 10.1890/130134 CrossRef Full Text | Google Scholar Acreman, M., Hughes, K. A., Arthington, A. H., Tickner, D., and Dueñas, M.-A. (2020). Protected areas and freshwater biodiversity: a novel systematic review distils eight lessons for effective conservation. Conserv. Lett. 13, e12684–e12020. doi: 10.1111/conl.12684 CrossRef Full Text | Google Scholar Anderson, E. P., Jackson, S., Tharme, R. E., Douglas, M., Flotemersch, J. E., Zwarteveen, M., et al. (2019). Understanding rivers and their social relations: a critical step to advance environmental water management. WIREs Water. 6:e1381. doi: 10.1002/wat2.1381 PubMed Abstract | CrossRef Full Text | Google Scholar Arthington, A. H., Bhaduri, A., Bunn, S. E., Jackson, S., Tharme, R. E., Tickner, D., et al. (2018). The brisbane declaration and global action agenda on environmental flows (2018). Front. Environ. Sci. 6:45. doi: 10.3389/fenvs.2018.00045 CrossRef Full Text | Google Scholar Booth, D. B., Roy, A. H., Smith, B., and Capps, K. A. (2016). Global perspectives on the urban stream syndrome. Freshw. Sci. 35, 412–420. doi: 10.1086/684940 CrossRef Full Text | Google Scholar Bunn, S. E. (2016). Grand Challenge for the Future of Freshwater Ecosystems. Front. Environ. Sci. 4:21. doi: 10.3389/fenvs.2016.00021 CrossRef Full Text | Google Scholar Buxton, R. T., Bennett, J. R., Reid, A. J, Shulman, C., Cooke, S. J., Francis, C. M., et al. (2021). Key information needs to move from knowledge to action for biodiversity conservation in Canada. Biol. Conserv. 256:108983. doi: 10.1016/j.biocon.2021.108983 CrossRef Full Text | Google Scholar Cantonati, M., Fensham, R. J., Stevens, L. E., Gerecke, R., Glazier, D. S., Goldscheider, N., et al. (2020). Urgent plea for global protection of springs. Conserv. Biol. 35, 378–382. doi: 10.1111/cobi.13576 PubMed Abstract | CrossRef Full Text | Google Scholar Castello, L. (2021). Science for conserving Amazon freshwater ecosystems. Aquatic Conserv. (in press). Google Scholar Craig, L. S., Olden, J. D., Arthington, A. H., Entrekin, S., Hawkins, C. P., Kelly, J. J., et al. (2017). Meeting the challenge of interacting threats in freshwater ecosystems: a call to scientists and managers. Elementa 5:72. doi: 10.1525/elementa.256 CrossRef Full Text | Google Scholar Darwall, W., Bremerich, V., De Wever, A., Dell, A. I., Freyhof, J., Gessner, M. O., et al. (2018). The Alliance for Freshwater Life: A global call to unite efforts for freshwater biodiversity science and conservation. Aquatic Conserv. 28, 1015–1022. doi: 10.1002/aqc.2958 CrossRef Full Text | Google Scholar Datry, T., Boulton, A. J., Bonada, N., Fritz, K., Leigh, C., Sauquet, E., et al. (2018). Flow intermittence and ecosystem services in rivers of the Anthropocene. J. Appl. Ecol. 55, 353–364. doi: 10.1111/1365-2664.12941 PubMed Abstract | CrossRef Full Text | Google Scholar Dudgeon, D., Arthington, A. H., Gessner, M. O., Kawabata, Z. I., Knowler, D. J., Leveque, C., et al. (2006). Freshwater biodiversity: importance, threats, status and conservation challenges. Biol. Rev. 81, 163–182. doi: 10.1017/S1464793105006950 PubMed Abstract | CrossRef Full Text | Google Scholar Duponchelle, F., Isaac, V. J., Doria, C., Van Damme, P. A., Herrera, -R G. A., et al. (2021). Conservation of migratory fishes in the Amazon basin. Aquatic Conserv. doi: 10.1002/aqc.3550 CrossRef Full Text | Google Scholar Finlayson, C.M., Arthington, A.H., and Pittock, J., (Eds.) (2018). Freshwater Ecosystems in Protected Areas: Conservation and Management. Earthscan Studies in Water Resource Management. London: Taylor and Francis Group; Routledge. doi: 10.4324/9781315226385 CrossRef Full Text | Google Scholar Flitcroft, R., Cooperman, M. S., Harrison, I. J., Juffe-Bignoli, D., and Boon, P. J. (2019). Theory and practice to conserve freshwater biodiversity in the Anthropocene. Aquatic Conserv. 29, 1013–1021. doi: 10.1002/aqc.3187 CrossRef Full Text | Google Scholar Geist, J. . (2011). Integrative freshwater ecology and biodiversity conservation. Ecol. Indic. 11, 1507–1516. doi: 10.1016/j.ecolind.2011.04.002 PubMed Abstract | CrossRef Full Text | Google Scholar Geist, J. (2015). Seven steps towards improving freshwater conservation. Aquatic Conserv. 25, 447–453. doi: 10.1002/aqc.2576 CrossRef Full Text | Google Scholar Geist, J., and Hawkins, S. J. (2016). Habitat recovery and restoration in aquatic ecosystems: current progress and future challenges. Aquatic Conserv. 26, 942–962 doi: 10.1002/aqc.2702 CrossRef Full Text | Google Scholar Grill, G., Lehner, B., Thieme, M., Geenen, B., Tickner, D., Antonelli, F., et al. (2019). Mapping the world's free-flowing rivers. Nature 569, 215–221. doi: 10.1038/s41586-019-1111-9 PubMed Abstract | CrossRef Full Text | Google Scholar Harper, M., Mejbel, H. S., Longert, D., Abell, R., Baird, T. D., Bennett, J. R., et al. (2021). Twenty-five essential research questions to enhance the protection and restoration of freshwater biodiversity. Aquatic Conserv. (in press). Google Scholar Hermoso, V., Pantus, F., Olley, J., Linke, S., Mugodo, J., and Lea, P. (2015). Prioritising catchment rehabilitation for multi objective management: an application from SE-Queensland, Australia. Ecol. Model. 316, 168–175. doi: 10.1016/j.ecolmodel.2015.08.017 CrossRef Full Text | Google Scholar Horne, A. C., Webb, J. A., Stewardson, M. J., Richter, B. D., and Acreman, M., (Eds). (2017). Water for the Environment: From Policy and Science to Implementation and Management. Cambridge, MA: Elsevier. Google Scholar Hurford, A. P., and Harou, J. J. (2014). Balancing ecosystem services with energy and food security – assessing trade-offs from reservoir operation and irrigation investments in Kenya's Tana Basin. Hydrol. Earth Syst. Sci. 18, 3259–3277. doi: 10.5194/hess-18-3259-2014 CrossRef Full Text | Google Scholar Keith, D.A., Ferrer-Paris, J.R., Nicholson, E., and Kingsford, R.T. (Editors),. (2021). IUCN Global Ecosystem Typology 2.0: descriptive profiles for biomes and ecosystem functional groups. IUCN, Gland, Switzerland. Available online at: https://portals.iucn.org/library/node/49250 Google Scholar Kingsford, R. T., McLoughlin, C. A., Brandle, R., Bino, G., Cockayne, B., Schmarr, D., et al. (2021). Adaptive management of Malkumba-Coongie Lakes ramsar site in Arid Australia-a free flowing river and Wetland System. Sustainability 13:3043. doi: 10.3390/su13063043 CrossRef Full Text | Google Scholar Langhans, S. D., Domisch, S., Balbi, S., Delacámara, G., Hermoso, V., Kuemmerlene, M., et al. (2019). Combining eight research areas to foster the uptake of ecosystem-based management in fresh waters. Aquatic Conserv. 29, 1161–1173. doi: 10.1002/aqc.3012 CrossRef Full Text | Google Scholar Lazarus, K. M., Corbett, M., Cardinale, P., Lin, N. S., Noeske, T., Kumar, V., et al. (2018). Strategic Environmental Assessment of the Myanmar Hydropower Sector. Washington, DC: International Finance Corporation. Available online at: www.elibrary.worldbank.org Google Scholar Leal, C. G., Lennox, G. D., Ferraz, S. F. B., Ferreira, J., Gardner, T. A., et al. (2020). Integrated terrestrial-freshwater planning doubles conservation of tropical aquatic species. Science 370, 117–121. doi: 10.1126/science.aba7580 PubMed Abstract | CrossRef Full Text | Google Scholar Linke, S., Hermoso, V., and Januchowski-Hartley, S. (2019). Toward process-based conservation prioritizations for freshwater ecosystems. Aquatic Conserv. 29, 1149–1160. doi: 10.1002/aqc.3162 CrossRef Full Text | Google Scholar Liu, J., Baulch, H. M., Macrae, M. L., Wilson, H. F., Elliott, J. A., Bergstrom, L., et al. (2019). Agricultural water quality in cold climates: processes, drivers, management options, and research needs. J. Env. Quality 48, 792–802. doi: 10.2134/jeq2019.05.0220 PubMed Abstract | CrossRef Full Text | Google Scholar Liu, J., Dietz, T., Carpenter, S. R., Taylor, W. W., Alberti, M., Deadman, P., et al. (2021). Coupled human and natural systems: the evolution and applications of an integrated framework. Ambio. doi: 10.1007/s13280-020-01488-5 PubMed Abstract | CrossRef Full Text | Google Scholar Lopes, P. F. M., de Freitas, C. T., Hallwass, G., Silvano, R. A. M., Begossi, A, and Campos-Silva, J.V. (2021). Just aquatic governance: the amazon basin as fertile ground for aligning participatory conservation with social justice. Aquatic Conserv. doi: 10.1002/aqc.3586 CrossRef Full Text | Google Scholar Lynch, A., and Liu, J. (2014). “Fisheries as coupled human and natural systems,” in Future of Fisheries: Perspectives for Emerging Professionals, ed. W.W. Taylor, A. Lynch, and N. Leonard, (Washington, DC: American Fisheries Society Press). Google Scholar Lynch, A. J., Cooke, S. J., Deines, A. M., Bower, S. D., Bunnell, D. B., Cowx, I.G., et al. (2016). The social, economic, and environmental importance of inland fish and fisheries. Environ. Rev. 24, 115–121. doi: 10.1139/er-2015-0064 PubMed Abstract | CrossRef Full Text | Google Scholar Maasri, A., Jähnig, S. C., Adamescu, M. C., Adrian, R., Baigun, C., Baird, D., et al. (2021). A Global Agenda for Advancing Freshwater Biodiversity Research. Authorea. doi: 10.22541/au.161640764.49902060/v1 CrossRef Full Text | Google Scholar Neeson, T. M., Smith, S. D. P., Allan, J. D., and McIntyre, P. B. (2016). Prioritizing ecological restoration among sites in multi-stressor landscapes. Ecol. Appl. 26, 1785–1796. doi: 10.1890/15-0948.1 PubMed Abstract | CrossRef Full Text | Google Scholar Oldekop, J. A., Holmes, G., Harris, W. E., and Evans, K. L. (2016). A global assessment of the social and conservation outcomes of protected areas. Cons. Biol. 30, 133–141. doi: 10.1111/cobi.12568 PubMed Abstract | CrossRef Full Text | Google Scholar Omerod, S. J., Dobson, M., Hildrew, A. G., and Townsend, C. R. (2010). Multiple stressors in freshwater ecosystems. Freshw. Biol. 55 (Suppl. 1); 1–4. doi: 10.1111/j.1365-2427.2009.02395.x CrossRef Full Text | Google Scholar Opperman, J. J., Kendy, E., and Barrios, E. (2019). Securing environmental flows through system reoperation and management: lessons from case studies of implementation. Front. Environ. Sci. 7:104. doi: 10.3389/fenvs.2019.00104 CrossRef Full Text | Google Scholar Palmer, M., Bernhardt, E., Allan, J. D., Lake, P. S., Alexander, G., Brooks, S., et al. (2005). Standards for ecologically successful river restoration. J. App. Ecol. 42, 208–217. doi: 10.1111/j.1365-2664.2005.01004.x CrossRef Full Text | Google Scholar Perry, D., Harrison, I., Fernandes, S., Burnham, S., and Nichols, A. (2021). Global analysis of durable policies for free-flowing river protections. Sustainability 13:2347. doi: 10.3390/su13042347 CrossRef Full Text | Google Scholar Poff, N. L., Allan, J. D., Palmer, M. A., Hart, D. D., Richter, B. D., Arthington, A. H., et al. (2003). River flows and water wars: emerging science for environmental decision making. Front. Ecol. Env. 1, 298–306. doi: 10.1890/1540-9295(2003)0010298:RFAWWE2.0.CO CrossRef Full Text | Google Scholar Poff, N. L., Tharme, R. E., and Arthington, A. H. (2017). “Evolution of environmental flows assessment science, principles, and methodologies,” in Water for the Environment, eds A. C. Horne, J. A. Webb, M. J. Stewardson, B. Richter, and M. Acreman (London: Academic Press), 203–236. doi: 10.1016/B978-0-12-803907-6.00011-5 CrossRef Full Text | Google Scholar Reid, A. J., Carlson, A. K., Creed, I. F., Eliason, E. J., Gell, P. A., Johnson, P. T., et al. (2019). Emerging threats and persistent conservation challenges for freshwater biodiversity. Biol. Rev. 94, 849–873. doi: 10.1111/brv.12480 PubMed Abstract | CrossRef Full Text | Google Scholar Reis, V., Hermoso, V., Hamilton, S. K., Ward, D., Fluet-Chouinard, E., Lehner, B., et al. (2017). A global assessment of inland wetland conservation status. Bioscience 67, 523–533. doi: 10.1093/biosci/bix045 CrossRef Full Text | Google Scholar Sabater, S., Elosegi, A., and Ludwig, R. (2018). Multiple Stressors in River Ecosystems: Status, Impacts and Prospects for the Future. Amsterdam: Elsevier. Google Scholar Sheldon, F., Peterson, E. E., Boone, E. L., Sippel, S., Bunn, S. E., and Harch, B. D. (2012). Identifying the spatial scale of land-use that most strongly influences overall river ecosystem health score. Ecol. Appl. 22, 2188–2203. doi: 10.1890/11-1792.1 PubMed Abstract | CrossRef Full Text | Google Scholar Tickner, D., Opperman, J. O., Abell, R., Acreman, A., Arthington, A. H., Bunn, S. E., et al. (2020). Bending the curve of global freshwater biodiversity loss: an emergency recovery plan. BioScience 70, 330–342, doi: 10.1093/biosci/biaa002 PubMed Abstract | CrossRef Full Text | Google Scholar van Rees, C. B., Waylen, K. A., Schmidt-Kloiber, A., Thackeray, S. J., Kalinkat, G., Martens, K., et al. (2020). Safeguarding freshwater life beyond 2020: recommendations for the new global biodiversity framework from the European experience. Conserv. Lett. 4:e12771. doi: 10.20944/preprints202001.0212.v1 CrossRef Full Text | Google Scholar Vörösmarty, C. J., McIntyre, P. B., Gessner, M. O., Dudgeon, D., Prusevich, A., Green, P. A., et al. (2010). Global threats to human water security and river biodiversity. Nature 467, 555–561. doi: 10.1038/nature09440 PubMed Abstract | CrossRef Full Text | Google Scholar Winemiller, K. O., McIntyre, P. B, Castello, L., Fluet-Chouinard, E., Giarrizzo, T., Nam, S., et al. (2016). Balancing hydropower and biodiversity in the Amazon, Congo, and Mekong. Science 351, 128–129. doi: 10.1126/science.aac7082 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: biodiversity, ecosystem services, multiple stressors, restoration, protected areas, socio-ecological governance, stakeholders Citation: Arthington AH (2021) Grand Challenges to Support the Freshwater Biodiversity Emergency Recovery Plan. Front. Environ. Sci. 9:664313. doi: 10.3389/fenvs.2021.664313 Received: 05 February 2021; Accepted: 06 April 2021; Published: 10 May 2021. Edited by: Reviewed by: Copyright © 2021 Arthington. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Angela H. Arthington, [email protected]
Junjun Li, Yanan Chen, Zhanxi Fan,
Frontiers in Energy Research, Volume 9; https://doi.org/10.3389/fenrg.2021.676876

Abstract:
Editorial on the Research TopicEmerging Technologies for Materials Design and Characterization in Energy Conversion and Storage The increasing consumption of fossil fuels leads to energy crisis and environmental issues, which seriously affects human daily life. To date, great efforts have been made to explore sustainable, eco-friendly and renewable energy alternatives to fossil fuels. In the past few decades, various energy conversion and storage technologies, such as water splitting (Zhang F. et al., 2019; Hu et al., 2021; Wu et al., 2021), proton exchange membrane fuel cells (Edwards et al., 2008; Park et al., 2012), nitrogen reduction reaction (NRR) (Wan et al., 2019; Zhang W. et al., 2019; Yang et al., 2020b; Li et al., 2021), CO2 reduction reaction (CO2RR) (Ozdemir et al., 2019; Liu et al., 2020; Yang et al., 2020a; Ma et al., 2021; Wang et al., 2021), and metal-air batteries (Cheng and Chen, 2012) have shown promising potential due to the high efficiency, energy security, and environmental protection. In these fields, more attention has been paid to preparing advanced materials with outstanding performance, and developing advanced technologies for prediction, characterization and detection (Centi, 2020). Electrocatalytic NRR to NH3 has been regarded as an attractive alternative to the traditional Haber-Bosch process owing to its lower energy consumption under ambient conditions (Tang and Qiao, 2019; Yang et al., 2020b). The development of advanced NRR catalysts with outstanding performance and low costs is highly desired. Recently, Wang et al. reported that the ringlike V2O3 nanostructures could effectively convert N2 to NH3 under ambient conditions. Scanning electron microscopy analysis shows that the ringlike structure is uniform with outer diameter of 350–500 nm. Transmission electron microscopy (TEM) analysis confirms that such nanoring possesses a rough surface, displaying more active sites. The high-resolution TEM image of an individual nanoring indicates a contracted interplanar distance of 0.211 nm, corresponding to the (113) plane. This work presents a facile strategy to fabricate the advanced non-noble-metal catalysts for NRR. It is believed that more effective and stable electrocatalysts would be developed for boosting the NRR in the future. Energy efficiency is another efficacious way to alleviate the energy crisis. In the field of energy-saving optoelectronics, electrochromic devices (ECDs) have shown great advantages. Among various fabrication materials for ECDs, coordination polymer (CP) shows a broad application prospect due to good cycle stability, high color rendering efficiency, and fast switching speed. Liu et al. present a comprehensive survey of the current achievements and progresses of CP in energy efficient ECDs from the aspect of influence of composition, coordination bonding and microstructure of pyridine-based CP on the performance of ECDs. This work is expected to provide the guideline for achieving a substantial enhancement in electrochromic and other optoelectronic fields. Nevertheless, one of the paramount challenges to develop new high-efficiency energy transformation materials is the long span from experiment to practical application, due to the complexity of research objects and methods, insufficient personal accumulated experience, etc. (Luo et al.) Artificial intelligence (AI) has potential for solving the problems mentioned above. Luo et al. investigated and summarized research works on energy storage materials for capacitors and Li-ion batteries. They pointed out that machine learning (ML), as a subset of AI, algorithms can reduce test number of cycles and required experiments, which greatly reduces time consumption and accelerates every stage of development. In addition, they summarized the status and progress of AI in energy storage materials and present solutions to relevant deficiencies, such as the establishment of a database, extracting data from unstructured literature with automaticity and high efficiency and accuracy, etc. Apart from saving time, AI can predict the performance of materials, monitor reaction processes, and explore reaction mechanisms (Luo et al.; Yang et al.). Focusing on the superiority of AI in predicting experiments, Yang et al. reviewed the situation and application of AI in respects of optoelectronic materials, hydrogen peroxidation catalysts, water electrolysis catalysts and microbial fuel cells. It indicates that the relationship between prediction and actual experiments is mutually facilitating. In other words, the efficiency of actual material processing can be promoted with accurate prediction, and the database for AI is extended. In conclusion, the development of advanced materials and technologies for energy conversion and storage are of vital importance. Until now, various promising materials with excellent performance have been prepared, such as carbon nanomaterials [nanofibers (Zhao et al., 2018; Lee et al., 2020), nanotubes (Ma et al., 2019; Sun et al., 2020; Tuo et al., 2020; Zhang et al., 2020), graphene (Chen et al., 2020), etc.], reticular structure [metal-organic framework (Nam et al., 2018), covalent organic framework (Lin et al., 2015)], and tandem catalyst (Morales-Guio et al., 2018), etc. It is worth mentioning that traditional technologies in detection and characterization are gradually substituted with new and advanced solutions, such as computer science (AI, ML, etc.), and in-situ characterization (In-situ/operando synchrotron radiation, in-situ/operando morphology/spectrum, etc.). It is believed the emerging technologies for materials design and characterization in energy conversion and storage will be greatly developed in the future. ZZ supervised the project. JL wrote the manuscript. ZZ, YC, and ZF revised the manuscript. All authors contributed to the article and approved the submitted version. This work was supported by the National Natural Science Foundation of China (22071172). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Centi, G. (2020). Smart catalytic materials for energy transition. SmartMat. 1:e1005. doi: 10.1002/smm2.1005 CrossRef Full Text | Google Scholar Chen, C, Yan, X., Liu, S., Wu, Y., Wan, Q., Sun, X., et al. (2020). Highly efficient electroreduction of CO2 to C2+ alcohols on heterogeneous dual active sites. Angew. Chem. Int. Ed. 59, 16459–16464. doi: 10.1002/anie.202006847 PubMed Abstract | CrossRef Full Text | Google Scholar Cheng, F., and Chen, J. (2012). Metal-air batteries: from oxygen reduction electrochemistry to cathode catalysts. Chem. Soc. Rev. 41, 2172–2192. doi: 10.1039/c1cs15228a PubMed Abstract | CrossRef Full Text | Google Scholar Edwards, P. P., Kuznetsov, V. L., David, W. I. F., and Brandon, N. P. (2008). Hydrogen and fuel cells: towards a sustainable energy future. Energy Policy 36, 4356–4362. doi: 10.1016/j.enpol.2008.09.036 CrossRef Full Text | Google Scholar Hu, H., Wang, Z., Cao, L., Zeng, L., Zhang, C., Lin, W., et al. (2021). Metal-organic frameworks embedded in a liposome facilitate overall photocatalytic water splitting. Nat. Chem. 13, 358–366. doi: 10.1038/s41557-020-00635-5 PubMed Abstract | CrossRef Full Text | Google Scholar Lee, J. C., Kim, J. Y., Joo, W. H., Hong, D., Oh, S. H., Kim, B., et al. (2020). Thermodynamically driven self-formation of copper-embedded nitrogen-doped carbon nanofiber catalysts for a cascade electroreduction of carbon dioxide to ethylene. J. Mater. Chem. A. 8, 11632–11641. doi: 10.1039/D0TA03322G CrossRef Full Text | Google Scholar Li, Y., Li, J., Huang, J., Chen, J., Kong, Y., Yang, B., et al. (2021). Boosting electroreduction kinetics of nitrogen to ammonia via tuning electron distribution of single-atomic iron sites. Angew. Chem. Int. Ed. 60, 9078–9085. doi: 10.1002/anie.202100526 PubMed Abstract | CrossRef Full Text | Google Scholar Lin, S., Diercks, C. S., Zhang, Y., Kornienko, N., Nichols, E. M., Zhao, Y., et al. (2015). Covalent organic frameworks comprising cobalt porphyrins for catalytic CO2 reduction in water. Science 349, 1208–1213. doi: 10.1126/science.aac8343 PubMed Abstract | CrossRef Full Text | Google Scholar Liu, H., Zhu, Y., Ma, J., Zhang, Z., and Hu, W. (2020). Recent advances in atomic-level engineering of nanostructured catalysts for electrochemical CO2 reduction. Adv. Func. Mater. 30:1910534. doi: 10.1002/adfm.201910534 CrossRef Full Text | Google Scholar Ma, C., Hou, P., Wang, X, Wang, Z., Li, W., and Kang, P. (2019). Carbon nanotubes with rich pyridinic nitrogen for gas phase CO2 electroreduction. Appl. Catal. B-Environ. 250, 347–354. doi: 10.1016/j.apcatb.2019.03.041 CrossRef Full Text | Google Scholar Ma, D., Han, S., Cao, C., Wei, W., Li, X., Chen, B., et al. (2021). Bifunctional single-molecular heterojunction enables completely selective CO2-to-CO conversion integrated with oxidative 3D nano-polymerization. Energy Environ. Sci. 14, 1544–1552. doi: 10.1039/D0EE03731A CrossRef Full Text | Google Scholar Morales-Guio, C. G., Cave, E. R., Nitopi, S. A., Feaster, J. T., Wang, L., Kuhl, K. P., et al. (2018). Improved CO2 reduction activity towards C2+ alcohols on a tandem gold on copper electrocatalys. Nat. Catal. 1, 764–771. doi: 10.1038/s41929-018-0139-9 CrossRef Full Text | Google Scholar Nam, D., Bushuyen, O. S., Li, J., Luna, P. D., Seifitokaldani, A., Dinh, C. T., et al. (2018). Metal-organic frameworks mediate Cu coordination for selective CO2 electroreduction. J. Am. Chem. Soc. 140, 11378–12386. doi: 10.1021/jacs.8b06407 PubMed Abstract | CrossRef Full Text | Google Scholar Ozdemir, J., Mosleh, I., Abolhassani, M., Greenlee, L. F., Beitle, R. R., and Beyzavi, M. H. (2019). Covalent organic frameworks for the capture, fixation, or reduction of CO2. Front. Energy Res. 7:77. doi: 10.3389/fenrg.2019.00077 CrossRef Full Text | Google Scholar Park, S., Shao, Y., Liu, J, and Wang, Y. (2012). Oxygen electrocatalysts for water electrolyzers and reversible fuel cells: status and perspective. Energy Environ. Sci. 5, 9331–9344. doi: 10.1039/c2ee22554a CrossRef Full Text | Google Scholar Sun, X., Zhang, Q., L, Q., Zhang, X., Shao, X., Yi, J., et al. (2020). Utilization of carbon nanotube and graphene in electrochemical CO2 reduction. Biointerface Res. Appl. Chem. 10, 5815–5827. doi: 10.33263/BRIAC104.815827 CrossRef Full Text | Google Scholar Tang, C., and Qiao, S. (2019). How to explore ambient electrocatalytic nitrogen reduction reliably and insightfully. Chem. Soc. Rev. 48, 3166–3180. doi: 10.1039/C9CS00280D PubMed Abstract | CrossRef Full Text | Google Scholar Tuo, J., Lin, Y., Zhu, Y., Jiang, H., Li, Y., Cheng, L., et al. (2020). Local structure tuning in Fe-N-C catalysts through support effect for boosting CO2 electroreduction. Appl. Catal. B-Environ. 272:118960. doi: 10.1016/j.apcatb.2020.118960 CrossRef Full Text | Google Scholar Wan, Y., Xu, J., and Lv, R. (2019). Heterogeneous electrocatalysts design for nitrogen reduction reaction under ambient conditions. Mater. Today 27, 69–90. doi: 10.1016/j.mattod.2019.03.002 CrossRef Full Text | Google Scholar Wang, N., Miao, R., Lee, G., Vomiero, A., Sinton, D., Ip, A. H., et al. (2021). Suppressing the liquid product crossover in electrochemical CO2 reduction. SmartMat. 2, 12–16. doi: 10.1002/smm2.1028 CrossRef Full Text | Google Scholar Wu, D., Kusada, K., Yoshioka, S., Yamamoto, T., Toriyama, T., Matsumura, S., et al. (2021). Efficient overall water splitting in acid with anisotropic metal nanosheets. Nat. Commun. 12:1145. doi: 10.1038/s41467-021-20956-4 PubMed Abstract | CrossRef Full Text | Google Scholar Yang, C., Li, S., Zhang, Z., Wang, H., Liu, H., Jiao, F., et al. (2020a). Organic-inorganic hybrid nanomaterials for electrocatalytic CO2 reduction. Small 16:2001847. doi: 10.1002/smll.202001847 PubMed Abstract | CrossRef Full Text | Google Scholar Yang, C., Zhu, Y., Liu, J., Qin, Y., Wang, H., Liu, H., et al. (2020b). Defect engineering for electrochemical nitrogen reduction reaction to ammonia. Nano Energy 77:105126. doi: 10.1016/j.nanoen.2020.105126 CrossRef Full Text | Google Scholar Zhang, F., Hu, Y., Sun, R., Fu, H., and Peng, K. (2019). Gold-sensitized silicon/ZnO core/shell nanowire array for solar water splitting. Front. Chem. 7:206. doi: 10.3389/fchem.2019.00206 PubMed Abstract | CrossRef Full Text | Google Scholar Zhang, T., Han, X., Yang, H., Han, A., Hu, E., Li, Y., et al. (2020). Atomically dispersed Nickel(I) on an alloy-encapsulated nitrogen-doped carbon nanotube array for high-performance electrochemical CO2 reduction reaction. Angew. Chem. Int. Ed. 59, 12055–12061. doi: 10.1002/anie.202002984 PubMed Abstract | CrossRef Full Text | Google Scholar Zhang, W., Low, J., Long, R., and Xiong, Y. (2019). Metal-free electrocatalysts for nitrogen reduction reaction. EnergyChem 2:100040. doi: 10.1016/j.enchem.2020.100040 CrossRef Full Text | Google Scholar Zhao, Y., Liang, J., Wang, C., Ma, J., and Wallace, G. G. (2018). Tunable and efficient Tin modified nitrogen-doped carbon nanofibers for electrochemical reduction of aqueous carbon dioxide. Adv. Energy. Mater. 8:1702524. doi: 10.1002/aenm.201702524 CrossRef Full Text | Google Scholar Keywords: energy conversion and storage, electrocatalysis, water splitting, CO2 reduction, nitrogen reduction reaction, fuel cell Citation: Li J, Chen Y, Fan Z and Zhang Z (2021) Editorial: Emerging Technologies for Materials Design and Characterization in Energy Conversion and Storage. Front. Energy Res. 9:676876. doi: 10.3389/fenrg.2021.676876 Received: 06 March 2021; Accepted: 12 April 2021; Published: 10 May 2021. Edited by: Reviewed by: Copyright © 2021 Li, Chen, Fan and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Zhicheng Zhang, [email protected]
Vijay Singh Gondil,
Frontiers in Pharmacology, Volume 12; https://doi.org/10.3389/fphar.2021.675440

Abstract:
Bacteriophages (phages) are bacterial predators which shape bacterial population dynamics in nature by rapidly killing their host bacterium (Abedon, 2008; Clokie et al., 2011). Phages were discovered at the beginning of the 20th century and explored for their therapeutic potential to treat human and animal infections (Duckworth and Gulig, 2002). However, the discovery of antibiotics and their rapid development in subsequent years declined the interest in the therapeutic application of phages (Sulakvelidze and Morris, 2001). In recent decades, the emergence of multidrug-resistant bacterial pathogens has spurred clinicians and researchers to look for alternative therapeutic options, which mainly includes phages. Phages have been evaluated in various animal models and clinical trials to establish their clinical relevance in treating drug-resistant bacterial infections (Abdelkader et al., 2019; Brix et al., 2020; Pirnay and Kutter, 2020). Based on the results of previous studies, these entities are considered as fascinating future antimicrobial agents. Interestingly, several pharmaceutical companies are actively engaged in commercializing phage-based therapeutics (Report, 2021). Along with bacteriophages, phage borne lytic proteins known as endolysins have also been extensively investigated in the past few years. Endolysins possess numerous advantageous properties over whole phage entities, which primarily are high specificity, rapid host lysis, modular structure, and low chances of resistance (Gondil et al., 2020b). Endolysins have also been evaluated in several animal infection models as well as phase I and phase II clinical trials to establish their efficacy for the treatment of drug-resistant bacterial infections (Abdelkader et al., 2019; Gondil et al., 2020b). Despite the therapeutic effects of phages and endolysins, these alternative agents face some empirical hurdles posed by the host system, which include low bioavailability, loss of activity, non-targeted delivery, rapid clearance by the reticuloendothelial system and antibody-mediated inactivation (Loh et al., 2020). Authors have also experienced and reported similar limitations in phage and endolysin mediated treatment of animal infection models in their studies (Singla et al., 2015; Singla et al., 2016; Gondil et al., 2021). Against this backdrop, a resurgent interest has been seen among researchers to evaluate the potential of delivery systems for encapsulation of bacteriophages and endolysins. A plethora of phage and endolysin encapsulation techniques have been reported in recent years (Loh et al., 2020). These delivery systems are being exploited for the treatment of acute and chronic infections in animal models by improving pharmacokinetic parameters and altered host immune response against therapeutic entities (Gondil and Chhibber, 2017). Several phage encapsulation studies have explored the potential of various drug delivery systems, which primarily include natural polymers, synthetic polymers, liposomes and electrospun fibers. Polymeric phage encapsulation has been extensively studied in the treatment of gastrointestinal tract infections. These polymers protect phages from harsh acidic conditions, potentially leading to phage inactivation or loss of phage titer. Other than extreme conditions, these polymeric encapsulation materials also protect encapsulated phages from digestive enzymes, bile juices and provide permeability to mucous lining where bacterial pathogens may reside (Malik et al., 2017). Numerous intrinsic properties of natural polymers such as limited sensitivity to enzymatic degradation, pH responsiveness, capable of crosslinking and tailor-made designing also render them a suitable candidate for phage delivery systems. In the gastrointestinal tract, natural polymeric materials such as chitosan, alginate, whey protein provide a safeguard to phages by improving their survival, which in turn increases their therapeutic efficacy (Ma et al., 2008; Dini et al., 2012; Tang et al., 2013; Colom et al., 2017). In pursuit of targeted delivery, Vinner and Malik (2018) demonstrated the alginate containing pH-responsive microencapsulation system triggered the controlled release of active Felix O1 phages in the gastrointestinal tract. Some natural cationic polymers, which primarily include chitosan, are also recognized to possess antiviral properties and reduces the bacteriophage concentration up to 1–2 logs in 1 min of phage-polymer coincubation (Ly-Chatain et al., 2013). These polymeric materials may interfere with phage receptors and modulate electrostatic interaction to impair their adsorption on their host cells, thus decreasing the infectivity and titer of phages. The polymer-phage interaction should be pre-evaluated in different physicochemical parameters to tackle the polymer mediated inactivation of bacteriophages. Similarly, synthetic polymers such as poly (lactic-co-glycolic acid and methacrylate-comethacrylic acid-based phage encapsulation delivery systems were also developed to augment gastrointestinal delivery (Puapermpoonsiri et al., 2009; Stanford et al., 2010). Encapsulated phage preparations exhibited high resistance to gastric acid and reduced fecal shedding of pathogenic bacteria in infection-induced animals. In the last few years, synthetic polymers are also being used for designing smart phage encapsulation systems in the treatment of topical infections, which can release the therapeutic payload on the specific physiochemical trigger. Such systems can be designed using thermo-responsive materials as Staphylococcus aureus phage K was encapsulated in poly (N-isopropylacrylamide)- allylamine nanospheres. These nanospheres undergo a temperature-dependent phase transition to release phage at an elevated temperature associated with bacterial infections (Hathaway et al., 2015). Another smart system class is “pH-sensitive formulations,” which allows the release of loaded phages at a specific pH. A pH-responsive polymer polymethyl methacrylate-co-methacrylic acid was employed to release Proteus mirabilis phage from urinary catheters in response to elevated urinary pH associated with bacterial infection (Milo et al., 2016). A similar system that uses the hyaluronidase enzyme as a trigger to release phages has also been reported in the literature. S. aureus phages were encapsulated in a layer of agarose and hyaluronic acid methacrylate (HAMA) polymer, which is sensitive to hyaluronidase. During infection state, HAMA is solubilized by hyaluronidase (produced from S. aureus) and release phages in the vicinity of pathogens to clear the infection (Bean et al., 2014). Synthetic polymers exert high control in the delivery of therapeutic entities in a range of physicochemical conditions. As compared to natural polymers, encapsulation in synthetic polymers involves the usage of organic solvents, which may decrease the phage concentration or infectivity over time. Phage and solvent choice must be compatible and needs to be pre-evaluated to ensure the therapeutic efficacy of encapsulated phages. In the treatment of gastrointestinal, respiratory, and intracellular pathogens, lipid-based encapsulation of phages in the form of liposomes has been studied extensively in the literature. In oral delivery, other than protection from gastric conditions, liposomes promote mucoadhesiveness for increased retention time in the intestine, which increases the efficacy of phage preparation (Colom et al., 2015). In our laboratory, Singla et al. established the efficacy of encapsulated phage liposome preparation in treating K. pneumoniae induced pneumonia in the murine model. Superior therapeutic efficacy and increased phage concentration was seen in the blood and other organs over a period of time. This observation supports the altered pharmacokinetic behavior and immunological response toward encapsulated over nonencapsulated phages (Singla et al., 2015; Singla et al., 2016). Liposomal phage preparation also showed 100% unresponsive to neutralizing antibodies and retained lytic activity into macrophage by intracellular localization, which is uncommon for native free phages (Singla et al., 2016). In other studies from our laboratory, liposomes and their modified counterparts transferosomes were also evaluated for successful treatment of burn wound and soft tissue infections in animal models (Chadha et al., 2017; Chhibber et al., 2017). From the clinical point of view, the intracellular delivery of phages is critical in treating drug-resistant intracellular pathogens as free phages have limited ability to move across the plasma membrane. Lipid-based encapsulation provides a “Trojan horse strategy,” which involves lipid carrier-mediated intracellular delivery of phages into the host cell to eradicate intracellular pathogens. The lipid-based phage cargos can be further explored for better therapeutic outcomes, including the use of other lipoidal delivery systems (ethosomes, niosomes, and transferosomes), targeted delivery, stimuli-responsive delivery, blending of hydrophilic polymers for increased retention, and positively charged polymers to aid mucosal adhesion. Organic solvents such as chlorinated solvents, ethanol, ethyl acetate, diethyl ether and methanol are generally used in the synthesis of liposomes. These solvents are removed from the formulations by evaporation; however, some traces of these solvents in the final preparation may lead to loss of phage titer, infectivity and presents a risk to human health. The use of advanced evaporation techniques, which includes reverse-phase evaporation, tangential flow filtration and rotatory evaporation methods, efficiently removes the solvent without influencing the stability of liposome formulations. Moreover, phage compatible and less toxic organic solvents such as todiethyl ether can be employed to maintain the therapeutic efficacy and limit the toxicity associated with the phage liposomal formulations. Electrospun phage delivery means encapsulation of phages in polymeric fibers (cellulose diacetate, polyethylene oxide, polyvinyl pyrrolidone and polyvinyl alcohol) using electrospinning (Malik et al., 2017). Electrospun phage preparations are being investigated in the in-vitro models and have shown retained activity of the encapsulated phages. Polycaprolactone electrospun nanofibers based phage encapsulation also showed a 99.99% decrement of Pseudomonas aeruginosa population in 2 h (Nogueira et al., 2017). In a recent study, Phagestaph and Fersis phages were encapsulated and evaluated for their biocompatibility and antibacterial activity against S. aureus and Streptococcus pyogenes (Díaz et al., 2018). However, the electrospun phage delivery system’s therapeutic applicability is limited to in-vitro experiments requiring more extensive validation, especially in animal infection models. In our experience, phage delivery systems are considered extremely effective in the early stages of infection, where only a single dose of phage formulation can significantly eradicate the infection. However, late administration of phage formulation requires co-administration of antibiotic or multiple doses of phage formulation to combat the infection progression. Endolysins, a class of bacteriolytic phage borne proteins, have also been encapsulated to enhance their therapeutic potential. However, unlike phage encapsulation, endolysin delivery systems are still in a very juvenile stage. Endolysin delivery strategies are considered more challenging than phage delivery because of the proteinaceous nature of endolysins and labile enzymatic activity. Organic solvents and harsh encapsulation conditions may affect the structure and function of these enzymatic entities. To date, limited studies have reported the delivery of endolysins in a truncated or full-length version for their controlled delivery and higher antibacterial activity. Hathaway et al. reported the truncated cysteine histidine-dependent amino hydrolase/peptidase (CHAPK) domain of LysK endolysin and lysostaphin in a thermally triggered Poly (N-isopropylacrylamide) (PNIPAM) nanoparticles for delivery of CHAPK cargo at a higher temperature, which is a standard indicator of infection (Hathaway et al., 2015). These delivery carriers can be designed for the delivery of antimicrobial cargos at a specific temperature which may be associated with a particular bacterial infection. Along with the development of phage delivery systems, our laboratory has extensively explored the delivery systems for anti-streptococcal and anti-staphylococcal endolysins. An anti-streptococcal full-length Cpl-1 endolysin was loaded into mucoadhesive chitosan nanoparticles for their pulmonary delivery and high antibacterial activity (Gondil et al., 2020a). Cpl-1 loaded into chitosan nanoparticles showed higher antibacterial efficacy than Cpl-1 alone in an in-vitro as well as animal infection model (Gondil et al., 2020a; Gondil et al., 2021). Encapsulation of Cpl-1 in chitosan nanoparticles enhanced its bioavailability and provided substantial mucoadhesiveness, which could be one of the major contributing factors in eliminating pulmonary bacterial infection. Portilla et al. reported the encapsulation of LysRODI endolysin in pH-sensitive liposomes, which demonstrate the ability of liposomes for targeted delivery of endolysin under mild acidic conditions. Encapsulated LysRODI was shown to significantly effective in reducing the cell count of S. aureus (planktonic and biofilm form) at pH 5 (Portilla et al., 2020). Recently, Kaur et al. also reported a chitosan-alginate based endolysin delivery system for efficient delivery of LysMR-5, an anti-staphylococcal endolysin that still needs to be evaluated in animal infection models (Kaur et al., 2020). In general, chitosan-based formulations are favorable choice over other type of delivery materials to deliver biotherapeutics. Chitosan-based delivery systems not only enhance the bioavailability of therapeutic agents but also clear safely from the host system after delivery of their loaded cargos. Despite the high therapeutic potential, phages and endolysin delivery systems face multiple challenges in their clinical implications. Ideal phage or endolysin concentration, their pharmacokinetics and immunomodulatory properties are critical knowledge gaps in the understanding and applications of phage and endolysin delivery systems. The comprehensive architectural range of phages (tailed and non-tailed) and endolysins (single modular and multi-modular) may lead to considerable variation in the formulation design, release kinetics and therapeutic outcome. Thus, delivery systems are needed to be critically designed and optimized for each phage or endolysin, which may increase the scaling-up efforts and cost in developing such formulations. Sterilization of prepared formulations is a major challenge in the scale-up of these delivery systems as thermosensitive phages, and endolysins cannot undergo high-temperature sterilization. The use of sterile conditions for deigning formulations or UV treatment is another potential alternative, but it also increases the cost as well as logistics difficulties in large-scale production settings. The natural origin of phages categorizes phages into non-patentable entities, making them less profitable and unappealing for pharmaceutical companies. Apart from technological limitations, a regulatory and legal framework is another major hurdle in the clinical progress of phage and endolysin delivery systems. The regulatory and legal framework for phages and endolysin applications is more limited to the local, nationwide boundaries and is yet to be standardized in a global manner (Furfaro et al., 2018). Phage and endolysins delivery systems are also expected to enter the clinical trials in the near future to establish their clinical acceptance. It is vital that scientists, clinicians, and regulatory bodies must work together to make the appearance of these therapeutic preparations in clinics sooner rather than later. The future of phage and endolysin encapsulation systems seems to be bright. With the emergence of antimicrobial resistance, the phage and their antibacterial products have emerged from a forgotten area to become a central research area for healthcare workers. There is a need to make these products more effective as therapeutic agent for treating human infections. Various delivery strategies have been devised to increase the efficacy and applicability of these antimicrobial products to clinical care. Phages have been evaluated in multiple encapsulation systems; however, there is a long road for the endolysins delivery system to prove their therapeutic applicability. Phage and endolysin delivery systems must undergo through a range of animal validation, followed by well-structured large clinical trials to establish their therapeutic outcome. The most obvious challenge for clinical applications of phage and endolysins delivery is the lack of a uniform regulatory framework across the globe. Progression of clinical trials and more scientific evidence may refine the current regulatory processes to a well-structured regulatory and legal framework. These future initiatives can further improve the understanding as well as clinical acceptance of phage and endolysin delivery systems to alleviate the burden of bacterial infections in healthcare. The current antibiotic crisis has driven the shifting of paradigm to phage-based therapies and countering delivery challenges. The next 1–2 decades could be an intriguing time to develop novel phage and endolysin delivery systems and investigate their potential in the healthcare system to combat multidrug-resistant infections. VG: Conceptualization and Writing-Original draft, SC: Conceptualization and Reviewing. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Abdelkader, K., Gerstmans, H., Saafan, A., Dishisha, T., and Briers, Y. (2019). The Preclinical and Clinical Progress of Bacteriophages and Their Lytic Enzymes: The Parts Are Easier than the Whole. Viruses. 11, 96. doi:10.3390/v11020096 PubMed Abstract | CrossRef Full Text | Google Scholar Abedon, S. T. (2008). Bacteriophage Ecology: Population Growth, Evolution, and Impact of Bacterial Viruses. Cambridge, United Kingdom: Cambridge University Press Bean, J. E., Alves, D. R., Laabei, M., Esteban, P. P., Thet, N. T., Enright, M. C., et al. (2014). Triggered Release of Bacteriophage K from Agarose/Hyaluronan Hydrogel Matrixes by Staphylococcus aureus Virulence Factors. Chem. Mater. 26, 7201–7208. doi:10.1021/cm503974g CrossRef Full Text | Google Scholar Brix, A., Cafora, M., Aureli, M., and Pistocchi, A. (2020). Animal Models to Translate Phage Therapy to Human Medicine. Int. J. Mol. Sci. 21, 3715. doi:10.3390/ijms21103715 PubMed Abstract | CrossRef Full Text | Google Scholar Chadha, P., Katare, O. P., and Chhibber, S. (2017). Liposome Loaded Phage Cocktail: Enhanced Therapeutic Potential in Resolving Klebsiella pneumoniae Mediated Burn Wound Infections. Burns 43, 1532–1543. doi:10.1016/j.burns.2017.03.029 PubMed Abstract | CrossRef Full Text | Google Scholar Chhibber, S., Shukla, A., and Kaur, S. (2017). Transfersomal Phage Cocktail Is an Effective Treatment against Methicillin-Resistant Staphylococcus aureus-Mediated Skin and Soft Tissue Infections. Antimicrob. Agents Chemother. 61, e02146–02116. doi:10.1128/aac.02146-16 PubMed Abstract | CrossRef Full Text | Google Scholar Clokie, M. R. J., Millard, A. D., Letarov, A. V., and Heaphy, S. (2011). Phages in Nature. Bacteriophage. 1, 31–45. doi:10.4161/bact.1.1.14942 PubMed Abstract | CrossRef Full Text | Google Scholar Colom, J., Cano-Sarabia, M., Otero, J., Aríñez-Soriano, J., Cortés, P., Maspoch, D., et al. (2017). Microencapsulation with alginate/CaCO(3): A Strategy for Improved Phage Therapy. Sci. Rep. 7, 41441. doi:10.1038/srep41441 PubMed Abstract | CrossRef Full Text | Google Scholar Colom, J., Cano-Sarabia, M., Otero, J., Cortés, P., Maspoch, D., and Llagostera, M. (2015). Liposome-Encapsulated Bacteriophages for Enhanced Oral Phage Therapy against Salmonella Spp. Appl. Environ. Microbiol. 81, 4841–4849. doi:10.1128/aem.00812-15 PubMed Abstract | CrossRef Full Text | Google Scholar Díaz, A., Del Valle, L., Rodrigo, N., Casas, M., Chumburidze, G., Katsarava, R., et al. (2018). Antimicrobial Activity of Poly(ester Urea) Electrospun Fibers Loaded with Bacteriophages. Fibers. 6, 33. doi:10.3390/fib6020033 CrossRef Full Text | Google Scholar Dini, C., Islan, G. A., De Urraza, P. J., and Castro, G. R. (2012). Novel Biopolymer Matrices for Microencapsulation of Phages: Enhanced Protection against Acidity and Protease Activity. Macromol. Biosci. 12, 1200–1208. doi:10.1002/mabi.201200109 PubMed Abstract | CrossRef Full Text | Google Scholar Duckworth, D. H., and Gulig, P. A. (2002). Bacteriophages. BioDrugs. 16, 57–62. doi:10.2165/00063030-200216010-00006 PubMed Abstract | CrossRef Full Text | Google Scholar Furfaro, L. L., Payne, M. S., and Chang, B. J. (2018). Bacteriophage Therapy: Clinical Trials and Regulatory Hurdles. Front. Cell Infect. Microbiol. 8, 376. doi:10.3389/fcimb.2018.00376 PubMed Abstract | CrossRef Full Text | Google Scholar Gondil, V. S., and Chhibber, S. (2017). Evading Antibody Mediated Inactivation of Bacteriophages Using Delivery Systems. Juniper Online J. Immuno Virol. 1, 555–574. doi:10.19080/JOJIV.2017.01.555574 CrossRef Full Text | Google Scholar Gondil, V. S., Dube, T., Panda, J. J., Yennamalli, R. M., Harjai, K., and Chhibber, S. (2020a). Comprehensive Evaluation of Chitosan Nanoparticle Based Phage Lysin Delivery System; a Novel Approach to Counter S. Pneumoniae Infections. Int. J. Pharmaceutics 573, 118850. doi:10.1016/j.ijpharm.2019.118850 PubMed Abstract | CrossRef Full Text | Google Scholar Gondil, V. S., Harjai, K., and Chhibber, S. (2020b). Endolysins as Emerging Alternative Therapeutic Agents to Counter Drug-Resistant Infections. Int. J. Antimicrob. Agents 55, 105844. doi:10.1016/j.ijantimicag.2019.11.001 PubMed Abstract | CrossRef Full Text | Google Scholar Gondil, V. S., Harjai, K., and Chhibber, S. (2021). Investigating the Potential of Endolysin Loaded Chitosan Nanoparticles in the Treatment of Pneumococcal Pneumonia. J. Drug Deliv. Sci. Technology 61, 102142. doi:10.1016/j.jddst.2020.102142 CrossRef Full Text | Google Scholar Hathaway, H., Alves, D. R., Bean, J., Esteban, P. P., Ouadi, K., Mark Sutton, J., et al. (2015). Poly(N-isopropylacrylamide-co-allylamine) (PNIPAM-Co-ALA) Nanospheres for the Thermally Triggered Release of Bacteriophage K. Eur J Pharm Biopharm. 96, 437–441. doi:10.1016/j.ejpb.2015.09.013 PubMed Abstract | CrossRef Full Text | Google Scholar Kaur, J., Kour, A., Panda, J. J., Harjai, K., and Chhibber, S. (2020). Exploring Endolysin-Loaded Alginate-Chitosan Nanoparticles as Future Remedy for Staphylococcal Infections. AAPS PharmSciTech. 21, 233. doi:10.1208/s12249-020-01763-4 PubMed Abstract | CrossRef Full Text | Google Scholar Loh, B., Gondil, V. S., Manohar, P., Khan, F. M., Yang, H., and Leptihn, S. (2020). Encapsulation and Delivery of Therapeutic Phages. Appl. Environ. Microbiol., 87, e01979–20. doi:10.1128/AEM.01979-20 CrossRef Full Text | Google Scholar Ly-Chatain, M. H., Moussaoui, S., Vera, A., Rigobello, V., and Demarigny, Y. (2013). Antiviral Effect of Cationic Compounds on Bacteriophages. Front. Microbiol. 4, 46. doi:10.3389/fmicb.2013.00046 PubMed Abstract | CrossRef Full Text | Google Scholar Ma, Y., Pacan, J. C., Wang, Q., Xu, Y., Huang, X., Korenevsky, A., et al. (2008). Microencapsulation of Bacteriophage Felix O1 into Chitosan-Alginate Microspheres for Oral Delivery. Appl Environ Microbiol. 74, 4799–4805. doi:10.1128/aem.00246-08 PubMed Abstract | CrossRef Full Text | Google Scholar Malik, D. J., Sokolov, I. J., Vinner, G. K., Mancuso, F., Cinquerrui, S., Vladisavljevic, G. T., et al. (2017). Formulation, Stabilisation and Encapsulation of Bacteriophage for Phage Therapy. Adv. Colloid Interf. Sci. 249, 100–133. doi:10.1016/j.cis.2017.05.014 CrossRef Full Text | Google Scholar Milo, S., Thet, N. T., Liu, D., Nzakizwanayo, J., Jones, B. V., and Jenkins, A. T. A. (2016). An In-Situ Infection Detection Sensor Coating for Urinary Catheters. Biosens. Bioelectron. 81, 166–172. doi:10.1016/j.bios.2016.02.059 PubMed Abstract | CrossRef Full Text | Google Scholar Nogueira, F., Karumidze, N., Kusradze, I., Goderdzishvili, M., Teixeira, P., and Gouveia, I. C. (2017). Immobilization of Bacteriophage in Wound-Dressing Nanostructure. Nanomedicine. 13, 2475–2484. doi:10.1016/j.nano.2017.08.008 PubMed Abstract | CrossRef Full Text | Google Scholar Pirnay, J. P., and Kutter, E. (2020). Bacteriophages: It's a Medicine, Jim, but Not as We Know it. Lancet Infect. Dis. 21, 309-311. doi:10.1016/S1473-3099(20)30464-3 PubMed Abstract | CrossRef Full Text | Google Scholar Portilla, S., Fernández, L., Gutiérrez, D., Rodríguez, A., and García, P. (2020). Encapsulation of the Antistaphylococcal Endolysin LysRODI in pH-Sensitive Liposomes. Antibiotics 9, 242. doi:10.3390/antibiotics9050242 PubMed Abstract | CrossRef Full Text | Google Scholar Puapermpoonsiri, U., Spencer, J., and Van Der Walle, C. F. (2009). A Freeze-Dried Formulation of Bacteriophage Encapsulated in Biodegradable Microspheres. Eur J Pharm Biopharm. 72, 26–33. doi:10.1016/j.ejpb.2008.12.001 PubMed Abstract | CrossRef Full Text | Google Scholar Report, B. T. M. (2021). Bacteriophages Therapy Market 2021 Industry Trends, Size Estimation, Price, Business Growth, Industry Outlook: Armata Pharmaceuticals, iNtODEWORLD, Phage International. Fixed-Phage Limited, https://www.pharmiweb.com/press-release/2021-02-08/bacteriophages-therapy-market-2021-industry-trends-size-estimation-price-business-growth-industr Singla, S., Harjai, K., Katare, O. P., and Chhibber, S. (2015). Bacteriophage-Loaded Nanostructured Lipid Carrier: Improved Pharmacokinetics Mediates Effective Resolution ofKlebsiella Pneumoniae-Induced Lobar Pneumonia. J. Infect. Dis. 212, 325–334. doi:10.1093/infdis/jiv029 PubMed Abstract | CrossRef Full Text | Google Scholar Singla, S., Harjai, K., Katare, O. P., and Chhibber, S. (2016). Encapsulation of Bacteriophage in Liposome Accentuates its Entry in to Macrophage and Shields it from Neutralizing Antibodies. PLoS One 11, e0153777. doi:10.1371/journal.pone.0153777 PubMed Abstract | CrossRef Full Text | Google Scholar Stanford, K., Mcallister, T. A., Niu, Y. D., Stephens, T. P., Mazzocco, A., Waddell, T. E., et al. (2010). Oral Delivery Systems for Encapsulated Bacteriophages Targeted at Escherichia coli O157:H7 in Feedlot Cattle. J. Food Prot. 73, 1304–1312. doi:10.4315/0362-028x-73.7.1304 PubMed Abstract | CrossRef Full Text | Google Scholar Sulakvelidze, A., and Morris, J. G. (2001). Bacteriophages as Therapeutic Agents. Ann. Med. 33, 507–509. doi:10.3109/07853890108995959 PubMed Abstract | CrossRef Full Text | Google Scholar Tang, Z., Huang, X., Baxi, S., Chambers, J. R., Sabour, P. M., and Wang, Q. (2013). Whey Protein Improves Survival and Release Characteristics of Bacteriophage Felix O1 Encapsulated in Alginate Microspheres. Food Res. Int. 52, 460–466. doi:10.1016/j.foodres.2012.12.037 CrossRef Full Text | Google Scholar Vinner, G. K., and Malik, D. J. (2018). High Precision Microfluidic Microencapsulation of Bacteriophages for Enteric Delivery. Res. Microbiol. 169, 522–530. doi:10.1016/j.resmic.2018.05.011 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: bacteriophages, endolysins, encapsulation, nanoparticles, antibiotic resistance, alternative therapy Citation: Gondil VS and Chhibber S (2021) Bacteriophage and Endolysin Encapsulation Systems: A Promising Strategy to Improve Therapeutic Outcomes. Front. Pharmacol. 12:675440. doi: 10.3389/fphar.2021.675440 Received: 03 March 2021; Accepted: 26 April 2021; Published: 07 May 2021. Edited by: Reviewed by: Copyright © 2021 Gondil and Chhibber. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Sanjay Chhibber, [email protected]
Silvia Adriana Mayer Lentz, Tanise Vendruscolo Dalmolin, Afonso Luís Barth,
Frontiers in Public Health, Volume 9; https://doi.org/10.3389/fpubh.2021.648940

Abstract:
In the last decade, polymyxins have been reintroduced in the therapeutic arsenal to treat severe infections by carbapenem-resistant Enterobacterales. At that time, reports of polymyxin resistance were all due to chromosomal mutations (1). These mechanisms included (i) modifications of the lipopolysaccharides (LPSs) moiety via the addition of cationic groups; (ii) mutations that lead to the loss of the LPS; (iii) porin mutations and overexpression of efflux pump systems; (iv) overproduction of capsular polysaccharide (CPS) in some Gram-negative bacteria (GNB) that hide the polymyxin-binding sites and the release of CPS-trapping polymyxins; and (v) enzymatic inactivation of polymyxins (2). Although some chromosomal resistance mechanisms have been studied since the 1960's, it was in the late 1990's, after the reintroduction of polymyxins in the therapeutic arsenal, that this problem became more important (3). In fact, this information is supported by the first report of colistin resistance among Acinetobacter baumannii clinical isolates from the Czech Republic in 1999 and Klebsiella pneumoniae from Athens in 2004 (4). However, in 2015, the mcr-1 gene, associated with IncI2-type plasmid, was identified in Escherichia coli resistant to colistin obtained from food animals and humans in China (1). This finding promoted a great concern in the international scientific community since the last therapeutic option to treat serious infections by multidrug-resistant GNB could be exhausted. With the horizontal transfer, the rapid spread of the mcr-1 gene would be inevitable. The mcr-1 gene carried by different plasmid types has already been identified in all five continents from different sources and hosts (1, 5). Surprisingly, Shen and colleagues, in a retrospective study, characterized the early occurrence of the mcr-1 gene in chicken isolates from 1980's (6). So far, a total of 10 different variants (7) of the mcr gene have been described mainly among the Enterobacterales, but with the mcr-1 gene remaining the most prevalent (1). To date, the sequences of 30 mcr-1 mutations (mcr-1.2 to mcr-1.30) have already been deposited in the GenBank database, differing from mcr-1 by one or few amino acids. Besides that, 10 mcr gene variants (mcr-1 to mcr-10) were deposited, with amino acid identity ranging from 31 to 83% (8). These variants were identified at the beginning in Enterobacterales isolates, including E. coli (mcr-1, mcr-2, and mcr-3 genes), Salmonella enterica (mcr-4, mcr-5 and mcr-9 genes), K. pneumoniae (mcr-7 and mcr-8 genes), and Enterobacter roggenkampii (mcr-10 gene). The exception is due to mcr-6 gene that was first identified in Moraxella spp. After that, some variants were identified in non-fermenter Gram-negative rods, as Acinetobacter spp. (mcr-1 and mcr-4) and Pseudomonas spp. (mcr-1 only) (9, 10). In general, the isolates carrying mcr genes were first isolated from animals such as pigs (mcr-1, mcr-2, mcr-3, mcr-4, mcr-6, and mcr-8 genes) and chickens (mcr-5 and mcr-7 genes), but mcr-9 and mcr-10 genes were identified, for the first time, from human patients (8). The resistance to polymyxins was attributed mainly to chromosomal mutations and is rare in human clinical isolates (0.67–1.6%) (11). Nevertheless, this differs among bacteria species, being higher in K. pneumoniae and A. baumannii (20–80%) (4) in contrast to lower rates in E. coli (0.2–0.6%) (11). The polymyxin resistance rate associated to plasmid, as mcr-1, is also low in humans (~1%) (4). On the other hand, according to a large US surveillance study, the association between mcr-1 and other antibiotic resistance genes, such as extended-spectrum β-lactamase (ESBL) and carbapenemases, may reach 32% of prevalence in K. pneumoniae (11). Regarding the mortality associated with infections caused by colistin-resistant isolates in humans, the rate is variable, and it is higher in critically ill patients (30–37%) including those previously exposed to colistin (4). The mortality rate may reach 100% in patients with nosocomial infections caused by pan–drug-resistant K. pneumoniae. It is important to emphasize that the prevalence of mcr-1 gene is higher among production animals, mainly in pig and chicken isolates (5). The data show colistin resistance rates of ~70% in E. coli isolates from China and ~90% among Enterobacterales in some European countries (8). So, these data corroborate with the scientific evidence that the worldwide spread of the mcr-1 gene is mainly associated with the large amounts of colistin use in production animals, and its emergence is a particular threat to public health as colistin is considered the last-resort antimicrobial for treatment of severe human infections, and its use in livestock production contributes to emerging resistance globally (1). In Latin America, a systematic review analysis showed that the prevalence of mcr-1 gene is higher in isolates from animals (8.7%) than in food (5.4%) and humans (2.0%) (12). To the best of our knowledge, the first reports of mcr-1 gene in Latin America dated from July and October 2012 when this gene was identified in E. coli isolates from two inpatients in different hospitals in Argentina (Table 1) (13). Patients presented neurological disease and diabetes, and the mcr-1–positive isolates were obtained from blood and urine, respectively. In this study, the authors evaluated the presence of the mcr-1 gene in 87 colistin-resistant clinical human isolates from 2008 to 2016 (28 E. coli, 19 K. pneumoniae, 36 of other members of the Enterobacterales, and 4 non-fermenter Gram-negative rods), and nine isolates of E. coli were mcr-1 positive. These isolates were associated with human infections, mainly in males, and the average age of the patients was 68.5 years. All mcr-1–positive E. coli isolates were genetically unrelated as determined by pulsed-field gel electrophoresis, and the resistance mechanism was horizontally transferable by conjugation (13). Still, in 2012, other studies reported mcr-1 harboring E. coli recovered from Kelp Guls in Argentina (14) and from swine in Brazil (Table 1) (15). Table 1. Summary of mainly studies reporting mcr-1 gene in Latin America. Since 2012, the mcr-1 gene has already been identified in bacteria from humans, animals, animal food products, and environmental sources in different countries in Latin America, including Brazil (15), Bolivia (16), Colombia (17), Chile (18), Uruguay (19), Paraguay (20), Peru (21), Mexico (22), Venezuela (23), and Ecuador (24). Brazil is the country with the highest number of mcr-1–positive bacteria reported in Latin America mainly from bacterial isolates obtained from poultry rectal swabs (15) (Table 1). It is important to consider that Brazil is the fourth largest pork producer and exporter and the largest chicken meat exporter in the world, which could contribute to the high prevalence of the mcr-1 gene in this country (25). As in other countries, the colistin was extensively used in Brazil as a growth promoter for many years. In 2016, the government published restrictions on the use of colistin in animal production (1, 26), which came into force in 2018. However, the use of colistin to treat or prevent infections in veterinary medicine including animal productions is still allowed. E. coli is the most common species harboring the mcr-1 gene in Latin America countries. However, many other Enterobacterales members such as K. pneumoniae, Salmonella spp., Citrobacter spp., and Enterobacter spp. were also reported as positive for the mcr-1 gene (17, 27). In addition to mcr-1, other variants of the gene were reported rarely in Latin America, such as mcr-3, mcr-5, mcr-7, and mcr-9 (28–30). E. coli isolates harboring mcr-1 gene belong to different sequence types (STs) (31, 32) (Table 1), indicating that the dissemination of the mcr-1 gene is associated with different clonal strains (1). Loayza-Villa and colleagues investigated the relationship between an E. coli carrying mcr-1 recovered from the gastrointestinal tract of a boy and an mcr-1–positive E. coli from fecal samples and rectal/cloacal swabs from his domestic animals. E. coli strains from domestic animals and from the boy were different; however, all plasmids harboring the mcr-1 gene shared 90% nucleotide identity and a highly conserved backbone, supporting the idea of horizontal dissemination of the mcr-1 gene (32). In Latin America, the E. coli belonging to CC10 clonal complex, known as the largest human clonal complex, was the most reported in previous studies, including the ST744 and ST10 (1, 17, 22, 33). E. coli CC10 strains are widely disseminated among humans, animals, meat products, and environmental sources (34, 35) and are designated as multidrug-resistant strains carrying frequently ESBL, among others (5, 31). The mcr-1 gene is carried by a wide range of conjugative and non-conjugative plasmid types, including IncX3, IncX4, an IncX3–X4 hybrid, IncH1, IncHI1, IncHI2, IncP, IncI2, IncF, IncFII, an IncI2–IncFIB hybrid, and IncY (5). The mcr-1 gene can also be integrated into the chromosome of some strains (17). However, in Latin America, only four plasmids have been described so far: IncX4 (36), IncP (22), IncI2 (31), and IncHI2 (37), of which the IncX4 plasmid is the most frequent in Brazil (38, 39) (Table 1). There is a clear association between the IncX4 plasmids and the insertion sequences associated with the dissemination of the mcr-1 gene (40). Plasmid analysis has revealed that the insertion sequence ISApl1 (which belongs to the IS30 family transposase), in a composite transposon (ISApl1-mcr-1-ISApl1), is usually present in IncHI2-type plasmids (size of 200 kb), being either present or absent in IncI2-type plasmids (60 kb), and completely absent in IncX4-type plasmids (30 kb) (Table 1). The role of ISApl1 in the mobilization of the mcr-1 gene was demonstrated in vitro by transposition. It was suggested that the recombination events associated with mobilization of the mcr-1 gene were initially mediated by two copies of ISApl1 from an unknown progenitor to a plasmid and subsequently transferred to Enterobacterales (41). Besides that, according to Snesrud et al., the presence of a single or two copies of ISApl1 indicates a recent acquisition of the mcr-1 gene, whereas the absence of this insertion sequence could be correlated with the adaptation of the mcr-1 gene to a new host (41). The regulation mechanism of mcr-1 gene expression is complex and remains unknown. In general, the gene expression is controlled by its promoter and the corresponding activators and/or inhibitors. Zhang et al. suspect that genes encoding activators and/or inhibitors in the host chromosome may affect the expression of the mcr-1 gene found on plasmids IncX4 and other plasmids. They may vary expressively in unlike genetic backgrounds of the different strains and/or mcr-1–harboring plasmids, despite that their promoters are remarkably similar (42). Although the mobility and dissemination of the mcr-1 gene are associated with ISApl1 and the pap2 gene in most plasmid types (43), the genetic context of the IncX4 plasmid type, in Latin America, is different. This context is characterized by lacking the ISApl1, but it preserves the pap2 sequence and a hypothetical protein (hp) around the mcr-1 gene (26, 44). What would be the explanation for that? Snesrud et al. analyzed the genetic environment of the mcr-1 gene associated or not with ISApl1 and concluded that the target site duplications generated by ISApl1 transposition are present even in lack of the ISApl1. This result suggests that the mechanism to mobilize the mcr-1 gene is the same as that observed in other plasmids, and after that, the loss of the insertion sequence by recombination events in IncX4 occurs (45). Furthermore, the IS26 mobile element upstream to the mcr-1 gene has been also associated with IncX4 plasmid types in Brazil, but there are no other reports in Latin America (26, 46) (Table 1). This Insertion Sequence (IS) plays an important role in the dissemination and evolution of the antimicrobial resistance genes on plasmids, including colistin resistance genes (1). In veterinary medicine, colistin is mainly administrated in pigs and poultry production, for prophylaxis or treatment. The spread of colistin resistance may lead to treatment failure, as well as increase the pathogen transmission reach with quality and economic loss in production animals. Strong scientific evidence indicates that the mcr-1 gene might have originated from animals because (i) colistin has been used extensively for decades in veterinary practices; (ii) mcr-1 gene was largely identified in several animals and animal food products; (iii) the identification of the mcr-1 gene in E. coli isolate recovered before 1980 in China suggests that the emergence of this gene may be linked to the use of colistin as a growth promoter in the poultry industry; and (iv) genetic features of mcr-1 gene associated with ISApl1 were first identified in Actinobacillus pleuropneumoniae, a common animal pathogen (43), which could be involved in recombination events leading to the mobilization of the mcr-1 cassette. Finally, a recent study has demonstrated that when colistin is banned from use in animal feed, there was a significant decrease of the mcr-1 gene prevalence in most sources, including pig farms, food, and environment samples (47). Given that the production animals can be a reservoir for mcr-1 gene and its dissemination can occur by food and environment, all countries should apply surveillance, monitoring, and restrictive measures to polymyxins use. In Latin America, Brazil, and Argentina (1) have already banned the use of colistin as a growth promoter, but the impact of this measure has not been evaluated yet. The problem of antimicrobial resistance is related to the use and abuse of antibiotics in humans, animals, and the environment. Besides that, the mcr-1 gene is disseminated mainly by E. coli clones, with a high capacity to survive in different ecological niches, some of them with pandemic and epidemic potential. So, it seems clear that the One Health approach should be adopted to integrate veterinary and human medicine to address antimicrobial resistance. SAML, TVD, and AFM: conception of the opinion, collected data, and wrote the paper. ALB and AFM: reviewed and edited. All authors contributed to the article and approved the submitted version. This study was funded by National Institute of Antimicrobial Resistance Research - INPRA (MCTI/CNPq/CAPES/FAPs n° 16/2014). SAML were supported by a grant from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 1. Anyanwu MU, Jaja IF, Nwobi OC. Occurrence and characteristics of mobile colistin resistance (Mcr) gene-containing isolates from the environment: a review. Int J Environ Res Public Health. (2020) 17:1028. doi: 10.3390/ijerph17031028 PubMed Abstract | CrossRef Full Text | Google Scholar 2. Olaitan AO, Morand S, Rolain J-M. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria. Front Microbiol. (2014) 5:643. doi: 10.3389/fmicb.2014.00643 PubMed Abstract | CrossRef Full Text | Google Scholar 3. Kai J, Wang S. Recent progress on elucidating the molecular mechanism of plasmid-mediated colistin resistance and drug design. Int Microbiol. (2020) 23:355–66. doi: 10.1007/s10123-019-00112-1 PubMed Abstract | CrossRef Full Text | Google Scholar 4. Li Z, Cao Y, Yi L, Liu J-H, Yang Q. Emergent polymyxin resistance: end of an Era? Open forum Infect Dis. (2019) 6:ofz368. doi: 10.1093/ofid/ofz368 PubMed Abstract | CrossRef Full Text | Google Scholar 5. Quiroga C, Nastro M, Di Conza J. Current scenario of plasmid-mediated colistin resistance in Latin America. Rev Argent Microbiol. (2019) 51:93–100. doi: 10.1016/j.ram.2018.05.001 PubMed Abstract | CrossRef Full Text | Google Scholar 6. Shen Z, Wang Y, Shen Y, Shen J, Wu C. Early emergence of mcr-1 in Escherichia coli from food-producing animals. Lancet Infect Dis. (2016) 16:293. doi: 10.1016/S1473-3099(16)00061-X PubMed Abstract | CrossRef Full Text | Google Scholar 7. Wang C, Feng Y, Liu L, Wei L, Kang M, Zong Z. Identification of novel mobile colistin resistance gene mcr-10. Emerg Microbes Infect. (2020) 9:508–16. doi: 10.1080/22221751.2020.1732231 PubMed Abstract | CrossRef Full Text | Google Scholar 8. Shen Y, Zhang R, Schwarz S, Wu C, Shen J, Walsh TR, et al. Farm animals and aquaculture: significant reservoirs of mobile colistin resistance genes. Environ Microbiol. (2020) 22:2469–84. doi: 10.1111/1462-2920.14961 PubMed Abstract | CrossRef Full Text | Google Scholar 9. Martins-Sorenson N, Snesrud E, Xavier DE, Cacci LC, Iavarone AT, McGann P, et al. A novel plasmid-encoded mcr-4.3 gene in a colistin-resistant Acinetobacter baumannii clinical strain. J Antimicrob Chemother. (2020) 75:60–4. doi: 10.1093/jac/dkz413 PubMed Abstract | CrossRef Full Text | Google Scholar 10. Caselli E, D'Accolti M, Soffritti I, Piffanelli M, Mazzacane S. Spread of mcr-1-driven colistin resistance on hospital surfaces, Italy. Emerg Infect Dis. (2018) 24:1752–3. doi: 10.3201/eid2409.171386 PubMed Abstract | CrossRef Full Text | Google Scholar 11. Sherry N, Howden B. Emerging gram negative resistance to last-line antimicrobial agents fosfomycin, colistin and ceftazidime-avibactam–epidemiology, laboratory detection and treatment implications. Expert Rev Anti Infect Ther. (2018) 16:289–306. doi: 10.1080/14787210.2018.1453807 PubMed Abstract | CrossRef Full Text | Google Scholar 12. Mendes Oliveira VR, Paiva MC, Lima WG. Plasmid-mediated colistin resistance in Latin America and Caribbean: a systematic review. Travel Med Infect Dis. (2019) 31:101459. doi: 10.1016/j.tmaid.2019.07.015 PubMed Abstract | CrossRef Full Text | Google Scholar 13. Rapoport M, Faccone D, Pasteran F, Ceriana P, Albornoz E, Petroni A, et al. First description of mcr-1-mediated colistin resistance in human infections caused by Escherichia coli in Latin America. Antimicrob Agents Chemother. (2016) 60:4412–3. doi: 10.1128/AAC.00573-16 PubMed Abstract | CrossRef Full Text | Google Scholar 14. Liakopoulos A, Mevius DJ, Olsen B, Bonnedahl J. The colistin resistance mcr-1 gene is going wild. J Antimicrob Chemother. (2016) 71:2335–6. doi: 10.1093/jac/dkw262 PubMed Abstract | CrossRef Full Text | Google Scholar 15. Fernandes MR, Moura Q, Sartori L, Silva KC, Cunha MP, Esposito F, et al. Silent dissemination of colistin-resistant Escherichia coli in South America could contribute to the global spread of the mcr-1 gene. Eurosurveillance. (2016) 21:30214. doi: 10.2807/1560-7917.ES.2016.21.17.30214 PubMed Abstract | CrossRef Full Text | Google Scholar 16. Sennati S, Di Pilato VD, Riccobono E, Maggio TD, Villagran AL, Pallecchi L, et al. Citrobacter braakii carrying plasmidborne mcr-1 colistin resistance gene from ready-to-eat food from a market in the Chaco region of Bolivia. J Antimicrob Chemother. (2017) 72:2127–9. doi: 10.1093/jac/dkx078 PubMed Abstract | CrossRef Full Text | Google Scholar 17. Saavedra SY, Diaz L, Wiesner M, Correa A, Arévalo SA, Reyes J, et al. Genomic and molecular characterization of clinical isolates of enterobacteriaceae harboring mcr-1 in Colombia, 2002 to 2016. Antimicrob Agents Chemother. (2017) 61:e00841–17. doi: 10.1128/AAC.00841-17 PubMed Abstract | CrossRef Full Text | Google Scholar 18. Gutiérrez C, Zenis J, Legarraga P, Cabrera-Pardo JR, García P, Bello-Toledo H, et al. Genetic analysis of the first mcr-1 positive Escherichia coli isolate collected from an outpatient in Chile. Brazil J Infect Dis. (2019) 23:203–6. doi: 10.1016/j.bjid.2019.05.008 PubMed Abstract | CrossRef Full Text | Google Scholar 19. Papa-Ezdra R, Grill Diaz F, Vieytes M, García-Fulgueiras V, Caiata L, Ávila P, Brasesco M, et al. First three Escherichia coli isolates harbouring mcr-1 in Uruguay. J Glob Antimicrob Resist. (2020) 20:187–90. doi: 10.1016/j.jgar.2019.07.016 PubMed Abstract | CrossRef Full Text | Google Scholar 20. Melgarejo Touchet N, Martínez M, Franco R, Falcón M, Busignani S, Espínola C, et al. Plasmid-mediated colistin resistance gene mcr-1 in Enterobacteriaceae in Paraguay. Rev Salud Publica Del Paraguay. (2018) 8:44–8. doi: 10.18004/rspp.2018.junio.44-48 CrossRef Full Text | Google Scholar 21. Ugarte Silva RG, Olivo López JM, Corso A, Pasteran F, Albornoz E, Sahuanay Blácido ZP. Resistencia a colistín mediado por el gen mcr-1 identificado en cepas de Escherichia coli y Klebsiella pneumoniae. Primeros reportes en el Perú. An Fac Med. (2018) 79:213. doi: 10.15381/anales.v79i3.15313 CrossRef Full Text | Google Scholar 22. Garza-Ramos U, Tamayo-Legorreta E, Arellano-Quintanilla DM, Rodriguez-Medina N, Silva-Sanchez J, Catalan-Najera J, et al. Draft genome sequence of a multidrug- and colistin-resistant mcr-1-producing Escherichia coli isolate from a swine farm in Mexico. Genome Announc. (2018) 6:e00102–18. doi: 10.1128/genomeA.00102-18 PubMed Abstract | CrossRef Full Text | Google Scholar 23. Delgado-Blas JF, Ovejero CM, Abadia-Patiño L, Gonzalez-Zorn B. Coexistence of mcr-1 and blaNDM-1 in Escherichia coli from Venezuela. Antimicrob Agents Chemother. (2016) 60:6356–8. doi: 10.1128/AAC.01319-16 PubMed Abstract | CrossRef Full Text | Google Scholar 24. Vinueza-Burgos C, Ortega-Paredes D, Narváez C, De Zutter L, Zurita J. Characterization of cefotaxime resistant Escherichia coli isolated from broiler farms in Ecuador. PLoS ONE. (2019) 14:e0207567. doi: 10.1371/journal.pone.0207567 PubMed Abstract | CrossRef Full Text | Google Scholar 25. Associação Brasileira de Proteína Animal. Relatório Anual. (2020). Available online at: http://abpa-br.org/wp-content/uploads/2020/05/abpa_relatorio_anual_2020_portugues_web.pdf (accessed November 20, 2020). Google Scholar 26. Rau RB, De Lima-Morales D, Wink PL, Ribeiro AR, Barth AL. Salmonella enterica mcr-1 positive from food in Brazil: detection and characterization. Foodborne Pathog Dis. (2020) 17:202–8. doi: 10.1089/fpd.2019.2700 PubMed Abstract | CrossRef Full Text | Google Scholar 27. Giani T, Sennati S, Antonelli A, Di Pilato V, di Maggio T, Mantella A, et al. High prevalence of carriage of mcr-1-positive enteric bacteria among healthy children from rural communities in the Chaco region, Bolivia, september to october 2016. Euro Surveill. (2018) 23:1800115. doi: 10.2807/1560-7917.ES.2018.23.45.1800115 PubMed Abstract | CrossRef Full Text | Google Scholar 28. dos Santos LDR, Furlan JPR, Ramos MS, Gallo IFL, de Freitas LVP, Stehling EG. Co-occurrence of mcr-1, mcr-3, mcr-7 and clinically relevant antimicrobial resistance genes in environmental and fecal samples. Arch Microbiol. (2020) 202:1795–800. doi: 10.1007/s00203-020-01890-3 PubMed Abstract | CrossRef Full Text | Google Scholar 29. Rocha IV, dos Santos Silva N, das Neves Andrade CA, de Lacerda Vidal CF, Leal NC, Xavier DE. Diverse and emerging molecular mechanisms award polymyxins resistance to Enterobacteriaceae clinical isolates from a tertiary hospital of Recife, Brazil. Infect Genet Evol. (2020) 85:104584. doi: 10.1016/j.meegid.2020.104584 PubMed Abstract | CrossRef Full Text | Google Scholar 30. Faccone D, Martino F, Albornoz E, Gomez S, Corso A, Petroni A. Plasmid carrying mcr-9 from an extensively drug-resistant NDM-1-producing Klebsiella quasipneumoniae subsp. quasipneumoniae clinical isolate. Infect Genet Evol. (2020) 81:104273. doi: 10.1016/j.meegid.2020.104273 PubMed Abstract | CrossRef Full Text | Google Scholar 31. Rumi M V, Mas J, Elena A, Cerdeira L, Muñoz ME, Lincopan N, et al. Co-occurrence of clinically relevant β-lactamases and MCR-1 encoding genes in Escherichia coli from companion animals in Argentina. Vet Microbiol. (2019) 230:228–34. doi: 10.1016/j.vetmic.2019.02.006 PubMed Abstract | CrossRef Full Text | Google Scholar 32. Loayza-Villa F, Salinas L, Tijet N, Villavicencio F, Tamayo R, Salas S, et al. Diverse Escherichia coli lineages from domestic animals carrying colistin resistance gene mcr-1 in an Ecuadorian household. J Glob Antimicrob Resist. (2020) 22:63–7. doi: 10.1016/j.jgar.2019.12.002 PubMed Abstract | CrossRef Full Text | Google Scholar 33. Dominguez JE, Faccone D, Tijet N, Gomez S, Corso A, Fernández-Miyakawa ME, et al. Characterization of Escherichia coli carrying mcr-1-plasmids recovered from food animals from Argentina. Front Cell Infect Microbiol. (2019) 9:41. doi: 10.3389/fcimb.2019.00041 PubMed Abstract | CrossRef Full Text | Google Scholar 34. Manges AR, Harel J, Masson L, Edens TJ, Portt A, Reid-Smith RJ, et al. Multilocus sequence typing and virulence gene profiles associated with Escherichia coli from human and animal sources. Foodborne Pathog Dis. (2015) 12:302–10. doi: 10.1089/fpd.2014.1860 PubMed Abstract | CrossRef Full Text | Google Scholar 35. Chen P-A, Hung C-H, Huang P-C, Chen J-R, Huang I-F, Chen W-L, et al. Characteristics of CTX-M extended-spectrum β-lactamase-producing Escherichia coli strains isolated from multiple rivers in Southern Taiwan. Appl Environ Microbiol. (2016) 82:1889–97. doi: 10.1128/AEM.03222-15 PubMed Abstract | CrossRef Full Text | Google Scholar 36. Fernandes MR, Sellera FP, Esposito F, Sabino CP, Cerdeira L, Lincopan N. Colistin-resistant mcr-1-positive Escherichia coli on public beaches, an infectious threat emerging in recreational waters. Antimicrob Agents Chemother. (2017) 61:e00234–17. doi: 10.1128/AAC.00234-17 PubMed Abstract | CrossRef Full Text | Google Scholar 37. Faccone D, Rapoport M, Albornoz E, Celaya F, De Mendieta J, De Belder D, et al. Plasmidic resistance to colistin mediated by mcr-1 gene in Escherichia coli clinical isolates in Argentina: a retrospective study, 2012-2018. Rev Panam Salud Pub. (2020) 44:e55. doi: 10.26633/RPSP.2020.55 CrossRef Full Text | Google Scholar 38. Perdigão Neto LV, Corscadden L, Martins RCR, Nagano DS, Cunha MPV, Neves PR, et al. Simultaneous colonization by Escherichia coli and Klebsiella pneumoniae harboring mcr-1 in Brazil. Infection. (2019) 47:661-−4. doi: 10.1007/s15010-019-01309-2 CrossRef Full Text | Google Scholar 39. Sacramento AG, Fernandes MR, Sellera FP, Muñoz ME, Vivas R, Dolabella SS, et al. Genomic analysis of MCR-1 and CTX-M-8 co-producing Escherichia coli ST58 isolated from a polluted mangrove ecosystem in Brazil. J Glob Antimicrob Resist. (2018) 15:288–9. doi: 10.1016/j.jgar.2018.10.024 PubMed Abstract | CrossRef Full Text | Google Scholar 40. Sun J, Fang L-X, Wu Z, Deng H, Yang R-S, Li X-P, et al. Genetic analysis of the IncX4 plasmids: implications for a unique pattern in the mcr-1 acquisition. Sci Rep. (2017) 7:424. doi: 10.1038/s41598-017-00095-x PubMed Abstract | CrossRef Full Text | Google Scholar 41. Snesrud E, He S, Chandler M, Dekker JP, Hickman AB, McGann P, et al. A model for transposition of the colistin resistance gene mcr-1 by ISApl1. Antimicrob Agents Chemother. (2016) 60:6973–6. doi: 10.1128/AAC.01457-16 PubMed Abstract | CrossRef Full Text | Google Scholar 42. Zhang H, Miao M, Yan J, Wang M, Tang Y-W, Kreiswirth BN, et al. Expression characteristics of the plasmid-borne mcr-1 colistin resistance gene. Oncotarget. (2017) 8:107596–602. doi: 10.18632/oncotarget.22538 PubMed Abstract | CrossRef Full Text | Google Scholar 43. Poirel L, Kieffer N, Nordmann P. In vitro study of ISApl1-mediated mobilization of the colistin resistance gene mcr-1. Antimicrob Agents Chemother. (2017) 61:e00127–17. doi: 10.1128/AAC.00127-17 PubMed Abstract | CrossRef Full Text | Google Scholar 44. Moreno LZ, Gomes VTM, Moreira J, de Oliveira CH, Peres BP, Silva APS, et al. First report of mcr-1-harboring Salmonella enterica serovar Schwarzengrund isolated from poultry meat in Brazil. Diagn Microbiol Infect Dis. (2019) 93:376–9. doi: 10.1016/j.diagmicrobio.2018.10.016 PubMed Abstract | CrossRef Full Text | Google Scholar 45. Snesrud E, McGann P, Chandler M. The birth and demise of the ISApl1-mcr-1-ISApl1 composite transposon: the vehicle for transferable colistin resistance. MBio. (2018) 9:e02381–17. doi: 10.1128/mBio.02381-17 PubMed Abstract | CrossRef Full Text | Google Scholar 46. Zamparette CP, Schorner M, Campos E, Moura Q, Cerdeira L, Tartari DC, et al. IncX4 plasmid-mediated mcr-1.1 in polymyxin-resistant Escherichia coli from outpatients in Santa Catarina, Southern Brazil. Microb Drug Resist. (2020) 26:1326–33. doi: 10.1089/mdr.2019.0203 PubMed Abstract | CrossRef Full Text | Google Scholar 47. Wang Y, Xu C, Zhang R, Chen Y, Shen Y, Hu F, et al. Changes in colistin resistance and mcr-1 abundance in Escherichia coli of animal and human origins following the ban of colistin-positive additives in China: an epidemiological comparative study. Lancet Infect Dis. (2020) 20:1161–71. doi: 10.1016/S1473-3099(20)30149-3 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: mcr-1 gene, IncX4 plasmid type, colistin resistance, Latin America, antimicrobial resistance Citation: Lentz SAM, Dalmolin TV, Barth AL and Martins AF (2021) mcr-1 Gene in Latin America: How Is It Disseminated Among Humans, Animals, and the Environment? Front. Public Health 9:648940. doi: 10.3389/fpubh.2021.648940 Received: 02 January 2021; Accepted: 22 February 2021; Published: 07 May 2021. Edited by: Reviewed by: Copyright © 2021 Lentz, Dalmolin, Barth and Martins. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Andreza Francisco Martins, [email protected]
Frontiers in Microbiology, Volume 12; https://doi.org/10.3389/fmicb.2021.680816

Abstract:
Editorial on the Research TopicShiga Toxin-Converting Bacteriophages Shiga toxin-producing Escherichia coli (STEC) strains, and particularly a subset of this group of bacteria called enterohemorrhagic E. coli (EHEC), are dangerous human pathogens (Newell and La Ragione, 2018). They cause bloody diarrhea and some other symptoms, but the most severe complication, causing the death of a relatively large fraction of patients, is hemolytic uremic syndrome (Karmali, 2018; Joseph et al., 2020). The major virulence factors of STEC are Shiga toxins that are complex proteins (composed of one A and five B subunits; AB5) which enter an eukaryotic cell mainly through the interaction of the toxin B subunit with the Gb3 and/or Gb4 receptor. After endocytosis, followed by retro-translocation from the Golgi apparatus and endoplasmic reticulum, the toxin A subunit, which is an actual toxin, is released into the cytoplasm where it inactivates ribosomes by specific cleavage of one of the adenine residues in 28S rRNA (Menge, 2020). The crucial point of virulence of STEC is the fact that Shiga toxins are not encoded in E. coli chromosomes but their genes, called stx genes, are located in the genomes of bacteriophages which occur in their hosts as prophages (Loś et al., 2011). These bacteriophages, called also Stx phages, belong to the group of lambdoid phages. After infection, Stx phages may lysogenize the host cell. Following integration of the phage DNA into the bacterial chromosome, it can be passively replicated together with the host genome. In lysogenic cells, the vast majority of phage genes are silent, including stx genes. Under conditions causing the appearance of single-stranded DNA fragments which induce the bacterial S.O.S. response, the phage-encoded major repressor (the cI protein) is cleaved. This leads to induction of the prophage, excision of phage DNA, and subsequent lytic development which consists of effective viral DNA replication, production of progeny virions, and their release after host cell lysis. This is accompanied with massive production and liberation of Shiga toxins. Although under laboratory conditions, UV irradiation or DNA-binding antibiotics, like mitomycin C, are routinely used to activate the S.O.S. response, it appears that oxidative stress may be one of major causes of Stx prophage induction in STEC-infected humans (Licznerska et al., 2016). Importantly, contrary to early predictions of uniformity in Stx phages, this group of viruses has been recognized as highly variable (Krüger and Lucchesi, 2015). An E. coli host can be lysogenized not only by one, but also by two or more Stx phages which may result in the production of different types of Stx toxins in one bacterial cell. In fact, it was demonstrated that understanding the biodiversity and evolution of Stx phages may have a great impact on both the accurate diagnostics of STEC-mediated infections and development of novel therapeutic approaches (Martínez-Castillo and Muniesa, 2014). The treatment of STEC-infected patients is particularly difficult as it was found that many antibiotics are potent inducers of prophages. Therefore, even if antibiotic treatment can kill bacterial cells or inhibit their growth, prophage induction would cause effective production of Shiga toxin in the meantime which could be more dangerous for patients than the presence of STEC cells. Although some antibiotics can kill bacteria without stimulating the S.O.S. response, the use of antibiotics in the treatment of STEC infections remains controversial (Bielaszewska et al., 2012). Such a medical procedure is formally forbidden in some countries if this type of disease is confirmed or even suspected (Kakoullis et al., 2019). In this light, development of alternative therapies appears crucial (Mühlen and Dersch, 2020). On the other hand, effective management of diseases caused by STEC requires an understanding of the details of pathogenicity mechanisms and epidemiology, and development of rapid and accurate diagnostic methods (Paletta et al., 2020). Cattle are considered to be the main reservoir of STEC (Kim et al., 2020). This is because bovine vascular cells do not contain the Gb3 receptor [though it may be present in some other tissues, as reported by Pruimboom-Brees et al. (2000)], therefore, Shiga toxins cannot enter them efficiently (Sapountzis et al., 2020). Nevertheless, despite enormous progress in studies on various aspects of STEC and Stx phages, it appears that we are still far from understanding the details of their diversity, evolution, distribution, and molecular mechanisms of functioning. In the light of the problems and open questions summarized above, a special issue of this journal has been devoted to present recent advances in studies on Stx phages. According to recent trends, articles published in this issue are focused on biodiversity and evolution of these phages, as well as on molecular mechanisms of their development, including genetic recombination, which seems to be of particular importance in increasing variability of viruses (general recombination) and virulence of E. coli strains (site-specific recombination). A novel view on evolution of Stx phages has been presented by Zuppi et al. They have isolated newly discovered Stx phages from STEC strains, analyzed phages' whole-genome sequences and compared them to previously sequenced genomes of other Stx phages. Considerable variability of these phages was noted, and multiple transduction events have been predicted. The crucial conclusion from this study was that “colonization of a specific reservoir by STEC strains could be influenced by the Stx phage that they carry.” This is an important contribution to our understanding of the distribution of STEC in animal carriers, and further attempts to recognize reservoirs of these bacteria more accurately. Genomic diversity of Stx phages has been indicated also in another article, by Zhang et al. Newly identified bacteriophages have been characterized after prophage induction from environmental STEC strains. This study provided further important evidence for Shiga toxin conversion among E. coli strains through Stx phage lysogenic infection. In this light, it appears crucial to estimate the efficiency of survival and persistence of Stx phages in food products, since they can spread virulence factors under favorable conditions, as stated by Spilsberg et al. These authors described an improved procedure for the detection of Stx phages in minced meat, employing real-time PCR and digital droplet PCR. Using this method, they demonstrated that these phages could survive and remain infective for at least 20 days during meat storage. Recombination events may significantly contribute to the variability of Stx phages, and the article by Greig et al. provides examples of the occurrence of such processes. Definitely, evolutionary analyses of Stx phages and STEC strains should take into consideration genetic recombination. The authors concluded that when studying the persistence and survival of STEC in the environment, microevolutionary processes and larger changes in genomes should be analyzed by using both short and long read technologies, in order to understand their mechanisms in more detail. The importance of such a research strategy has been underlined in another study by Greig et al. in which they analyzed biological material derived from a STEC-caused outbreak of foodborne disease. Epidemiological data suggested that the outbreak strains originated from one country while phylogenetic studies indicated that they were most related to isolates from geographically very distinct regions. As proposed by the authors, early warning of emerging threats to public health can be facilitated by monitoring transmission of STEC and Stx phages. As indicated above, Stx phages belong to lambdoid bacteriophages, as classified on the basis of the organization of their genomes. However, two articles published in this special issue indicated that the molecular mechanisms of development of various Stx phages might be very different from those found in bacteriophage λ. Mohaisen et al. studied the site-specific recombination system of the Stx bacteriophage ϕ24B and found that att regions in genomes of this phage and its host differ significantly from those occurring in λ and between gal and bio loci in E. coli chromosomes. Moreover, the integration of the ϕ24B genome appeared independent on the integration host factor (the IHF protein), contrary to the strict dependence on IHF in the case of analogous reaction in λ. Similarly, a DNA replication system of Eru bacteriophages (a newly identified group of Stx phages), which is completely unrelated to that of λ, has been described by Llarena et al. The authors proposed a novel classification of Stx phages, based on their replication regions, in addition to the type of stx genes they carry. In summary, recent studies have brough unexpected and fascinating results which are important to understand the biology of Stx phages in more detail. Therefore, we encourage researchers to read the articles included in this special issue, and to contribute to our collection of knowledge on these important viruses. GW and MM contributed to the analysis of the reviewed articles and to preparing the manuscript. The work of GW on Stx phages was supported by the National Science Center (Poland), with Grant No. 2018/29/B/NZ1/00549, and that of MM was supported by the Spanish Ministerio de Innovación y Ciencia (AGL2016-75536-P) AEI/ERF, EU. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Bielaszewska, M., Idelevich, E. A., Zhang, W., Bauwens, A., Schaumburg, F., Mellmann, A., Peters, G., and Karch, H. (2012). Effects of antibiotics on Shiga toxin 2 production and bacteriophage induction by epidemic Escherichia coli O104:H4 strain. Antimicrob. Agents Chemother. 56, 3277–3282. doi: 10.1128/AAC.06315-11 PubMed Abstract | CrossRef Full Text | Google Scholar Joseph, A., Cointe, A., Mariani Kurkdjian, P., Rafat, C., and Hertig, A. (2020). Shiga toxin-associated hemolytic uremic syndrome: a narrative review. Toxins 12:67. doi: 10.3390/toxins12020067 PubMed Abstract | CrossRef Full Text | Google Scholar Kakoullis, L., Papachristodoulou, E., Chra, P., and Panos, G. (2019). Shiga toxin-induced haemolytic uraemic syndrome and the role of antibiotics: a global overview. J. Infect. 79, 75–94. doi: 10.1016/j.jinf.2019.05.018 PubMed Abstract | CrossRef Full Text | Google Scholar Karmali, M. A. (2018). Factors in the emergence of serious human infections associated with highly pathogenic strains of Shiga toxin-producing Escherichia coli. Int. J. Med. Microbiol. 308, 1067–1072. doi: 10.1016/j.ijmm.2018.08.005 PubMed Abstract | CrossRef Full Text | Google Scholar Kim, J. S., Lee, M. S., and Kim, J. H. (2020). Recent updates on outbreaks of Shiga toxin-producing Escherichia coli and its potential reservoirs. Front. Cell Infect. Microbiol. 10:273. doi: 10.3389/fcimb.2020.00273 PubMed Abstract | CrossRef Full Text | Google Scholar Krüger, A., and Lucchesi, P. M. (2015). Shiga toxins and stx phages: highly diverse entities. Microbiology 161, 451–462. doi: 10.1099/mic.0.000003 PubMed Abstract | CrossRef Full Text | Google Scholar Licznerska, K., Nejman-Faleńczyk, B., Bloch, S., Dydecka, A., Topka, G., Gasior, T., et al. (2016). Oxidative stress in Shiga toxin production by enterohemorrhagic Escherichia coli. Oxid. Med. Cell. Longev. 2016:3578368. doi: 10.1155/2016/3578368 PubMed Abstract | CrossRef Full Text | Google Scholar Loś, J. M., Loś, M., and Wegrzyn, G. (2011). Bacteriophages carrying shiga toxin genes: genomic variations, detection and potential treatment of pathogenic bacteria. Future Microbiol. 6, 909–924. doi: 10.2217/fmb.11.70 PubMed Abstract | CrossRef Full Text | Google Scholar Martínez-Castillo, A., and Muniesa, M. (2014). Implications of free Shiga toxin-converting bacteriophages occurring outside bacteria for the evolution and the detection of Shiga toxin-producing Escherichia coli. Front. Cell. Infect. Microbiol. 4:46. doi: 10.3389/fcimb.2014.00046 PubMed Abstract | CrossRef Full Text | Google Scholar Menge, C. (2020). Molecular biology of Escherichia coli Shiga toxins' effects on mammalian cells. Toxins 12:345. doi: 10.3390/toxins12050345 PubMed Abstract | CrossRef Full Text | Google Scholar Mühlen, S., and Dersch, P. (2020). Treatment strategies for infections with Shiga toxin-producing Escherichia coli. Front. Cell. Infect. Microbiol. 10:169. doi: 10.3389/fcimb.2020.00169 PubMed Abstract | CrossRef Full Text | Google Scholar Newell, D. G., and La Ragione, R. M. (2018). Enterohaemorrhagic and other Shiga toxin-producing Escherichia coli (STEC): where are we now regarding diagnostics and control strategies? Transbound. Emerg. Dis. 65, 49–71. doi: 10.1111/tbed.12789 PubMed Abstract | CrossRef Full Text | Google Scholar Paletta, A. C. C., Castro, V. S., and Conte-Junior, C. A. (2020). Shiga toxin-producing and enteroaggregative Escherichia coli in animal, foods, and humans: pathogenicity mechanisms, detection methods, and epidemiology. Curr. Microbiol. 77, 612–620. doi: 10.1007/s00284-019-01842-1 PubMed Abstract | CrossRef Full Text | Google Scholar Pruimboom-Brees, I. M., Morgan, T. W., Ackermann, M. R., Nystrom, E. D., Samuel, J. E., Cornick, N. A., et al. (2000). Cattle lack vascular receptors for Escherichia coli O157:H7 shiga toxins. Proc. Natl. Acad. Sci. U.S.A. 97, 10325–10329. doi: 10.1073/pnas.190329997 PubMed Abstract | CrossRef Full Text | Google Scholar Sapountzis, P., Segura, A., Desvaux, M., and Forano, E. (2020). An overview of the elusive passenger in the gastrointestinal tract of cattle: the shiga toxin producing Escherichia coli. Microorganisms 8:877. doi: 10.3390/microorganisms8060877 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: Shiga toxin, bacteriophages, enterohemorrhagic Escherichia coli, phage development, phage evolution Citation: Węgrzyn G and Muniesa M (2021) Editorial: Shiga Toxin-Converting Bacteriophages. Front. Microbiol. 12:680816. doi: 10.3389/fmicb.2021.680816 Received: 15 March 2021; Accepted: 06 April 2021; Published: 04 May 2021. Edited by: Reviewed by: Copyright © 2021 Węgrzyn and Muniesa. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Grzegorz Węgrzyn, [email protected]; Maite Muniesa, [email protected]
Frontiers in Cellular and Infection Microbiology, Volume 11; https://doi.org/10.3389/fcimb.2021.670677

Abstract:
The recently published original article by Mortaji et al. (2020) characterized for the first time the function of a type I toxin-antitoxin (TA) system in the gastric pathogen Helicobacter pylori. It was found that the high expression of an AapA1 toxin, which is part of this system, causes a drastic decrease in the amount of culturable H. pylori cells and their transformation from a spiral to a coccoid morphotype. It was also established that AapA1 is a hydrophobic peptide disrupting cell division and that oxidative stress is an inducer of the toxin expression. The development of genomics and bioinformatics in recent years has contributed to the discovery of a high frequency of TA systems in microorganisms, which was a strong stimulus for the intensification of research on their structure and function (Lee and Lee, 2016; Yang and Walsh, 2017). Prokaryotic TA modules are genetic elements that encode information about a toxin involved in inhibiting growth of the bacterial producer and an antitoxin that counteracts the activity of the former. Toxins belonging to TA systems restrict microbial replication by targeting key processes for cell physiology, including replication, transcription, translation and/or cell wall synthesis (Harms et al., 2018). Attention is being paid increasingly to the participation of these systems in suppressing the microbial multiplication and the stimulatory effect on adaptation to stressful conditions, i.e., nutritional starvation, exposure to antimicrobial substances or immune system cells’ attack (Lee and Lee, 2016; Yang and Walsh, 2017). To date, five type II TA systems of H. pylori have been identified. These include chromosomally encoded HP0892-HP0893 (Han et al., 2013), HP0894-HP0895 (Han et al., 2011), HP0315-HP0316 (Kwon et al., 2012), and HP0967-HP0968 (Cárdenas-Mondragón et al., 2016), and the newly identified TfiT-TfiA (Boampong et al., 2020), which is encoded on mobile genetic fragments. The expression of toxins belonging to the above modules arrest the growth of bacterial producers and cause the reduction of their number (expressed in CFU/mL). Similar observations were made in 2017 by Arnion et al. (2017), who first identified the existence of the type I TA system in H. pylori (called AapA1-IsoA1), and noted that the expression of the toxin significantly decreases the amount of culturable H. pylori cells. At this point it is worth mentioning that Mortaji et al. (2020) deepened the knowledge related to the above phenomenon. They proved in their next original article that this decline was caused by a reduction in the culturability (observed as the optical density of the culture) but not the viability of H. pylori (preserved cell membrane integrity and a stable ATP level), and was accompanied by the transition of morphology from spiral to coccoidal (Mortaji et al., 2020). This observation is very valuable from the scientific point of view and confirms the postulates presented by our research group, pointing to difficulties in the correct interpretation of the H. pylori viability (understood as the sum of various cell parameters suggesting its physiological activity) and frequent mistakes made by scientists taking the culturability (detected by culture optical density or CFU/mL) as the only determinant of the viability of this pathogen (Krzyżek and Grande, 2020). An additional valuable cognitive element shown by Mortaji et al. (2020) was a proof that oxidative stress was an inducer of the aapA1 expression in H. pylori, and thus a trigger for the spiral-to-coccoid transition. Exposure to high concentrations of oxygen, understood here as oxidative stress, is a well-known stress factor for H. pylori determining its intensive transformation into spherical forms (Chuang et al., 2005; Zeng et al., 2008). Thus, Mortaji et al. (2020) neatly revealed a possible molecular mechanism governing this process. In regard to this, it is also worth paying attention to the results presented by many research teams that have shown that bactericidal antibiotics, unlike bacteriostatic ones, stimulate the formation of oxygen free radicals and oxidative stress in bacterial cells, regardless of their target site (Kohanski et al., 2007; Brynildsen et al., 2013; Dwyer et al., 2014; Belenky et al., 2015; Lobritz et al., 2015; Li et al., 2017). According to Lobritz et al. (2015), this effect was particularly visible with the use of antibiotics acting on the microbial cell wall and DNA, but neither translation nor transcription. The above information, in conjunction with the results provided by Mortaji et al. (2020), seem to be extremely interesting, as they may explain why bactericidal antibiotics (amoxicillin, levofloxacin or metronidazole) induce morphological transformation into spherical forms in H. pylori significantly faster than bacteriostatic antibiotics (Sörberg et al., 1997; Sörberg et al., 1998; Akada et al., 1999; Faghri et al., 2014; Krzyżek et al., 2019a; Krzyżek et al., 2019b). Still, it should be remembered that the process of cell death and/or formation of coccoids by H. pylori during the exposure to bactericidal antibiotics may depend on many factors simultaneously or be independent of oxidative stress. In the original article by Mortaji et al. (2020), H. pylori was exposed to one of two antibiotics: rifampicin or tetracycline targeting transcription or translation, respectively. The authors did not observe any significant increase in the aapA1 expression in rifampicin- or tetracycline-treated cells, concluding that exposure of H. pylori to antibiotics did not affect the expression of this toxin. In the light of the above presented deduction, however, it seems that divergent results may arise for other antibiotics used in the therapy of H. pylori, especially those with a strong bactericidal activity, e.g., amoxicillin, levofloxacin or metronidazole. Extending research to include these antibiotics would allow it to be established whether the hypothesis presented by an author of this commentary about the inducing effect of bactericidal antibiotics and their oxidative stress-dependent generation of morphological transition into spherical forms by H. pylori is correct (Figure 1). Figure 1 Schematic drawing presenting a hypothetical model describing differences in the potential of antibiotics to generate the spiral-to-coccoid transformation in H. pylori. In the routine therapy of H. pylori, the following antibiotics are used: rifampicin (transcription), tetracycline and clarithromycin (translation), metronidazole and levofloxacin (the DNA structure or replication), and amoxicillin (the cell wall) (Jones et al., 2008; Francesco, 2011; Nishizawa and Suzuki, 2014). Based on reports showing the ability of bactericidal antibiotics to stimulate oxidative stress in microbial cells (Kohanski et al., 2007; Brynildsen et al., 2013; Dwyer et al., 2014; Belenky et al., 2015; Lobritz et al., 2015; Li et al., 2017) and the results of Mortaji et al. (2020), demonstrating the oxidative stress-dependent induction of the toxin-antitoxin system in H. pylori, a hypothetical model integrating the above observations has been proposed. Antibiotics acting on transcription and translation (rifampicin, tetracycline or clarithromycin) have a marginal effect on the oxido-reductive state of bacterial cells and therefore do not significantly affect the toxin-antitoxin balance. The opposite situation is suggested for antibiotics targeting the cell wall or DNA (amoxicillin, levofloxacin or metronidazole), all of which stimulate the accumulation of reactive oxygen species in bacterial cells and the oxidative stress-related disturbance of the toxin-antitoxin balance in favor of the former. The increased production of this toxin is accompanied by the conversion of H. pylori into spherical forms. Finally, it is worth noting that the results presented by Mortaji et al. (2020) may have clinically significant implications, especially in the context of the eradication of difficult-to-treat, recurrent H. pylori infections. Recently, Morales-Espinosa et al. (2020) showed that the expression of HP0315, one of the components of the type II TA systems, is expressed significantly higher in intracellular H. pylori subpopulations and that the expression of this gene was accompanied by the formation of coccoid forms by these bacteria. Therefore, it seems very interesting to determine whether this type of relationship can also be demonstrated for other TA modules, including AapA1-IsoA1, and whether lowering the expression of the toxin or increasing the expression of the antitoxin would positively influence the frequency of H. pylori eradication. The author confirms being the sole contributor of this work and has approved it for publication. The study was supported by the Wroclaw Medical University grant No: SUB.A130.21.031. The funder had no role in the preparation of the manuscript. The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Akada, J. K., Shirai, M., Fujii, K., Okita, K., Nakazawa, T. (1999). In Vitro Anti-Helicobacter pylori Activities of New Rifamycin Derivatives, KRM-1648 and KRM-1657. Antimicrob. Agents Chemother. 43, 1072–1076. doi: 10.1128/aac.43.5.1072 PubMed Abstract | CrossRef Full Text | Google Scholar Arnion, H., Korkut, D. N., Gelo, S. M., Chabas, S., Reignier, J., Iost, I., et al. (2017). Mechanistic Insights Into Type I Toxin Antitoxin Systems in Helicobacter pylori: The Importance of mRNA Folding in Controlling Toxin Expression. Nucleic Acids Res. 45, 4782–4795. doi: 10.1093/nar/gkw1343 PubMed Abstract | CrossRef Full Text | Google Scholar Belenky, P., Ye, J. D., Porter, C. B. M., Cohen, N. R., Lobritz, M. A., Ferrante, T., et al. (2015). Bactericidal Antibiotics Induce Toxic Metabolic Perturbations That Lead to Cellular Damage. Cell Rep. 13, 968–980. doi: 10.1016/j.celrep.2015.09.059 PubMed Abstract | CrossRef Full Text | Google Scholar Boampong, K., Smith, S. L., Delahay, R. M. (2020). Rapid Growth Inhibitory Activity of a YafQ-Family Endonuclease Toxin of the Helicobacter pylori Tfs4 Integrative and Conjugative Element. Sci. Rep. 10, 18171. doi: 10.1038/s41598-020-72063-x PubMed Abstract | CrossRef Full Text | Google Scholar Brynildsen, M. P., Winkler, J. A., Spina, C. S., MacDonald, I. C., Collins, J. J. (2013). Potentiating Antibacterial Activity by Predictably Enhancing Endogenous Microbial ROS Production. Nat. Biotechnol. 31, 160–165. doi: 10.1038/nbt.2458 PubMed Abstract | CrossRef Full Text | Google Scholar Cárdenas-Mondragón, M. G., Ares, M. A., Panunzi, L. G., Pacheco, S., Camorlinga-Ponce, M., Girón, J. A., et al. (2016). Transcriptional Profiling of Type II Toxin-Antitoxin Genes of Helicobacter pylori Under Different Environmental Conditions: Identification of HP0967-HP0968 System. Front. Microbiol. 7, 1872. doi: 10.3389/fmicb.2016.01872 PubMed Abstract | CrossRef Full Text | Google Scholar Chuang, M.-H., Wu, M.-S., Lin, J.-T., Chiou, S.-H. (2005). Proteomic Analysis of Proteins Expressed by Helicobacter pylori Under Oxidative Stress. Proteomics 5, 3895–3901. doi: 10.1002/pmic.200401232 PubMed Abstract | CrossRef Full Text | Google Scholar Dwyer, D. J., Belenky, P. A., Yang, J. H., Cody MacDonald, I., Martell, J. D., Takahashi, N., et al. (2014). Antibiotics Induce Redox-Related Physiological Alterations as Part of Their Lethality. Proc. Natl. Acad. Sci. U. S. A. 111, E2100–E2109. doi: 10.1073/pnas.1401876111 PubMed Abstract | CrossRef Full Text | Google Scholar Faghri, J., Poursina, F., Moghim, S., Zarkesh Esfahani, H., Nasr Esfahani, B., Fazeli, H., et al. (2014). Morphological and Bactericidal Effects of Different Antibiotics on Helicobacter pylori. Jundishapur. J. Microbiol. 7, e8704. doi: 10.5812/jjm.8704 PubMed Abstract | CrossRef Full Text | Google Scholar Francesco, V. (2011). Mechanisms of Helicobacter pylori Antibiotic Resistance: An Updated Appraisal. World J. Gastrointest. Pathophysiol. 2, 41. doi: 10.4291/wjgp.v2.i3.35 CrossRef Full Text | Google Scholar Han, K. D., Ahn, D. H., Lee, S. A., Min, Y. H., Kwon, A. R., Ahn, H. C., et al. (2013). Identification of Chromosomal HP0892-HP0893 Toxin-Antitoxin Proteins in Helicobacter pylori and Structural Elucidation of Their Protein-Protein Interaction. J. Biol. Chem. 288, 6004–6013. doi: 10.1074/jbc.M111.322784 PubMed Abstract | CrossRef Full Text | Google Scholar Han, K. D., Matsuura, A., Ahn, H. C., Kwon, A. R., Min, Y. H., Park, H. J., et al. (2011). Functional Identification of Toxin-Antitoxin Molecules From Helicobacter pylori 26695 and Structural Elucidation of the Molecular Interactions. J. Biol. Chem. 286, 4842–4853. doi: 10.1074/jbc.M109.097840 PubMed Abstract | CrossRef Full Text | Google Scholar Harms, A., Brodersen, D. E., Mitarai, N., Gerdes, K. (2018). Toxins, Targets, and Triggers: An Overview of Toxin-Antitoxin Biology. Mol. Cell 70, 768–784. doi: 10.1016/j.molcel.2018.01.003 PubMed Abstract | CrossRef Full Text | Google Scholar Jones, K. R., Cha, J.-H., Merrell, D. S. (2008). Who’s Winning the War? Molecular Mechanisms of Antibiotic Resistance in Helicobacter pylori. Curr. Drug Ther. 3, 190–203. doi: 10.2174/157488508785747899 PubMed Abstract | CrossRef Full Text | Google Scholar Kohanski, M. A., Dwyer, D. J., Hayete, B., Lawrence, C. A., Collins, J. J. (2007). A Common Mechanism of Cellular Death Induced by Bactericidal Antibiotics. Cell 130, 797–810. doi: 10.1016/j.cell.2007.06.049 PubMed Abstract | CrossRef Full Text | Google Scholar Krzyżek, P., Franiczek, R., Krzyżanowska, B., Łaczmański, Ł., Migdał, P., Gościniak, G. (2019a). In Vitro Activity of 3-Bromopyruvate, an Anti-Cancer Compound, Against Antibiotic-Susceptible and Antibiotic-Resistant Helicobacter pylori Strains. Cancers (Basel) 11, 229. doi: 10.3390/cancers11020229 CrossRef Full Text | Google Scholar Krzyżek, P., Franiczek, R., Krzyżanowska, B., Łaczmański, Ł., Migdał, P., Gościniak, G. (2019b). In Vitro Activity of Sertraline, an Antidepressant, Against Antibiotic-Susceptible and Antibiotic-Resistant Helicobacter pylori Strains. Pathogens 8, 228. doi: 10.3390/pathogens8040228 CrossRef Full Text | Google Scholar Krzyżek, P., Grande, R. (2020). Transformation of Helicobacter pylori Into Coccoid Forms as a Challenge for Research Determining Activity of Antimicrobial Substances. Pathogens 9, 184. doi: 10.3390/pathogens9030184 CrossRef Full Text | Google Scholar Kwon, A. R., Kim, J. H., Park, S. J., Lee, K. Y., Min, Y. H., Im, H., et al. (2012). Structural and Biochemical Characterization of HP0315 From Helicobacter pylori as a VapD Protein With an Endoribonuclease Activity. Nucleic Acids Res. 40, 4216–4228. doi: 10.1093/nar/gkr1305 PubMed Abstract | CrossRef Full Text | Google Scholar Lee, K. Y., Lee, B. J. (2016). Structure, Biology, and Therapeutic Application of Toxin-Antitoxin Systems in Pathogenic Bacteria. Toxins (Basel) 8, 305. doi: 10.3390/toxins8100305 CrossRef Full Text | Google Scholar Li, Z., Tan, J., Shao, L., Dong, X., Ye, R. D., Chen, D. (2017). Selenium-Mediated Protection in Reversing the Sensitivity of Bacterium to the Bactericidal Antibiotics. J. Trace Elem. Med. Biol. 41, 23–31. doi: 10.1016/j.jtemb.2017.02.007 PubMed Abstract | CrossRef Full Text | Google Scholar Lobritz, M. A., Belenky, P., Porter, C. B. M., Gutierrez, A., Yang, J. H., Schwarz, E. G., et al. (2015). Antibiotic Efficacy is Linked to Bacterial Cellular Respiration. Proc. Natl. Acad. Sci. U. S. A. 112, 8173–8180. doi: 10.1073/pnas.1509743112 PubMed Abstract | CrossRef Full Text | Google Scholar Morales-Espinosa, R., Delgado, G., Serrano, L. R., Castillo, E., Santiago, C. A., Hernández-Castro, R., et al. (2020). High Expression of Helicobacter pylori VapD in Both the Intracellular Environment and Biopsies From Gastric Patients With Severity. PLoS One 15, e0230220. doi: 10.1371/journal.pone.0230220 PubMed Abstract | CrossRef Full Text | Google Scholar Mortaji, L., Tejada-Arranz, A., Rifflet, A., Boneca, I. G., Pehau-Arnaudet, G., Radicella, J. P., et al. (2020). A Peptide of a Type I Toxin-Antitoxin System Induces Helicobacter pylori Morphological Transformation From Spiral Shape to Coccoids. Proc. Natl. Acad. Sci. U. S. A. 117, 31398–31409. doi: 10.1073/pnas.2016195117 PubMed Abstract | CrossRef Full Text | Google Scholar Nishizawa, T., Suzuki, H. (2014). Mechanisms of Helicobacter pylori Antibiotic Resistance and Molecular Testing. Front. Mol. Biosci. 1:19. doi: 10.3389/fmolb.2014.00019 PubMed Abstract | CrossRef Full Text | Google Scholar Sörberg, M., Hanberger, H., Nilsson, M., Björkman, A., Nilsson, L. E. (1998). Risk of Development of In Vitro Resistance to Amoxicillin, Clarithromycin, and Metronidazole in Helicobacter pylori. Antimicrob. Agents Chemother. 42, 1228. doi: 10.1128/AAC.42.5.1222 CrossRef Full Text | Google Scholar Sörberg, M., Hanberger, H., Nilsson, M., Nilsson, L. E. (1997). Pharmacodynamic Effects of Antibiotics and Acid Pump Inhibitors on Helicobacter pylori. Antimicrob. Agents Chemother. 41, 2218–2223. doi: 10.1128/aac.41.10.2218 PubMed Abstract | CrossRef Full Text | Google Scholar Yang, Q. E., Walsh, T. R. (2017). Toxin-Antitoxin Systems and Their Role in Disseminating and Maintaining Antimicrobial Resistance. FEMS Microbiol. Rev. 41, 343–353. doi: 10.1093/femsre/fux006 PubMed Abstract | CrossRef Full Text | Google Scholar Zeng, H., Guo, G., Mao, X. H., De Tong, W., Zou, Q. M. (2008). Proteomic Insights Into Helicobacter pylori Coccoid Forms Under Oxidative Stress. Curr. Microbiol. 57, 281–286. doi: 10.1007/s00284-008-9190-0 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: Helicobacter pylori, toxin-antitoxin system, coccoid forms, morphological transformation, stress response Citation: Krzyżek P (2021) Toxin-Antitoxin Systems - A New Player in Morphological Transformation of Antibiotic-Exposed Helicobacter pylori? Front. Cell. Infect. Microbiol. 11:670677. doi: 10.3389/fcimb.2021.670677 Received: 22 February 2021; Accepted: 06 April 2021; Published: 26 April 2021. Edited by: Reviewed by: Copyright © 2021 Krzyżek. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Paweł Krzyżek, [email protected]
Dongna Zou, Haitao Yu, Feifei Li
Published: 16 April 2021
Frontiers in Pharmacology, Volume 12; https://doi.org/10.3389/fphar.2021.647564

Abstract:
As we all know, Coronavirus Disease 2019 spread all over the world and had became a public international event of global concern. Among Coronavirus Disease 2019 patients in China, two doctors, Yi Fan and Weifeng Hu, were noticed with their skin pigmentary disorder due to polymyxin B. However, we found that polymyxin E has almost no reports of skin hyperpigmentation, but polymyxin B was reported about skin hyperpigmentation, although the number of relevant reports was small, what causes the difference between the polymyxin B and polymyxin E? Polymyxin is a general term for a group of basic peptide antibiotics, including A, B, C, D and E mainly. At present, the two commonly used clinically are polymyxin B and polymyxin E. The mechanism of polymyxin B causing skin hyperpigmentation may include the following aspects: 1. Polymyxin B activates mast cells to release histamine, which binds to the H2 receptor on the surface of melanocytes inducing the stimulation of melanin synthesis Polymyxin B is a mast cell activator that binds to non-selective G protein-coupled receptors on the surface of mast cells, activates subsequent intracellular signaling pathways, and induces mast cells to degranulate and release histamine (Bushby and Green, 1955; Ferry et al., 2002; Morrison et al., 1974; Morrison et al., 1978; Zhan et al., 2019). Histamine is an inflammatory mediator involved in stimulating melanin production. Histamine binds to the H2 receptor on the surface of melanocytes in the basal layer to induce the production of cyclic adenosine monophosphate and the activation of protein kinase a in melanocytes, which leads to phosphorylation of members of the cyclic adenylate response element binding protein transcription factor family. Cyclic AMP response element binding protein activates a variety of genes and induces the transcription of a variety of enzymes and proteins related to melanin synthesis. Eventually leads to increased melanin synthesis in the cytoplasm (Yoshida et al., 2020). Polymyxin E can also release histamine equivalent to polymyxin B (Bushby and Green, 1955), the reason why polymyxin E does not lead to skin hyperpigmentation is not very clear, the difference of amino acid at position 6 between the two drugs and the lower conversion rate of polymyxin E in vivo are possible explanation. According to the research, colistin methanesulfonate (CMS) as the prodrug is predominantly cleared by renal excretion, with only a relatively small fraction of the dosage converted to the active antibacterial in renally healthy individuals (Li et al., 2004; Li et al., 2006), it is evident that in patients with moderate to good renal function, administration of a daily dose of colistin base activity (CBA) at the upper limit of the current product-recommended dose range (300 mg CBA per day) was not able to generate plasma colistin concentrations that would be expected to be reliably efficacious (Garonzik et al., 2011). Is the concentration of polymyxin E not enough to activate mast cells to release enough histamine for inducing the stimulation of melanin synthesis? Further study is worthy. 2. The skin inflammation process is related to the activation of melanocytes Histological and immunohistochemical results of pigmented skin in patients with polymyxin B treatment showed an abundant melanocyte-pigmented dendritic network. Langerhans cells’ hyperplasia and dermal IL-6 overexpression were also found, presumably for an inflammatory process due to polymyxin B use (Mattos et al., 2016; Li et al., 2020; Wen et al., 2020); At the same time, although hyperpigmented skin did not show signs of inflammationby clinical inspection, under microscopic view the dermis contained mild-to- moderate perivascular inflammatory infiltrate of lymphocytes and histiocytes. Langerhans cells are antigen-presenting cells, which play an important role in skin immunity and inflammation. The proliferation of Langerhans cells in the epidermis of patients with polymyxin B induced-pigmentation indicates that polymyxin B can induce the inflammatory process of the skin. In addition to its known inflammatory effects, IL-6 also inhibits the proliferation and melanogenesis of human melanocytes, When the skin is hyperpigmented, IL-6 is often feedback overexpression which may be for regulation (Jawdat et al., 2004; Mattos et al., 2017). At the same time, studies have shown that mast cell-derived factors (including histamine) can stimulate Langerhans cell migration and are related to the melanin production pathway (Miori et al., 1990). Matzneller et al. reported that polymyxin E could decrease inflammatory cytokines, including IL-6, in the blood of L lipopolysaccharide-challenged healthy volunteers in a model of human endotoxiemia (Matzneller et al., 2017). And according to the newest report, it was showed that polymyxin E can’t regulate the expression of the inflammatory cytokine IL6, IL6 mRNA expression levels were not changed after administration of polymyxin E (Ubagai et al., 2021). According to these findings, the effects of polymyxin B and polymyxin E on IL6 are different. We speculate that it may explain the difference in pigmentation between the two drugs. 3. Oxidative stress is also considered to be one of the mechanisms of pigmentation (Zavascki et al., 2016) Ahmed et al. (2017) studied the effect of polymyxin B on human lung epithelial cells A549, and the results showed that polymyxin B induced oxidative stress and loss of mitochondrial membrane potential. Compared to untreated control cells, after 8 h treatment, the cellular oxidative stress increased around 1.9-fold and 3.8-fold for 1.0 and 2.0 mM of polymyxin B, respectively, which increased up to 2.6-fold and 4.7-fold at 24 h correspondingly. The article also mentioned that the hydrophobicity of the 6-phenylalanine in the structure of polymyxin B and the cationic form under physiological conditions play a key role in cytotoxicity and mitochondrial oxidative stress. This may explain the reason that why the pigmentation had not been found in the patient who used polymyxin E. In the results of polymyxin B nephrotoxicity studies, it was found that mitochondrial stress response and the production of reactive oxygen species were found in renal tubular cells treated with polymyxin B (Azad et al., 2015). Reactive oxygen species such as NO can induce the activation of guanylate cyclase and enhance the expression of tyrosinase gene to increase melanin production (Sasaki et al., 2000). 4. Phenylalanine increases melanin synthesis (Martindale, 2014) Compared with polymyxin E, polymyxin B has a different structure at position 6. Polymyxin B is phenylalanine at position 6, and polymyxin E is leucine. According to Martindale records, vitiligo can be treated with phenylalanine, and more than 60% of patients have skin pigmentation during the treatment process (Kopple et al., 2007). This could be explained by that phenylalanine is hydroxylated in the body to form tyrosine (Chang et al., 2009), which is a non-essential amino acid in the human body and the main raw material for the synthesis of melanin (Rzepka et al., 2016). And we have not seen any reports of leucine-induced skin hyperpigmentation, that maybe another reason why polymyxin E-related pigmentation has not been reported. The nephrotoxicity and neurotoxicity induced by polymyxin B have been proved to be dose-dependent (John et al., 2017), but there is no direct evidence that polymyxin B-induced skin hyerpigmentation is a dose-dependent adverse reaction. But it was speculated that acute kidney injury (AKI) with lower creatinine clearance may be an important factor for polymyxin B-induced pigmentary disorder (Zheng et al., 2018; Lu and Hou, 2020). In addition, neonates and infants were more likely to suffered from skin hyerpigmentation after polymyxin B administration than adults, it was reported that 16 infants in ICU generalized skin hyperpigmentation in premature infants receiving polymyxin B (Shih and Gaik, 2014), and it was also noted generalized skin hyperpigmentation among neonates receiving IV polymyxin B (Gothwal et al., 2016), according to these findings, we speculate that it may be related to their immature kidney function leading to the cumulation of polymyxin B (Gothwal et al., 2016; Li et al., 2020), as polymyxin B is excreted through the kidney, while the incidence of skin hyperpigmentation were 15% or 8% of adult patients (Mattos et al., 2016; Mattos et al., 2017). It should be pointed out that there have been no reports of inhalation of polymyxin B-induced skin hyperpigmentation so far, the administration for all patients suffered from skin hyerpigmentation induced by polymyxin B were intravenous (Knueppel and Rahimian, 2007; Shih and Gaik, 2014; Zavascki et al., 2015; Gothwal et al., 2016; Mattos et al., 2016; Zavascki et al., 2016; Lahiry et al., 2017; Mattos et al., 2017; Zheng et al., 2018), which is worthy of further discussion on the relationship between the administration and adverse drug reaction. According to the findings above, we speculate boldly that polymyxin B needs to reach a certain concentration in blood to cause pigmentation and that reducing the dosage may be an effective way to prevent skin hyperpigmentation, however, reducing the dosage of polymyxin B may be likely to reduce the efficacy and even lead to bacterial resistance. It should be point out that there is no definite evidence that the occurrence of pigmentation is related to the increased concentration of polymyxin B in blood, and there is no report of dosage adjustment after the occurrence of skin pigmentation. Regarding the difference between polymyxin B and polymyxin E in causing skin pigmentation, further research is needed in the future, and further research is needed on how to prevent and treat polymyxin B-induced skin pigmentation. However, the current related reports can remind us that we should pay attention to monitoring related adverse reactions when applying polymyxin B. If skin hyerpigmentation occurs, provide corresponding psychological counseling to the patient, or take corresponding treatment measures such as laser cosmetic therapy and topical whitening agents, or adjust the dosing regimen if necessary. FL was responsible for the study conception and design. DZ drafted the manuscript. HY Searched the literature. All authors contributed to the article and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ahmed, M. U., Velkov, T., Lin, Y.-W., Yun, B., Nowell, C. J., Zhou, F., et al. Potential toxicity of polymyxins in human lung epithelial cells. Antimicrob. Agents Chemother. 2017;61(6):e02690-16. Published 2017 May 24. doi:10.1128/AAC.02690-16 PubMed Abstract | CrossRef Full Text | Google Scholar Azad, M. A. K., Akter, J., Rogers, K. L., Nation, R. L., Velkov, T., and Li, J. Major pathways of polymyxin-induced apoptosis in rat kidney proximal tubular cells. Antimicrob. Agents Chemother. 2015;59(4):2136–2143. doi:10.1128/AAC.04869-14 PubMed Abstract | CrossRef Full Text | Google Scholar Bushby, S. R. M., and Green, A. F. The release of histamine by polymyxin B and polymyxin E. Br. J. Pharmacol. Chemother. 1955;10(2):215–219. doi:10.1111/j.1476-5381.1955.tb00085.x PubMed Abstract | CrossRef Full Text | Google Scholar Chang, T.-S. An updated review of tyrosinase inhibitors. Ijms. 2009;10(6):2440–2475. Published 2009 May 26. doi:10.3390/ijms10062440 PubMed Abstract | CrossRef Full Text | Google Scholar Ferry, X., Brehin, S., Kamel, R., and Landry, Y. G protein-dependent activation of mast cell by peptides and basic secretagogues. Peptides. 2002;23(8):1507–1515. doi:10.1016/s0196-9781(02)00090-6 PubMed Abstract | CrossRef Full Text | Google Scholar Garonzik, S. M., Li, J., Thamlikitkul, V., Paterson, D. L., Shoham, S., Jacob, J., et al. Population pharmacokinetics of colistin methanesulfonate and formed colistin in critically ill patients from a multicenter study provide dosing suggestions for various categories of patients. Antimicrob. Agents Chemother. 2011;55(7):3284–3294. doi:10.1128/AAC.01733-10 PubMed Abstract | CrossRef Full Text | Google Scholar Gothwal, S., Meena, K., and Sharma, S. D. (2016). Polymyxin B induced generalized hyperpigmentation in neonates. Indian J. Pediatr. 83, 179–180. doi:10.1007/s12098-015-1798-z PubMed Abstract | CrossRef Full Text | Google Scholar Jawdat, D. M., Albert, E. J., Rowden, G., Haidl, I. D., and Marshall, J. S. IgE-mediated mast cell activation induces Langerhans cell migration in vivo. J. Immunol. 2004;173(8):5275–5282. doi:10.4049/jimmunol.173.8.5275 PubMed Abstract | CrossRef Full Text | Google Scholar John, J. F., Falci, D. R., Rigatto, M. H., Oliveira, R. D., Kremer, T. G., and Zavascki, A. P. Severe infusion-related adverse events and renal failure in patients receiving high-dose intravenous polymyxin B. Antimicrob. Agents Chemother. 2017;62(1):e01617-17. Published 2017 Dec 21. doi:10.1128/AAC.01617-17 PubMed Abstract | CrossRef Full Text | Google Scholar Knueppel, R. C., and Rahimian, J. Diffuse cutaneous hyperpigmentation due to tigecycline or polymyxin B. Clin. Infect. Dis. 2007, 45(1): 136. doi:10.1086/518706 PubMed Abstract | CrossRef Full Text | Google Scholar Kopple, J. D. Phenylalanine and tyrosine metabolism in chronic kidney failure. J. Nutr. 2007;137(6 Suppl. 1):1586S–1590S. doi:10.1093/jn/137.6.1586S PubMed Abstract | CrossRef Full Text | Google Scholar Lahiry, S., Choudhury, S., Mukherjee, A., Bhunya, P. K., and Bala, M. Polymyxin B-induced diffuse cutaneous hyperpigmentation. J. Clin. Diagn. Res. 2017, 11(2): FD01. doi:10.7860/JCDR/2017/24278.9213 PubMed Abstract | CrossRef Full Text | Google Scholar Li, J., Milne, R. W., Nation, R. L., Turnidge, J. D., Smeaton, T. C., and Coulthard, K. Pharmacokinetics of colistin methanesulphonate and colistin in rats following an intravenous dose of colistin methanesulphonate. J. Antimicrob. Chemother. 2004;53(5):837–840. doi:10.1093/jac/dkh167 PubMed Abstract | CrossRef Full Text | Google Scholar Li, J., Nation, R. L., Turnidge, J. D., Milne, R. W., Coulthard, K., Rayner, C. R., et al. Colistin: the re-emerging antibiotic for multidrug-resistant Gram-negative bacterial infections. Lancet Infect. Dis. 2006;6(9):589–601. doi:10.1016/S1473-3099(06)70580-1 PubMed Abstract | CrossRef Full Text | Google Scholar Li, Y. M., Milikowski, C., Selvaggi, G., Abbo, L. M., Skiada, D., and Galimberti, F. Polymyxin B‐induced skin hyperpigmentation. Transpl. Infect. Dis. 2020;22(5):e13312. doi:10.1111/tid.13312 PubMed Abstract | CrossRef Full Text | Google Scholar Lu, C., and Hou, N. (2020) Skin hyperpigmentation in Coronavirus Disease 2019 patients: is polymyxin B the culprit? Front. Pharmacol. 11:01304. doi:10.3389/fphar.2020.01304 PubMed Abstract | CrossRef Full Text | Google Scholar Martindale (2014):The complete drug reference[M].38 th ed,London, United Kingdom,Pharmaceutical Press:2086 Mattos, K. P. H., Cintra, M. L., Gouvêa, I. R., Ferreira, L. Á., Velho, P. E. N. F., and Moriel, P. Skin hyperpigmentation following intravenous polymyxin B treatment associated with melanocyte activation and inflammatory process. J. Clin. Pharm. Ther. 2017;42(5):573–578. doi:10.1111/jcpt.12543 PubMed Abstract | CrossRef Full Text | Google Scholar Mattos, K. P. H., Lloret, G. R., Cintra, M. L., Gouvêa, I. R., Betoni, T. R., Mazzola, P. G., et al. Acquired skin hyperpigmentation following intravenous polymyxin B treatment: a cohort study. Pigment Cel Melanoma Res. 2016;29(3):388–390. doi:10.1111/pcmr.12468 CrossRef Full Text | Google Scholar Matzneller, P., Strommer, S., Drucker, C., Petroczi, K., Schörgenhofer, C., Lackner, E., et al. Colistin reduces LPS-triggered inflammation in a human sepsis ModelIn vivo:A randomized controlled trial. Clin. Pharmacol. Ther. 2017;101(6):773–781. doi:10.1002/cpt.582 PubMed Abstract | CrossRef Full Text | Google Scholar Miori, L., Vignini, M., and Rabbiosi, G. Flagellate dermatitis after bleomycin. The Am. J. Dermatopathology. 1990;12(6):598–602. doi:10.1097/00000372-199012000-00011 CrossRef Full Text | Google Scholar Morrison, D. C., Roser, J. F., Henson, P. M., and Cochrane, C. G. Activation of rat mast cells by low molecular weight stimuli. J. Immunol. 1974;112(2):573–582. doi:10.1115/PVP2004-2756 PubMed Abstract Google Scholar Morrison, D. C., Roser, J. F., Curry, B. J., Henson, P. M., and Ulevitch, R. J. Two distinct mechanisms for the initiation of mast cell degranulation. Inflammation. 1978;3(1):7–25. doi:10.1007/BF00917318 PubMed Abstract | CrossRef Full Text | Google Scholar Rzepka, Z., Buszman, E., Beberok, A., and Wrześniok, D. From tyrosine to melanin: signaling pathways and factors regulating melanogenesis. Postepy Hig Med. Dosw 2016;70(0):695–708. Published 2016 Jun 30. doi:10.5604/17322693.1208033 PubMed Abstract | CrossRef Full Text | Google Scholar Sasaki, M., Horikoshi, T., Uchiwa, H., and Miyachi, Y. Up-regulation of tyrosinase gene by nitric oxide in human melanocytes. Pigment Cel Res. 2000;13(4):248–252. doi:10.1034/j.1600-0749.2000.130406.x CrossRef Full Text | Google Scholar Shih, L. K., and Gaik, C. L. Polymyxin B induced generalized skin hyperpigmentation in infants. Jps. 2014, 6: e215. doi:10.17334/jps.10375 CrossRef Full Text | Google Scholar Ubagai, T., Sato, Y., Kamoshida, G., Unno, Y., and Ono, Y. Immunomodulatory gene expression analysis in LPS-stimulated human polymorphonuclear leukocytes treated with antibiotics commonly used for multidrug-resistant strains. Mol. Immunol. 2021;129:39–44. doi:10.1016/j.molimm.2020.11.012 PubMed Abstract | CrossRef Full Text | Google Scholar Wen, X., Luo, C., and Lyu, W. Polymyxin B-induced skin hyperpigmentation. Case Rep. Med. 2020; 2020:1. Published 2020 Sep 18. doi:10.1155/2020/6461329 CrossRef Full Text | Google Scholar Yoshida, M., Takahashi, Y., and Inoue, S. Histamine induces melanogenesis and morphologic changes by protein kinase A activation via H2 receptors in human normal melanocytes. J. Invest. Dermatol. 2000;114(2):334–342. doi:10.1046/j.1523-1747.2000.00874.x PubMed Abstract | CrossRef Full Text | Google Scholar Zavascki, A. P., Manfro, R. C., Maciel, R. A., and Falci, D. R. Head and neck hyperpigmentation probably associated with polymyxin B therapy. Ann. Pharmacother. 2015, 49(10): 1171. doi:10.1177/1060028015595643 PubMed Abstract | CrossRef Full Text | Google Scholar Zavascki, A. P., Schuster, L. F., and Duquia, R. P. Histopathological findings of pigmented lesion and recovery of natural skin colour in a patient with polymyxin B-associated diffuse hyperpigmentation. Int. J. Antimicrob. Agents. 2016;48(5):579–580. doi:10.1016/j.ijantimicag.2016.08.010 PubMed Abstract | CrossRef Full Text | Google Scholar Zhan, Y., Ma, N., Liu, R., Wang, N., Zhang, T., and He, L. Polymyxin B and polymyxin E induce anaphylactoid response through mediation of Mas-related G protein-coupled receptor X2. Chemico-Biological Interactions. 2019;308:304–311. doi:10.1016/j.cbi.2019.05.014 PubMed Abstract | CrossRef Full Text | Google Scholar Zheng, G., Cao, L., Che, Z., Mao, E., Chen, E., and He, J. Polymyxin B-induced skin hyperpigmentation: a rare case report and literature review. BMC Pharmacol. Toxicol. 2018; 19(1):41. Published 2018 Jul 4. doi:10.1186/s40360-018-0226-1 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: polymyxin B, polymyxin E, skin hyerpigmentation, histamine, melanocytes, oxidative stress, phenylalanine Citation: Zou D, Yu H and Li F (2021) The Difference Between Polymyxin B and Polymyxin E in Causing Skin Hyerpigmentation. Front. Pharmacol. 12:647564. doi: 10.3389/fphar.2021.647564 Received: 30 December 2020; Accepted: 24 March 2021; Published: 16 April 2021. Edited by: Reviewed by: Copyright © 2021 Zou, Yu and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Feifei Li, [email protected]
Carolina Osuna-Mascaró, Rafael Rubio de Casas, Jacob B. Landis, Francisco Perfectti
Frontiers in Ecology and Evolution, Volume 9; https://doi.org/10.3389/fevo.2021.620601

Abstract:
Erysimum (Brassicaceae) is a genus of more than 200 species (Al-Shehbaz, 2012). It is widely distributed in the Northern Hemisphere and has been the focus of active research in ecology, evolution, and genetics (Gómez and Perfectti, 2010; Gómez, 2012; Valverde et al., 2016). Despite long-standing interest in Erysimum, its taxonomy has yet to be properly established, partly due to a complex and reticulated evolutionary history that renders phylogenetic reconstructions highly challenging (Ancev, 2006; Marhold and Lihová, 2006; Abdelaziz et al., 2014; Gomez et al., 2014; Moazzeni et al., 2014; Züst et al., 2020). The Baetic Mountains (South-Eastern Iberia) are among the most critical glacial refugia in Europe. The waxing and waning of plant populations following climatic fluctuations have likely complicated the distribution and genetic variation of extant diversity in this region. Isolation and posterior secondary contact between taxa may have favored hybridization and introgression (Médail and Diadema, 2009). The Erysimum species that inhabit these mountains have been a particularly fruitful system for plant evolutionary ecology [e.g., Gómez et al., 2006, 2008; Gómez and Perfectti, 2010; Gómez, 2012; Valverde et al., 2016]. However, the relationships among these species remain unresolved, hampering comparative and evolutionary studies. Genome duplications, incomplete lineage sorting, and hybridization have compromised the phylogenetic reconstructions within Erysimum (Marhold and Lihová, 2006; Osuna-Mascaró, 2020). Additionally, clarifying this group's complex evolution requires extensive genomic resources, which are currently being produced but are mostly lacking. The fast development of high-throughput sequencing technologies has led to a rapid increase in genomic and transcriptomic for many plant species (Dong et al., 2004; Duvick et al., 2007; Sundell et al., 2015; Boyles et al., 2019). However, obtaining complete genome sequencing remains a challenge with large, repetitive-DNA enriched genomes. Transcriptome sequencing is comparatively more accessible, providing a relatively cheap and fast method to obtain large amounts of functional genomic data (Timme et al., 2012; Yang and Smith, 2013; Wickett et al., 2014; Léveillé-Bourret et al., 2017). Accordingly, global initiatives such as the 1,000 plants (1KP) project have generated transcriptomic resources for over 1,000 plant species (Matasci et al., 2014; Leebens-Mack et al., 2019). In addition, the use of RNA-Seq could be useful in obtaining complete chloroplast genomes in a reliable and accessible way, making possible the use of complete molecules in phylogenomic analyses (Smith, 2013; Osuna-Mascaró et al., 2018; Morales-Briones et al., 2021). Here, we report the annotation of 18 floral transcriptomes assembled de novo from total RNA-Seq libraries and nine chloroplast genomes from seven Erysimum species inhabiting the Baetic Mountains. The chloroplast genomes were assembled from total RNA-Seq data following a previously-validated reference assemble approach (Osuna-Mascaró et al., 2018). The data presented here represent reliable genomic resources for transcriptomic, proteomic, and phylotranscriptomic studies. These data contribute to the ecological and genetic resources available for Brassicaceae in general and the genus Erysimum in particular, being the only genomic resources for these species coming from flower buds. We sampled flower buds at the same development stage (completely developed non-open buds) from three different populations of Erysimum mediohispanicum, E. nevadense, E. popovii, and E. baeticum, four populations of E. bastetanum, and one population of E. lagascae, and E. fitzii (see Supplementary Table 1 for details). We stored the samples in liquid nitrogen and maintained them in an ultra-freezer (−80°C) until RNA extraction. Then, we extracted RNA from the buds under highly sterile conditions. The buds were snap-frozen in liquid nitrogen and ground with mortar and pestle. We used the Qiagen RNeasy Plant Mini Kit, following the manufacturer's protocol, to extract total RNA and their quality and quantity were checked using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, United States) and with an Agilent 2100 Bioanalyzer system (Agilent Technologies Inc). Library preparation and RNA sequencing were conducted by Macrogen Inc. (Seoul, Korea). We used rRNA-depletion (Ribo-Zero) for mRNA enrichment and to avoid sequencing rRNAs. Library preparation was performed using the TruSeq Stranded Total RNA LT Sample Preparation Kit (Plant). The sequencing of the 18 libraries was carried out using the Hiseq 3000-4000 sequencing protocol and TruSeq 3000-4000 SBS Kit v3 reagent, following a paired-end 150 bp strategy on the Illumina HiSeq 4000 platform. A summary of sequencing statistics appears in Supplementary Table 2. We analyzed the fastq files for each library using FastQC v 0.11.5 (Andrews, 2010). Then, we trimmed the adapters using cutadapt v 1.1540 (Martin, 2011), specifying the “-b” option for trimming the adapters in 5′ and 3′ and the “-n” option to search repeatedly for the adapter sequences (28 iterations). This option ensures that the correct adapters were detected by searching in loops until any adapter match is found or until the specified number of rounds is reached. Following, we trimmed the reads by quality using Sickle v 1.3341 (Joshi and Fass, 2011), using the “pe” option for paired-end reads and the “-t” to use Illumina quality values (see https://github.com/najoshi/sickle). This trimming software uses sliding-window analyses and quality and length thresholds to cut and discard the reads that do not fit the selected threshold values. We specified the “pe” option for paired-end reads and the “-t” to use Illumina quality values, setting a threshold quality value of Q20 (see https://github.com/najoshi/sickle). After trimming, we used FastQC (Andrews, 2010) again to verify the trimming efficiency. The summary of the number of reads after the quality trimming is represented in Supplementary Table 3. To assemble contigs from the resulting high-quality cleaned reads, we followed a de novo approach using Trinity v 2.8.4 (Grabherr et al., 2011), due to the absence of an available published assembled genome for Erysimum at the time of the analyses. Each library was normalized in silico before assembly to validate and reduce the number of reads using the “insilico_read_normalization.pl” function in Trinity (Haas et al., 2013). Then we used the parameter “min_kmer_cov 2” to eliminate single-occurrence k-mers that are heavily enriched in sequencing errors, following Haas et al. (2013). Thus, only k-mers that occur more than once were considered for contigs. Candidate open reading frames (ORF) within transcript sequences were predicted and translated using TransDecoder v 5.2.0 (Haas et al., 2013). We performed functional annotation of Trinity transcripts with ORFs using Trinotate v 3.0.1 (Haas, 2015), an annotation suite designed for automatic functional annotation of de novo assembled transcriptomes. Sequences were searched against UniProt (UniProt Consortium, 2014), using SwissProt databases (Bairoch and Apweiler, 2000) (with BLASTX and BLASTP searching and an e-value cutoff of 10). We then used the Pfam database (Bateman et al., 2004) to annotate protein domains for each predicted protein sequence. We also annotated the transcripts using the databases eggnog (Jensen et al., 2007), GO (Gene Ontology Consortium, 2004), and Kegg (Kanehisa and Goto, 2000). We obtained between 104K and 382K different Trinity transcripts after assembling, producing between 66K and 235K Trinity isogenes. The total assembled bases ranged from 92 Mbp (in Em21 population of E. mediohispanicum) to 319 Mbp (in En10 population of E. nevadense). The summary statistics of the assembled transcriptomes appear in Supplementary Table 4. Among the annotated unigenes, the highest proportion was annotated using BLASTX search against the SwissProt reference database, and the number of genes annotated ranges between 71,606 (E. nevadense, En12) and 197,069 (E. baeticum, Ebb10); mean value 146,314.35. The unigenes from the assembled sequences using different databases are shown in Supplementary Table 5. Lastly, we used BUSCO v 2.0 (Seppey et al., 2019) to validate the quality of all the assemblies, using the plant database brassicales_odb10.2019-11-20. Overall, a high level of single-copy orthologous retrieval was noted for the 18 assemblies, as shown in Figure 1. Specifically, we found a completeness ratio ranging from 72.29 to 85.62% for E. popovii, from 73.06 to 83.26% for E. nevadense, from 71.60 to 84.65% to E. mediohispanicum, from 63. 12 to 86.74% for E. bastetanum, from 79.31 to 72.5% for E. baeticum, a completeness ratio of 86.46% for E. lagascae, and 75.07% for E. fitzii. The least complete case was for E. bastetanum, Ebt13, exhibiting a 63.12% ratio, with 8% missing orthologs and a 28.88% partial completeness. Figure 1. BUSCO assessment results for the 18 assembled transcriptomes. The transcriptome annotations, the set of assembled unigenes and their annotations, and the predicted amino acid sequences can be found in Data citation 2, Data citation 3, and Data citation 4, respectively. We assembled the chloroplast genome from nine Erysimum RNA-Seq libraries. Specifically, we assembled E. bastetanum (Ebt01, Ebt10, Ebt12, Ebt22), E. fitzii (Ef), E. lagascae (Ela07), and E. popovii (Ep16, Ep20, Ep27). Our team (Osuna-Mascaró et al., 2018) previously assembled the remaining chloroplast genomes (the ones corresponding to E. baeticum, E. mediohispanicum, and E. nevadense RNA-Seq libraries). Here, we used a reference assembly approach, using Geneious R.11 (Kearse et al., 2012) with the A. thaliana chloroplast genome as reference (NC_000932.1 (Sato et al., 1999)). This method has been previously validated with the chloroplast genomes of E. mediohispanicum, E. nevadense, and E. baeticum (Osuna-Mascaró et al., 2018). We annotated the chloroplast genomes using cpGAVAS (Liu et al., 2012). The annotations were manually curated using Geneious R.11 (Kearse et al., 2012). All transfer RNA sequences (tRNA) encoded in the chloroplast genomes were verified using tRNAscan-SE 2.0 (Schattner et al., 2005) and ARAGORN v1.2.38 (Laslett and Canback, 2004) with the default search settings. The annotation summary is presented in Supplementary Table 6. We obtained almost complete chloroplast genomes, with some missing genes detected (Supplementary Table 7). Most of the missing genes were related to photosynthesis such as the group of subunits of NADH-dehydrogenase (e.g., ndhA), genes with conserved open reading frame (e.g., ycf1, and ycf5), or genes related with subunit od Acetyl-CoA carboxylase (aacD), or the elongation factor (tuf). The assemblies of chloroplast genomes and their annotations are shown in Data citation 5. Chloroplast genome resources including trn's, rrn's, mrn's, genes, tRNA validation results, and annotation report files are shown in Data citation 6. We uploaded the nine chloroplast genome sequences to GenBank with accession numbers showed in Data citation 7. We aligned the chloroplast sequences using MAFFT v.7 with default parameters (Katoh and Standley, 2013). Then, we reconstructed a time-calibrated phylogeny using Beast 2.0 (Bouckaert et al., 2014), with Arabidopsis thaliana chloroplast genome sequence (NC_000932.1 (Sato et al., 1999)) as an outgroup. We made three different partitions: one for coding regions, one for non-coding regions, and the last for the third positions of the coding regions, for which substitutions are synonymous. We calibrated using an average mutation rate reported for synonymous sites of chloroplast genes of seed plants (1.2–1.7 × 109 substitutions/site/year) (Graur and Li, 2000) applying it for the partition of the third position region. In addition, we included a timed-calibration obtained from the literature. In Moazzeni et al. (2014) the divergence of Western European Erysimum species was estimated in the middle Pleistocene (2.43 – 0.74 Mya, using a fast substitution rate; or 8.48 – 2.15 Mya, using a slow substitution rate). Here, we used the average of these dating intervals (2.43 and 2.15 Mya) as a calibration point (2.29 Mya). The Bayesian search for tree topologies and node ages was conducted during 20,000,000 generations in BEAST using a strict clock model and a Yule process as prior. MCMC was sampled every 1,000 generations, discarding a burn-in of 10%. We checked the MCMC trace files generated using Tracer v1.6.1 (Rambaut et al., 2014). The time-calibrated phylogeny is shown in Figure 2. Figure 2. A time-calibrated phylogeny for the chloroplast DNA of the different populations of the Erysimum species analyzed here. Note the reticulated position of some populations, probably due to hybridization events. The raw sequence read data for all the transcriptomes were deposited in the NCBI Sequence Read Archive (Data citation 1). Furthermore, for the free download of the generated data, we have created a project on figshare containing: the assembled transcriptomes (Data citation 2), the transcriptome annotations (Data citation 3), the set of assembled unigenes, their annotations, and the predicted amino acid sequences (Data citation 4), the chloroplast genomes assemblies and their annotations (Data citation 5), and chloroplast genomic resources including trn's, rrn's, mrn's, genes, trna validation results, and annotation report files (Data citation 6). The chloroplast genome sequences were deposited in GenBank. The accession numbers can be found in Data citation 7. Data citation 1: NCBI Sequence Read Archive, BioProject PRJNA607615 under the following accession numbers: E. popovii: Ep27 (SRX7756239), Ep20 (SRX7756238), Ep16 (SRX7756237); E. lagascae: Ela07 (SRX7756236); E. fitzii: Ef01 (SRX7756235); E. bastetanum: Ebt22 (SRX7756234), Ebt13 (SRX7756233), Ebt12 (SRX7756232), Ebt01 (SRX7756231), and BioProject PRJNA473238 under the following accession numbers: E. baeticum: Ebb12 (SRX4130243), Ebb10 (SRX4130242), Ebb07 (SRX4130235); E. mediohispanicum: Em39 (SRX4130241), Em71 (SRX4130240), Em21 (SRX4130233); E. nevadense: En12 (SRX4130237), En10 (SRX4130236), En05 (SRX4130234). Data citation 2: Osuna-Mascaró, C., de Casas, R. R., Landis, J.B., & Perfectti, F., figshare https://doi.org/10.6084/m9.figshare.11877786.v3 (2020). Data citation 3: Osuna-Mascaró, C., de Casas, R. R., Landis, J.B., & Perfectti, F., figshare https://doi.org/10.6084/m9.figshare.11866389.v3 (2020). Data citation 4: Osuna-Mascaró, C., de Casas, R. R., Landis, J.B., & Perfectti, F., figshare https://doi.org/10.6084/m9.figshare.11873937.v1 (2020). Data citation 5: Osuna-Mascaró, C., de Casas, R. R., Landis, J.B., & Perfectti, F., figshare https://doi.org/10.6084/m9.figshare.11881656.v2 (2020). Data citation 6: Osuna-Mascaró, C., de Casas, R. R., Landis, J.B., & Perfectti, F., figshare https://doi.org/10.6084/m9.figshare.11881419.v2 (2020). Data citation 7: Chloroplast genome sequences deposited in GenBank under the following accession numbers: E. bastetanum: Ebt01 (MT150122), Ebt12 (MT150121), Ebt13 (MT150114), Ebt22 (MT150115); E. fitzii: Ef01 (MT150118); E. lagascae: Ela07 (MT150116); E. popovii: Ep16 (MT150117), Ep20 (MT150119), Ep27 (MT150120). Erysimum is a genus for which phylogenetic relationships have not yet been fully established. Therefore, the primary use of this dataset will likely lie in molecular evolution analyses aimed at disentangling the taxonomy and biogeography of these and related species. Moreover, since the primary data are transcriptomes, they could be useful in plant evo-devo and physiological studies. They can also be incorporated into comparative studies aimed at identifying the differential expression of the genes expressed in the tissues sequenced in this work (i.e., flower buds). Although we expect our dataset to be essentially free of contamination, caution is advised when using this Data Report as we did not filter the reads for the presence of alien (i.e., bacterial or fungi) sequences. The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material. COM, RR, and FP conceived and designed the study. COM analyzed the data, with the help of FP and JL and wrote the first draft. The final version of the M.S. was redacted with the contribution of all the authors. Funding was provided by the Spanish Ministry of Science and Competitiveness (CGL2016- 79950-R; CGL2017-86626-C2-2-P), including FEDER funds. This research was also funded by the Consejería de Economía, Conocimiento, Empresas y Universidad, and European Regional Development Fund (ERDF), ref. SOMM17/ 6109/UGR and A-RNM-505-UGR18. COM was supported by the Ministry of Economy and Competitiveness (BES-2014-069022). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We are grateful to Modesto Berbel Cascales and José M. Gómez for their help in sampling and DNA/RNA extractions. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fevo.2021.620601/full#supplementary-material Abdelaziz, M., Munoz-Pajares, A. J., Lorite, J., Herrador, M. B., Perfectti, F., and Gómez, J. M. (2014). Phylogenetic relationships of “Erysimum” (Brassicaceae) from the Baetic Mountains (SE Iberian Peninsula). Anales del Jardín Botánico de Madrid 71:5. doi: 10.3989/ajbm.2377 CrossRef Full Text | Google Scholar Al-Shehbaz, I. A. (2012). A generic and tribal synopsis of the Brassicaceae (Cruciferae). Taxon 61, 931–954. doi: 10.1002/tax.615002 CrossRef Full Text | Google Scholar Ancev, M. (2006). Polyploidy and hybridization in Bulgarian Brassicaceae: distribution and evolutionary role. Phytol. Balcanica 12, 357–366. Google Scholar Andrews, S. (2010). FastQC: A Quality Control Tool for High Throughput Sequence Data. Available online at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (accessed September 26, 2020). Google Scholar Bairoch, A., and Apweiler, R. (2000). The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000. Nucl. Acids Res. 28, 45–48. doi: 10.1093/nar/28.1.45 PubMed Abstract | CrossRef Full Text | Google Scholar Bateman, A., Coin, L., Durbin, R., Finn, R. D., Hollich, V., Griffiths-Jones, S., et al. (2004). The Pfam protein families database. Nucl. Acids Res. 32(suppl_1), D138–D141. doi: 10.1093/nar/gkh121 CrossRef Full Text | Google Scholar Bouckaert, R., Heled, J., Kühnert, D., Vaughan, T., Wu, C. H., Xie, D., et al. (2014). BEAST 2: a software platform for Bayesian evolutionary analysis. PLoS Comput. Biol. 10:e1003537. doi: 10.1371/journal.pcbi.1003537 PubMed Abstract | CrossRef Full Text | Google Scholar Boyles, R. E., Brenton, Z. W., and Kresovich, S. (2019). Genetic and genomic resources of sorghum to connect genotype with phenotype in contrasting environments. Plant J. 97, 19–39. doi: 10.1111/tpj.14113 PubMed Abstract | CrossRef Full Text | Google Scholar Dong, Q., Schlueter, S. D., and Brendel, V. (2004). PlantGDB, plant genome database and analysis tools. Nucl. Acids Res. 32(suppl_1), D354–D359. doi: 10.1093/nar/gkh046 PubMed Abstract | CrossRef Full Text | Google Scholar Duvick, J., Fu, A., Muppirala, U., Sabharwal, M., Wilkerson, M. D., Lawrence, C. J., et al. (2007). PlantGDB: a resource for comparative plant genomics. Nucl. Acids Res. 36(suppl_1), D959–D965. doi: 10.1093/nar/gkm1041 PubMed Abstract | CrossRef Full Text | Google Scholar Gene Ontology Consortium (2004). The Gene Ontology (GO) database and informatics resource. Nucl. Acids Res. 32(suppl_1), D258–D261. doi: 10.1093/nar/gkh036 CrossRef Full Text | Google Scholar Gómez, J. M. (2012). Herbivory reduces the strength of pollinator-mediated selection in the Mediterranean herb Erysimum mediohispanicum: consequences for plant specialization. Am. Naturalist 162, 242–256. doi: 10.1086/376574 PubMed Abstract | CrossRef Full Text | Google Scholar Gómez, J. M., Bosch, J., Perfectti, F., Fernández, J. D., Abdelaziz, M., and Camacho, J. P. M. (2008). Association between floral traits and rewards in Erysimum mediohispanicum (Brassicaceae). Ann. Botany 101, 1413–1420. doi: 10.1093/aob/mcn053 PubMed Abstract | CrossRef Full Text | Google Scholar Gomez, J. M., Munoz-Pajares, A. J., Abdelaziz, M., Lorite, J., and Perfectti, F. (2014). Evolution of pollination niches and floral divergence in the generalist plant Erysimum mediohispanicum. Ann. Botany 113, 237–249. doi: 10.1093/aob/mct186 PubMed Abstract | CrossRef Full Text | Google Scholar Gómez, J. M., and Perfectti, F. (2010). Evolution of complex traits: the case of Erysimum corolla shape. Int. J. Plant Sci. 171, 987–998. doi: 10.1086/656475 CrossRef Full Text | Google Scholar Gómez, J. M., Perfectti, F., and Camacho, J. P. M. (2006). Natural selection on Erysimum mediohispanicum flower shape: insights into the evolution of zygomorphy. Am. Naturalist 168, 531–545. doi: 10.1086/507048 PubMed Abstract | CrossRef Full Text | Google Scholar Grabherr, M. G., Haas, B. J., Yassour, M., Levin, J. Z., Thompson, D. A., Amit, I., et al. (2011). Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29:644. doi: 10.1038/nbt.1883 PubMed Abstract | CrossRef Full Text | Google Scholar Graur, D., and Li, W. H. (2000). Fundamentals of molecular evolution. Dynamics 20, 160–229. doi: 10.1017/s0016672300030032 CrossRef Full Text | Google Scholar Haas, B. J. (2015). Trinotate: Transcriptome Functional Annotation and Analysis. Available online at: http://trinotate.github.io (accessed September 26, 2020). Google Scholar Haas, B. J., Papanicolaou, A., Yassour, M., Grabherr, M., Blood, P. D., Bowden, J., et al. (2013). De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat. Protocols 8, 1494–1512. doi: 10.1038/nprot.2013.084 CrossRef Full Text | Google Scholar Jensen, L. J., Julien, P., Kuhn, M., von Mering, C., Muller, J., Doerks, T., et al. (2007). eggNOG: automated construction and annotation of orthologous groups of genes. Nucl. Acids Res. 36(suppl_1), D250–D254. doi: 10.1093/nar/gkm796 PubMed Abstract | CrossRef Full Text | Google Scholar Joshi, N. A., and Fass, J. N. (2011). Sickle: A Sliding-Window, Adaptive, Quality-Based Trimming Tool for FastQ Files. Available online at: https://github.com/najoshi/sickle (accessed September 26, 2020). Google Scholar Kanehisa, M., and Goto, S. (2000). KEGG: kyoto encyclopedia of genes and genomes. Nucl. Acids Res. 28, 27–30. doi: 10.1093/nar/28.1.27 PubMed Abstract | CrossRef Full Text | Google Scholar Katoh, K., and Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780. doi: 10.1093/molbev/mst010 PubMed Abstract | CrossRef Full Text | Google Scholar Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., et al. (2012). Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28, 1647–1649. doi: 10.1093/bioinformatics/bts199 PubMed Abstract | CrossRef Full Text | Google Scholar Laslett, D., and Canback, B. (2004). ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucl. Acids Res. 32, 11–16. doi: 10.1093/nar/gkh152 PubMed Abstract | CrossRef Full Text | Google Scholar Leebens-Mack, J. H., Barker, M. S., Carpenter, E. J., Deyholos, M. K., Gitzendanner, M. A., Graham, S. W., et al. (2019). One thousand plant transcriptomes and the phylogenomics of green plants. Nature 574, 679–685. doi: 10.1038/s41586-019-1693-2 PubMed Abstract | CrossRef Full Text | Google Scholar Léveillé-Bourret, É., Starr, J. R., Ford, B. A., Moriarty Lemmon, E., and Lemmon, A. R. (2017). Resolving rapid radiations within angiosperm families using anchored phylogenomics. Systematic Biol. 67, 94–112. doi: 10.1093/sysbio/syx050 PubMed Abstract | CrossRef Full Text | Google Scholar Liu, C., Shi, L., Zhu, Y., Chen, H., Zhang, J., Lin, X., et al. (2012). CpGAVAS, an integrated web server for the annotation, visualization, analysis, and GenBank submission of completely sequenced chloroplast genome sequences. BMC Genom. 13, 1–7. doi: 10.1186/1471-2164-13-715 PubMed Abstract | CrossRef Full Text | Google Scholar Marhold, K., and Lihová, J. (2006). Polyploidy, hybridization and reticulate evolution: lessons from the Brassicaceae. Plant Systematics Evol. 259, 143–174. doi: 10.1007/s00606-006-0417-x CrossRef Full Text | Google Scholar Martin, M. (2011). Cutadapt removes adapter sequences from highthroughput sequencing reads. EMBnet J. 17, 10–12. doi: 10.14806/ej.17.1.200 CrossRef Full Text | Google Scholar Matasci, N., Hung, L. H., Yan, Z., Carpenter, E. J., Wickett, N. J., Mirarab, S., et al. (2014). Data access for the 1,000 Plants (1KP) project. Gigascience 3, 2047–217X. doi: 10.1186/2047-217X-3-17 PubMed Abstract | CrossRef Full Text | Google Scholar Médail, F., and Diadema, K. (2009). Glacial refugia influence plant diversity patterns in the Mediterranean Basin. J. Biogeogr. 36, 1333–1345. doi: 10.1111/j.1365-2699.2008.02051.x CrossRef Full Text | Google Scholar Moazzeni, H., Zarre, S., Pfeil, B. E., Bertrand, Y. J., German, D. A., Al-Shehbaz, I. A., et al. (2014). Phylogenetic perspectives on diversification and character evolution in the species-rich genus Erysimum (Erysimeae; Brassicaceae) based on a densely sampled ITS approach. Botanical J. Linnean Soc. 175, 497–522. doi: 10.1111/boj.12184 CrossRef Full Text | Google Scholar Morales-Briones, D. F., Kadereit, G., Tefarikis, D. T., Moore, M. J., Smith, S. A., Brockington, S. F., et al. (2021). Disentangling sources of gene tree discordance in phylogenomic data sets: testing ancient hybridizations in amaranthaceae sl. Systematic Biol. 70, 219–235. doi: 10.1093/sysbio/syaa066 CrossRef Full Text | Google Scholar Osuna-Mascaró, C. (2020). Hybridization as an Evolutionary Driver for Speciation: A Case in the Southern European Erysimum species. (Ph.D. Thesis), Universidad de Granada, Granada, Spain. Google Scholar Osuna-Mascaró, C., de Casas, R. R., and Perfectti, F. (2018). Comparative assessment shows the reliability of chloroplast genome assembly using RNA-seq. Sci. Rep. 8:17404. doi: 10.1038/s41598-018-35654-3 PubMed Abstract | CrossRef Full Text | Google Scholar Rambaut, A., Suchard, M. A., Xie, D., and Drummond, A. J. (2014). Tracer v1. 6. Avaialble online at: http://beast.community/tracer.html (accessed September 26, 2020). Google Scholar Sato, S., Nakamura, Y., Kaneko, T., Asamizu, E., and Tabata, S. (1999). Complete structure of the chloroplast genome of Arabidopsis thaliana. DNA Res. 6, 283–290. doi: 10.1093/dnares/6.5.283 PubMed Abstract | CrossRef Full Text | Google Scholar Schattner, P., Brooks, A. N., and Lowe, T. M. (2005). The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucl. Acids Res. 33(suppl_2), W686–W689. doi: 10.1093/nar/gki366 PubMed Abstract | CrossRef Full Text | Google Scholar Seppey, M., Manni, M., and Zdobnov, E. M. (2019). “BUSCO: assessing genome assembly and annotation completeness,” in Gene Prediction (New York, NY: Humana), 227–245. doi: 10.1007/978-1-4939-9173-0_14 PubMed Abstract | CrossRef Full Text | Google Scholar Smith, D. R. (2013). RNA-Seq data: a goldmine for organelle research. Briefings Funct. Genom. 12, 454–456. doi: 10.1093/bfgp/els066 PubMed Abstract | CrossRef Full Text | Google Scholar Sundell, D., Mannapperuma, C., Netotea, S., Delhomme, N., Lin, Y. C., Sjödin, A., et al. (2015). The Plant genome integrative explorer resource: PlantGen IE.org. New Phytol. 208, 1149–1156. doi: 10.1111/nph.13557 CrossRef Full Text | Google Scholar Timme, R. E., Bachvaroff, T. R., and Delwiche, C. F. (2012). Broad phylogenomic sampling and the sister lineage of land plants. PLoS ONE 7:e29696. doi: 10.1371/journal.pone.0029696 PubMed Abstract | CrossRef Full Text | Google Scholar UniProt Consortium. (2014). UniProt: a hub for protein information. Nucl. Acids Res. 43, D204–D212. doi: 10.1093/nar/gku989 CrossRef Full Text | Google Scholar Valverde, J., Gómez, J. M., and Perfectti, F. (2016). The temporal dimension in individual-based plant pollination networks. Oikos 125, 468–479. doi: 10.1111/oik.02661 CrossRef Full Text | Google Scholar Wickett, N. J., Mirarab, S., Nguyen, N., Warnow, T., Carpenter, E., Matasci, N., et al. (2014). Phylotranscriptomic analysis of the origin and early diversification of land plants. Proc. Natl. Acad. Sci. 111, E4859–E4868. doi: 10.1073/pnas.1323926111 PubMed Abstract | CrossRef Full Text | Google Scholar Yang, Y., and Smith, S. A. (2013). Optimizing de novo assembly of short-read RNA-seq data for phylogenomics. BMC Genom. 14:328. doi: 10.1186/1471-2164-14-328 PubMed Abstract | CrossRef Full Text | Google Scholar Züst, T., Strickler, S. R., Powell, A. F., Mabry, M. E., An, H., Mirzaei, M., et al. (2020). Independent evolution of ancestral and novel defenses in a genus of toxic plants (Erysimum, Brassicaceae). Elife 9:e51712. doi: 10.7554/eLife.51712.sa2 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: transcriptomes, de novo assembly, phylotranscriptomic, Brassicaceae, chloroplast, time calibrated phylogeny Citation: Osuna-Mascaró C, Rubio de Casas R, Landis JB and Perfectti F (2021) Genomic Resources for Erysimum spp. (Brassicaceae): Transcriptome and Chloroplast Genomes. Front. Ecol. Evol. 9:620601. doi: 10.3389/fevo.2021.620601 Received: 23 October 2020; Accepted: 18 March 2021; Published: 13 April 2021. Edited by: Reviewed by: Copyright © 2021 Osuna-Mascaró, Rubio de Casas, Landis and Perfectti. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Francisco Perfectti, [email protected]; Carolina Osuna-Mascaró, [email protected]
Erika M. Timpe, , Cathy Binger, , Nancy Harrington, Jamie B. Schwartz
Augmentative and Alternative Communication, Volume 37, pp 113-128; https://doi.org/10.1080/07434618.2021.1921025

Abstract:
Three parents of preschool-aged children with Down syndrome using mobile augmentative and alternative communication (AAC) technologies to communicate participated indirect, systematic communication-partner instruction. Intervention featured an adaptation of the ImPAACT Program (Improving Partner Applications of Augmentative Communication Techniques; Kent-Walsh, Binger, & Malani, 2010) that included six face-to-face and three telepractice sessions. Parents learned to use the evidence-based Read–Ask–Answer (RAA) instructional strategy (Kent-Walsh, Binger, & Hasham, 2010 Kent-Walsh, J. , Binger, C. , & Hasham, Z. (2010). Effects of parent instruction on the symbolic communication of children using augmentative and alternative communication during storybook reading. American Journal of Speech-Language Pathology, 19(2), 97–107. doi:10.1044/1058-0360(2010/09-0014) [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] ) during shared storybook reading with their children. A single-case, multiple-probe across participants design was used to assess parents’ accurate implementation of the instructional strategy and children’s multimodal communicative turns. All three parents increased their use of the RAA strategy and maintained strategy use over time, and all three children increased their frequency of communicative turns taken and maintained higher turn-taking rates. Results support the use of the ImPAACT Program with parents of children with complex communication needs, including the integration of hybrid learning as part of the instructional approach.
Sulaiman Ali Al Yousef
Acta fytotechnica et zootechnica, Volume 24; https://doi.org/10.15414/afz.2021.24.01.1-8

Abstract:
Article Details: Received: 2020-07-09 | Accepted: 2020-10-14 | Available online: 2021-03-31 https://doi.org/10.15414/afz.2021.24.01.1-8 Extended-spectrum β-lactamases (ESBL) are enzymes produced by Gram-negative microorganisms, which may be resistant to commonly used antibiotics. The purpose of this research was to estimate the bactericidal effects of cinnamon oil on ESBLproducing bacteria. In this study, 227 water samples were collected from wells in Hafr Al-Batin, Saudi Arabia. The samples were cultured on a cystine lactose electrolyte-deficient (CLED) medium. A MicroScan system was used to identify bacteria and also for antimicrobial susceptibility test. Activity of crud cinnamon oil and its fractions were detected by determining the minimum inhibitory concentration (MIC) against the ESBL-producing bacteria. Morphological changes of the treated bacteria were observed and oil compounds was investigated. The culture was positive on the CLED medium in 170 out of 227 water samples. In 170 CLED-positive isolates, E. coli was the most common organism, followed by K. pneumoniae. The results showed that 100% of K. pneumoniae isolates were completely resistant to ampicillin (100%), then by mezlocillin (92.5%), cefazolin, and cefuroxime (77.5%). Also, 86.9% of E. coli isolates were the most resistant to ampicillin, followed by mezlocillin (83%). 82% of K. pneumoniae and 89% of E. coli isolates were confirmed by phenotypic confirmatory disc diffusion test (PCDDT) as ESBL-producers. The cinnamon oil activity was only concentrated in the oxygenated fraction. The MICs of the oxygenated fraction were 80 and 20 µl/mL at 105 CFU of ESBL-producing E. coli and K. pneumoniae, respectively. This study indicated the antibacterial effects of cinnamon essential oil to eliminate some antibiotic-resistant bacteria from water. Keywords: water, Escherichia coli, Klebsiella pneumoniae, antibiotic resistance, essential oil References ADEYEMI, A.O. et al. (2014). Antibiotics susceptibility patterns of some uropathogens to nitrofurantoin and nalidixic acid among pregnant women with urinary tract infections in federal medical centre, Bida, Niger-State, North Central, Nigeria. American Journal of Epidemiology and Infectious Disease, 2, 88–92. http://dx.doi.org/10.12691/ajeid-2-4-1 AL YOUSEF, S. A. et al. (2016). Control. Detection of extended spectrum beta-lactamase producing Escherichia coli on water at Hafr Al Batin, Saudi Arabia. Journal of Pollution Effects & Control, 4(01). http://dx.doi.org/10.4172/2375-4397.1000155 BRADFORD, P.A. (2001). Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clinical Microbiology Reviews, 14(4), 933–951. http://dx.doi.org/10.1128/cmr.14.4.933-951.2001 BRENES, A. and ROURA, E. (2010). Essential oils in poultry nutrition: Main effects and modes of action. Animal Feed Science and Technology, 158, 1–14. http://dx.doi.org/10.1016/j.anifeedsci.2010.03.007 BURT, S. (2004). Essential oils: their antibacterial properties and potential applications in foods – a review. International Journal of Food Microbiology, 94(2), 223–253. http://dx.doi.org/10.1016/j.ijfoodmicro.2004.03.022 CHANG, C.W. et al. (2008). Antibacterial activities of plant essential oils against Legionella pneumophila. Water Research, 42, 278–286. http://dx.doi.org/10.1016/j.watres.2007.07.008 DIAO, W.R. et al. (2013). Chemical composition and antibacterial activity of the essential oil from green huajiao (Zanthoxylum schinifolium) against selected foodborne pathogens. J. Agric. Food Chem., 61(25), 6044–6049. http://dx.doi.org/10.1021/jf4007856 DOI, Y. et al. (2007). Community-acquired extended-spectrum β-lactamase producers, United States. Emerging Infectious Diseases, Centers for Disease Control and Prevention (CDC), 13(7),1121–1123. http://dx.doi.org/10.3201/eid1307.070094 DORMAN, H. and DEANS, S.G. (2000). Antimicrobial agents from plants: antibacterial activity of plant volatile oils. Journal of Applied Microbiology, 88(2), 308–416. http://dx.doi.org/10.1046/j.1365-2672.2000.00969.x GASPARI, R.J. et al. (2005). Antibiotic resistance trends in paediatric uropathogens. International Journal of Antimicrobial Agents, 26(4), 267–271. https://doi.org/10.1016/j.ijantimicag.2005.07.009 JONES R.N. (1986). NCCLS standards: approved methods for dilution antimicrobial susceptibility tests. Antimicrobic Newsletter, 3(1), 1–3. http://dx.doi.org/10.1016/0738-1751(86)90022-5 KOHANSKI, M.A. et al. (2007). A common mechanism of cellular death induced by bactericidal antibiotics. Cell, 130(5), 797–810. http://dx.doi.org/10.1016/j.cell.2007.06.049 LAL, P. et al. (2007). Occurrence of TEM & SHV gene in extended spectrum β-lactamases (ESBLs) producing Klebsiella sp. isolated from a tertiary care hospital. Indian J. Med. Res., 125, 173–178. LIN, L. et al. (2017). Antibacterial poly (ethylene oxide) electrospun nanofibers containing cinnamon essential oil/ beta-cyclodextrin proteoliposomes. Carbohydrate Polymers, 178, 131–140. http://dx.doi.org/10.1016/j.carbpol.2017.09.043 MAcKENZIE, F. et al. (2002). Comparison of screening methods for TEM-and SHV-derived extended-spectrum β-lactamase detection. Clinical Microbiology and Infection, 8(11), 715–724. http://dx.doi.org/10.1046/j.1469-0691.2002.00473.x MOLAND, E.S et al. (2002). Occurrence of newer β-lactamases in Klebsiella pneumoniae isolates from 24 US hospitals. 2002. American Society for Microbiology, 46(12), 3837–3842. http://dx.doi.org/10.1128/aac.46.12.3837-3842.2002 NARAYANASWAMY, A. and MALLIKA M.E. (2011). Prevalence and Susceptibility of extended spectrum beta-lactamases in urinary isolates of Escherichia coli in a Tertiary Care Hospital, Chennai-South India. Internet Journal of Medical Update, 6(1), 39–43. http://dx.doi.org/10.4314/ijmu.v6i1.63975 OJAGH, S.M. et al. (2010). Development and evaluation of a novel biodegradable film made from chitosan and cinnamon essential oil with low affinity toward water. Food Chemistry, 122(1), 161–166. http://dx.doi.org/10.1016/j.foodchem.2010.02.033 PATEL, J. et al. (2001). M100-S25, Performance standards for antimicrobial susceptibility testing. Clinical Microbiology Newsletter, 23, 35–49. http://dx.doi.org/10.1016/s0196-4399(01)88009-0 PESAVENTO, G. et al. (2015). Antibacterial activity of Oregano, Rosmarinus and Thymus essential oils against Staphylococcus aureus and Listeria monocytogenes in beef meatballs. Food Control, 54, 188–199. http://dx.doi.org/10.1016/j.foodcont.2015.01.045 RAEISI, M. et al. (2015). Antimicrobial effect of cinnamon essential oil against Escherichia coli and Staphylococcus aureus. Health Scope, 4(4), e21808. http://dx.doi.org/10.17795/jhealthscope-21808 RAMESH, N. et al. (2019). Extended Spectrum Beta-lactamase (ESBL)-mediated resistance to third generation cephalosporins and conjugative transfer of resistance in Gram-negative bacteria isolated from hospitals in Tamil Nadu, India. Preprints, 2019100103. http://dx.doi.org/10.20944/preprints201910.0103.v1 REDDY, N. and YANG, Y. (2015). Coconut Husk Fibers, Natural Cellulose Fibers from Renewable Resources. In Innovative Biofibers from Renewable Resources, 24, 31–34. RODRIGUES, C. et al. (2004). Detection of-lactamases in nosocomial gram negative clinical isolates. Indian J. Med. Microbiol, 22, 247–250. ROSENTHAL, V.D. et al. (2010). International nosocomial infection control consortium (INICC) report, data summary for 2003–2008, issued June 2009–2010. American Journal of Infection Control, 38(2), 95–104. http://dx.doi.org/10.1016/j.ajic.2009.12.004 SHAABAN, H.A. et al. (2012). Bioactivity of essential oils and their volatile aroma components. Journal of Essential Oil Research, 24(2), 203–212. http://dx.doi.org/10.1080/10412905.2012.659528 SHAKIBAIE, M.R. et al. (2014). Antimicrobial susceptibility pattern and ESBL production among uropathogenic Escherichia coli isolated from UTI children in pediatric unit of a hospital in Kerman, Iran. British Microbiology Research Journal, 4(3), 262– 271. http://dx.doi.org/10.9734/bmrj/2014/6563 STURENBURG, E. and Mack, D.J. (2003). Extended-spectrum β-lactamases: implications for the clinical microbiology laboratory, therapy, and infection control. Journal of Infection, 47(4), 273–395. https://doi.org/10.1016/S0163-4453(03)00096-3 ZORC, J.J. et al. (2005). Diagnosis and management of pediatric urinary tract infections. Clinical Microbiology Reviews, 18(2), 417–422. http://dx.doi.org/10.1128/cmr.18.2.417-422.2005
, Miroslav Vosátka, Christopher Rensing, Helena Freitas
Published: 25 March 2021
Frontiers in Microbiology, Volume 12; https://doi.org/10.3389/fmicb.2021.634891

Abstract:
Editorial on the Research TopicAdvanced Microbial Biotechnologies for Sustainable Agriculture Plant responses to various environmental or climatic stresses are immensely complex and implicate changes at the transcriptome, cellular, and physiochemical levels, consequently hindering crop growth as well as yield quantity and quality. Agriculture has been considered a complex network of plant-microbe interactions. The use of microorganisms of agricultural importance [e.g., plant growth-promoting microorganisms (PGPM)] represent a major ecological strategy for integrated agricultural practices such as nutrient addition, biological control, abiotic stress (e.g., drought, salinity, heavy metals) alleviation to minimize the use of agrichemicals (e.g., fertilizers and pesticides) in agriculture as well as to improve crop performance (Ma et al., 2011, 2016a,b). While the immense diversity of soil microorganisms represents a tremendous opportunity for selecting PGPM, the interactions with plants and the cooperative and competitive interactions among microbes themselves make it extremely challenging to determine which microbes are responsible for synergistic ecosystem functions. The essential aspects for the effectiveness of PGPM application biotechnology are the utilization of a proper inocula formulation and a suitable carrier, as well as delivery methods. Thus, considering the detrimental effects of biotic and abiotic stresses on agricultural production and food security, the development of technologies for exploring the microbial microenvironment and improving our understanding of how microbes communicate/interact with plants to enhance nutrient use-efficiency could pay substantial dividends. Therefore, this Research Topic “Advanced Microbial Biotechnologies for Sustainable Agriculture” was launched to advance our knowledge of underlying mechanisms of plant-microbe interactions and review recent progress on the relationship between the microbiome and crop productivity and health that is at the frontier of agricultural sciences, with the potential to progress and transform agricultural systems in the field. The cogent review and synthesis embodying all the scattered information about drought and salinity stress responses and microbe-induced tolerance in crop plants were provided by Ma G. et al. They also provide insight as a means to develop an understanding of the mechanisms strongly involved in plants respond/adapt to the selected environmental stresses (e.g., drought and salinity) at the morphological, physiological, biochemical, and metabolomic levels, as well as plant-microbe interactions that confer abiotic stress tolerance in plants. So far, in most of the topics, the literature references regarding drought and salt stress are simply listed close to each other, without an integrated approach. In this review, the comparison between plant responses to drought and salinity was thoroughly highlighted and explained. Salinity stress has been the main restraint to agriculture, limiting crop growth and productivity. Many studies have focused on utilizing PGPM to improve plant tolerance to salinity (Ma Y. et al.). It has been demonstrated that inoculation of plants with 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing PGPB can enhance plant growth under salinity stress (Ma et al., 2019b). Orozco-Mosqueda et al. assessed ACC deaminase activity and trehalose accumulation of PGPB Pseudomonas sp. UW4, using constructed mutant strains (acdS, treS, or both) and a trehalose over-expressing strain (OxtreS). The findings indicate the synergistic action of ACC deaminase and trehalose in Pseudomonas sp. UW4 plays an essential role in protecting host plants against salinity stress. To cope with drought stress, PGPM were found to secrete osmolytes to alleviate drought stress, which act synergistically with plants internal osmolytes stimulating plant growth (Paul et al., 2008). The trehalose accumulation is an adaptive mechanism in microbes in response to drought stress to protect cells and proteins from osmotic shock and desiccation (Reina-Bueno et al., 2012). Sharma et al. explore the biological importance and the role of trehalose in the tripartite symbiotic relationship between plants, rhizobia, and AMF, as well as physiological functions and molecular investigations using omics-based approaches. The review provides a critical discussion on the role of microbe-mediated trehalose accumulation in improving stress tolerance. The use of PGPM has been considered an alternative to protect plants from diseases and improve crop productivity, reducing the amount of chemical pesticides needed (Ma Y. et al.). The ability to deconstruct fungal cell walls is a defining characteristic of fungal antagonism and anti-fungal biocontrol (Mesa-Arango et al., 2016). Schönbichler et al. explore the ability of B. subtilis natto to use complex fungal fruiting body and cell wall as a carbon source by secreting chitinases and proteases. The findings show that chitin does not allow bacterial growth nor induce the secretion of chitinolytic enzymes, and protease secretion might be the key mechanism for nutrient scavenging and depredating fungal cell walls by B. subtilis natto. There have been thousands of scientific papers published that contribute to our knowledge on individual features of microbes, their behavior in soil, and after all their interaction with plants both in natural ecosystems and agroecosystems. Numerous papers revealed potentially huge positive effects of soil microorganisms on plant tolerance to stress and resulting ability to produce more biomass or other target yields (Reina-Bueno et al., 2012; Ma et al., 2019b; Ma Y. et al.). Nevertheless, a further step is needed to bring this knowledge closer to practice that would allow to formulate the new products and implement new biotechnologies of crop cultivation. The present Research Topic shows important advances in the understanding of the mechanisms behind plant beneficial microbial activities that help host plants cope with environmental stresses and fills the gap to translate scientific knowledge into sustainable applications. As examples, Rocha et al. and Ferreira et al. show that the knowledge transfer to real agriculture can be feasible since there are numerous beneficial microbes that we know and have isolated and even established effective procedures for their mass production. However, the delivery systems for their large-scale applications in the field represent the most common bottleneck. Seed coating has been considered a precise and cost-effective method to deliver microbial inoculants. A delivery system based on seed coating of various microbes seems to be economically feasible and applicable even in broad-acre agriculture (Ma et al., 2019a). As discussed in Rocha et al., there are still numerous considerable factors that hamper the wider use of microbial seed coating and in general the use of microbes as bioagents in general agriculture practice. The most important ones are the self-life of microbes after coating, their compatibility among themselves, and their ultimate efficacy when they are used in mixtures. There is also a crucial factor in production and application costs. The seed companies are not always keen to change their long-term practices and use biologicals instead of chemical treatment of their seeds (also taking into account that those two treatments are unlikely to be compatible with each other). Moreover, farmers are usually not equipped to do the seed treatment themselves and they have little incentives to ask seed companies for microbially coated seeds (charged premium price) until they see significant evidence of better-coated seed performance. Last but not least there is a general issue of final cost per hectare and especially for low-value crops like cereals where there are generally low-profit margins, it is difficult to accommodate any extra costs. Moreover, the special issue for biocontrol microbes mandates a very strict and costly registration (in particular within the EU). Nevertheless, with proceeding soil degradation, increasing effects of global climatic change and after all growing awareness and demands for agrochemical reduction, healthy and secure food crops, the microbial seed coating technology will certainly be growing in its implementation and wider use. The transition of scientific knowledge to a real commercial application has been happening at a large scale already for some important crops and it holds a strong potential for the near future for other microbes and crops worldwide. A very good example of a fundamental science study conducted by Ferreira et al. that can be transformed into real agriculture is the testing of bacteria-based fertilizer that can alleviate iron-deficiency-induced chlorosis (IDIC). In this work, the ability of two new Fe freeze-dried fertilizer products, prepared from the filtrate cultures of A. vinelandii and B. subtilis was tested using an important soybean crop. Plants treated with A. vinelandii Fe fertilizer developed a dry mass comparable to that of o,o-EDDHA and the A. vinelandii-treated plants had higher Fe content. The results indicated that the freeze-dried product, prepared from A. vinelandii, represents a very promising, sustainable, and environment-friendly Fe-fertilizer alternative for application in the IDIC amendment in calcareous soils. Similar calcareous soils that are naturally alkaline or are being threatened by increasing salinity are becoming more abundant globally (Yadav et al., 2011). Also, the Fe deficiency is generally increasing in arable soils of numerous regions of the world and therefore similar fertilizers can be a biologically-based solution for that. Interactions between plants and microbes including fungi are mediated by a chemical language containing multiple compounds, infochemicals, such as terpenes (Schmidt et al., 2017). We are slowly deciphering this communication that could be called signalomics (Mhlongo et al., 2018) to understand the interplay between environment, plants, and not only microbes but also other organisms. Phytohormones produced not only by plants but also by microbes play a crucial role in these interactions as described in a review by Kudoyarova et al.. Auxin-producing bacteria were shown to influence processes such as root elongation but both root elongation and inhibition of root elongation could be observed depending on the plant, the environment, and the dosage and the auxins. Other phytohormones include cytokinins such as zeatin riboside produced by certain bacteria also influence plant physiology but is so far even less elucidated. Other important phytohormones generated by microbes include ACC deaminase and abscisic acid (ABA). ACC deaminase lowers ethylene production but the effect of ABA accumulation is not often defined by a clear-cut physiological effect. In a paper dealing with a related topic, Luziatelli et al. examine the effect of plant growth-promoting Pantoea agglomerans on the rooting of Pyrus communis. It could be shown that exometabolites such as indole-3-acetic acid (IAA) of P. agglomerans promoted adventitious rooting. Of interest is that the synergy between auxin-related compounds such as IAA and other metabolites produced by P. agglomerans such as cinnamic-related compounds was shown to be very delicate and concentration-dependent. As previously shown for IAA, there is an optimal concentration and more is not necessarily better. Here it was demonstrated that the optimal concentration of auxin-like products is also dependent on the simultaneous production of other yet to be defined products. In another article, Xu et al. examine the relationship between soybean genotype, arbuscular mycorrhiza fungi, and rhizobium inoculation. The soybean genotype directly influenced the establishment of the rhizosphere fungal community and additionally, rhizobium inoculation also determined the composition of the rhizosphere fungal community. We are only at the beginning of understanding these complicated interkingdom dynamics. In conclusion, there is great potential for near future enhancements in the use of PGPM in world agriculture. A paramount need is to bridge the gap between fundamental, applied science, and agricultural practice. As early as a possible transition of knowledge to the farmers as end-users of innovative products and biotechnologies can ensure efficient commercialization of scientific results and can also fuel new research on the way to achieve more sustainable use of natural resources and more efficient biologically/ecologically based agriculture. YM, MV, and CR drafted the editorial text. YM and HF revised and approved the final version of the editorial text. All authors contributed to the article and approved the submitted version. This work is carried out at the R&D Unit Center for Functional Ecology—Science for People and the Planet (CFE), with reference UIDB/04004/2020, financed by FCT/MCTES through national funds (PIDDAC). The FCT supported the research contract of YM (SFRH/BPD/76028/2011). Research in the lab of CR was funded by National Natural Science Foundation of China (NSFC) (Grant number: 31770123). Technology Agency of the Czech Republic – National Center of Competence BIOCIRTECH (No. TN010000048) and the project of the Czech Academy of Sciences (RVO 67985939). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ma, Y., Látr, A., Rocha, I., Freitas, H., Vosátka, M., and Oliveira, R. S. (2019a). Delivery of inoculum of Rhizophagus irregularis via seed coating in combination with Pseudomonas libanensis for cowpea production. Agronomy 9:33. doi: 10.3390/agronomy9010033 CrossRef Full Text | Google Scholar Ma, Y., Oliveira, R. S., Freitas, H., and Zhang, C. (2016a). Biochemical and molecular mechanisms of plant-microbe-metal interactions: relevance for phytoremediation. Front. Plant Sci. 7:918. doi: 10.3389/fpls.2016.00918 PubMed Abstract | CrossRef Full Text | Google Scholar Ma, Y., Prasad, M. N. V., Rajkumar, M., and Freitas, H. (2011). Plant growth promoting rhizobacteria and endophytes accelerate phytoremediation of metalliferous soils. Biotechnol. Adv. 29, 248–258. doi: 10.1016/j.biotechadv.2010.12.001 PubMed Abstract | CrossRef Full Text | Google Scholar Ma, Y., Rajkumar, M., Oliveira, R. S., Zhang, C., and Freitas, H. (2019b). Potential of plant beneficial bacteria and arbuscular mycorrhizal fungi in phytoremediation of metal-contaminated saline soils. J. Hazard. Mater. 379:120813. doi: 10.1016/j.jhazmat.2019.120813 PubMed Abstract | CrossRef Full Text | Google Scholar Ma, Y., Rajkumar, M., Zhang, C., and Freitas, H. (2016b). Beneficial role of bacterial endophytes in heavy metal phytoremediation. J. Environ. Manag. 174, 14–25. doi: 10.1016/j.jenvman.2016.02.047 PubMed Abstract | CrossRef Full Text | Google Scholar Mesa-Arango, A. C., Rueda, C., Román, E., Quintin, J., Terrón, M. C., Luque, D., et al. (2016). Cell Wall changes in amphotericin B-resistant strains from Candida tropicalis and relationship with the immune responses elicited by the host. Antimicrob. Agents Chemother. 60, 2326–2335. doi: 10.1128/AAC.02681-15 PubMed Abstract | CrossRef Full Text | Google Scholar Mhlongo, M. I., Piater, L. A., Madala, N. E., Labuschagne, N., and Dubery, I. A. (2018). The chemistry of plant-microbe interactions in the rhizosphere and the potential for metabolomics to reveal signaling related to defense priming and induced systemic resistance. Front. Plant Sci. 9:112. doi: 10.3389/fpls.2018.00112 PubMed Abstract | CrossRef Full Text | Google Scholar Paul, M. J., Primavesi, L. F., Jhurreea, D., and Zhang, Y. (2008). Trehalose metabolism and signaling. Annu. Rev. Plant Biol. 59, 417–441. doi: 10.1146/annurev.arplant.59.032607.092945 CrossRef Full Text | Google Scholar Reina-Bueno, M., Argandoña, M., Nieto, J. J., Hidalgo-García, A., Iglesias-Guerra, F., Delgado, M. J., et al. (2012). Role of trehalose in heat and desiccation tolerance in the soil bacterium Rhizobium etli. BMC Microbiol. 12:207. doi: 10.1186/1471-2180-12-207 PubMed Abstract | CrossRef Full Text Schmidt, R., Jager, V., Zühlke, D., Wolff, C., Bernhardt, J., Cankar, K., et al. (2017). Fungal volatile compounds induce production of the secondary metabolite Sodorifen in Serratia plymuthica PRI-2C. Sci. Rep. 7:862. doi: 10.1038/s41598-017-00893-3 PubMed Abstract | CrossRef Full Text Yadav, S., Irfan, M., Ahmad, A., and Hayat, S. (2011). Causes of salinity and plant manifestations to salt stress: a review. J. Environ. Biol. 32, 667–685. PubMed Abstract | Google Scholar Keywords: plant growth-promoting microorganisms, behavior of inoculated microorganisms, biotechnological interventions, biotic and abiotic stresses, inoculum delivery, colonization pattern, sustainable agriculture Citation: Ma Y, Vosátka M, Rensing C and Freitas H (2021) Editorial: Advanced Microbial Biotechnologies for Sustainable Agriculture. Front. Microbiol. 12:634891. doi: 10.3389/fmicb.2021.634891 Received: 29 November 2020; Accepted: 03 March 2021; Published: 25 March 2021. Edited by: Reviewed by: Copyright © 2021 Ma, Vosátka, Rensing and Freitas. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Ying Ma, [email protected]; [email protected]
Published: 18 March 2021
Frontiers in Immunology, Volume 12; https://doi.org/10.3389/fimmu.2021.635522

Abstract:
We celebrate the 30th anniversary of the “hygiene hypothesis”, which has been a cornerstone for research into asthma and allergic diseases for many of us. It appeared while we witnessed the rapid increase of these conditions in the westernized world by the end of last century (1). It has stimulated thought of many researchers resulting in numerous more or less modified hypotheses meandering in diverse gestalt through the scientific landscape. It all started with an epidemiological observation about significantly decreased risk of allergic sensitization and hay fever in subjects having many siblings. This observation was counterintuitive at that time when the prevalent paradigm stated that viral infections cause asthma. However, the observation was confirmed many times in independent populations and is one of the most robust epidemiological findings in the context of allergy (2). Over the years the epidemiological gestalt changed from siblings to day care, oro-fecal and other infections and then to farm exposures. Interestingly, the farm effect is independent of the “sibling effect” (3). The gestalt also took on various immunological garments from a Th1-Th2 dichotomy to regulatory networks. Lately, the technological progress allowing exploration of the world of microbiomes has revitalized the debates around the “hygiene hypothesis” with tantalizing findings from mouse experiments and population-based studies. The “hygiene hypothesis” has also been a cornerstone of my scientific life resulting in my continued interest in the farm populations that I have been following with many colleagues since the beginning of this century. In this modified gestalt, the farm exposures may be considered strong support for a hypothesis that may be rephrased as pointing to the importance of microbial exposures for the development of childhood asthma and allergies. The concept is intuitively easily understandable which has resulted in a widespread perception by the lay press. Yet, we still wrestle with the identification and mechanistic understanding of the relevant building blocks that may allow translation into prevention of childhood asthma and allergies. In the following, I attempt to distil some lessons from the farm studies. The protective effect of a traditional farm exposure on the development of childhood asthma and allergies as documented in numerous studies is very robust. Similar to the allergy protective “sibling effect” and the asthma risk by exposure to moulds and active/passive smoking it is a remarkably reproducible finding across populations and continents. Moreover, the effects are strong. The most consistent finding, which relates to allergic sensitization and hay fever, shows odds ratios around 0.5 suggesting halving of risk (4). Findings for asthma seem somewhat weaker and less reproducible which may be attributable to the many facets of the asthma syndrome. These observations may suggest a strong extrinsic factor that once identified could serve as novel prevention strategy for these illnesses. We have identified two main pillars of the protective farm effect, one being the exposure to animal sheds, in particular cowsheds and the second the consumption of unprocessed cow’s milk (5). It is tempting to speculate that one unifying exposure may underlie these seemingly distinct exposures. The working group of Erika Jensen-Jarolim proposes that ß-lactoglobulin, which is found in cow’s milk and urine and thus also in ambient air of cowsheds, carries farm-specific ligands which render this lipocalin tolerogenic rather than allergenic (6). Thereby ß-lactoglobulin could act as important transport protein presenting its allergy- and potentially asthma-protective cargo to competent immune cells. This concept awaits however, confirmation in mouse studies of experimental allergic asthma and allergy. If substantiated the nature of the cargo needs further investigation and the relative contribution of the transporter versus the cargo (and the diversity of the cargo) must be resolved. It seems conceivable that the farm environment confers not only the protective exposures, but also the transporters that enhance the protective effects by optimizing presentation to competent immune cells. From the epidemiological observations, diversity of exposures in the farm environment has however been a central theme. We have so far not found one single component conferring protection. One must bear in mind that cowsheds and unprocessed cow’s milk are “soups” containing myriads of potentially relevant elements. We have shown that an increased diversity of food introduction protects from food allergy, atopic dermatitis and asthma (7). Moreover, the diversity of farm animal exposure during pregnancy has been associated with lower risk of atopic dermatitis and higher IFN-y and TNF-α levels in supernatants of cord blood mononuclear cells stimulated with LPS (8, 9). Finally, the diversity of the environmental and human nasal microbiome, respectively, have been associated with lower risk of asthma in the farm populations (10). A strong signal with diversity results in a low likelihood to find the one “magic bullet” explaining the protective associations. It can in turn be interpreted as a multitude of additive (weak) effects interacting with a multitude of host factors in the general population which is made up of subjects with very diverse genetic and immune response backgrounds. Alternatively, diversity may harbour a limited number of relevant, necessary and sufficient elements or hubs in exposure networks which drive the protective effects. We have some evidence that these necessary elements exist because in experimental studies of farm exposure, i.e. extracts from cowshed dust extracts do no longer protect mice from allergic asthma when they are devoid of MYD88/TRIF and epithelial A20 signalling (11, 12). Thereby, innate immune responses may be essential elements for the protective effect, but the precise nature of these elements stills awaits elucidation. The complexity of the interplay of protective elements may be further increased by the multitude of exposure routes that may matter. Environmental exposures such as the indoor microbiome or the stay in cowsheds may be inhaled or ingested. In fact, we have seen that both the nasal and gut microbiome are influenced by these external exposures because young children breathe in airborne matter and put their contaminated fingers in their mouth thereby ingesting external compounds. In addition, ingestion of relevant exposures such as unprocessed cow’s milk or a diverse introduction of solid foods further shapes the gut microbiome and its development (13). We have unfortunately not investigated skin exposures, but these may also add to the complex interplay of farm exposure routes. We are therefore left with the impression that key elements of protection within the farm environment affect a number of body compartments (upper and lower airways, gut and skin) in probably redundant and overlapping pathways, which may in turn confer protection for a large majority of exposed subjects with rather diverse genetic and immune response backgrounds. If this notion was correct, then translation into novel asthma preventive approaches will have to be multifaceted. As discussed in a recent review (14), it seems unlikely that asthma phenotypes are distinct conditions with distinct underlying pathologies resulting in exclusive unambiguous disease categories. Complex diseases such as asthma are more likely to consist of combinations of various traits and underlying redundant mechanisms given the many weak genetic and environmental effects and their interactions on disease development. In such a scenario the one and only causal mechanism can neither be found nor successfully targeted for prevention. Then only some facets of the disease will be addressed which however results in weak effects on a population level. In a complex disease a combination of a multitude of involved mechanisms matters which should preferably all be targeted if all of asthma should be prevented. Given that the “farm effect” on asthma is strong across multiple populations it seems more likely that the multitude and diversity of exposures across several routes of exposures (upper and lower airways, gut, skin) contributes to the overall protective effect. The author confirms being the sole contributor of this work and has approved it for publication. Personal fees from Pharmaventures, OM Pharma S. A., Peptinnovate Ltd., Böhringer Ingelheim International GmbH. Patent LU101064: Barn dust extract for the prevention and treatment of diseases pending, Patent EP2361632: Specific environmental bacteria for the protection from and/or the treatment of allergic, chronic inflammatory and/or autoimmune disorders with royalties paid to ProtectImmun GmbH, Patent EP 1411977: Composition containing bacterial antigens used for the prophylaxis and the treatment of allergic diseases. licensed to ProtectImmun GmbH, Patent EP1637147: Stable dust extract for allergy protection licensed to ProtectImmun GmbH, Patent EP 1964570: Pharmaceutical compound to protect against allergies and inflammatory diseases licensed to ProtectImmun GmbH. 1. Eder W, Ege MJ, von Mutius E. The asthma epidemic. N Engl J Med (2006) 355(21):2226–35. doi: 10.1056/NEJMra054308 PubMed Abstract | CrossRef Full Text | Google Scholar 2. von Mutius E. The environmental predictors of allergic disease. J Allergy Clin Immunol (2000) 105:9–19. doi: 10.1016/S0091-6749(00)90171-4 PubMed Abstract | CrossRef Full Text | Google Scholar 3. Genuneit J, Strachan DP, Büchele G, Weber J, Loss G, Sozanska B, et al. The combined effects of family size and farm exposure on childhood hay fever and atopy. Pediatr Allergy Immunol (2013) 24:293–8. doi: 10.1111/pai.12053 PubMed Abstract | CrossRef Full Text | Google Scholar 4. Genuneit J. Exposure to farming environments in childhood and asthma and wheeze in rural populations: a systematic review with meta-analysis. Pediatr Allergy Immunol (2012) 23(6):509–18. doi: 10.1111/j.1399-3038.2012.01312.x PubMed Abstract | CrossRef Full Text | Google Scholar 5. Illi S, Depner M, Genuneit J, Horak E, Loss G, Strunz-Lehner C, et al. Protection from childhood asthma and allergy in Alpine farm environments-the GABRIEL Advanced Studies. J Allergy Clin Immunol (2012) 129(6):1470–7.e6. doi: 10.1016/j.jaci.2012.03.013 PubMed Abstract | CrossRef Full Text | Google Scholar 6. Roth-Walter F, Afify SM, Pacios LF, Blokhuis BR, Redegeld F, Regner A, et al. Cow’s milk protein b-lactoglobulin confers resilience against allergy by targeting complexed iron into immune cells. J Allergy Clin Immunol (2020) 147(1):321–34.e4. doi: 10.1016/j.jaci.2020.05.023. PubMed Abstract | CrossRef Full Text | Google Scholar 7. Roduit C, Frei R, Depner M, Schaub B, Loss G, Genuneit J, et al. Increased food diversity in the first year of life is inversely associated with allergic diseases. J Allergy Clin Immunol (2014) 133(4):1056–64. doi: 10.1016/j.jaci.2013.12.1044 PubMed Abstract | CrossRef Full Text | Google Scholar 8. Roduit C, Wohlgensinger J, Frei R, Bitter S, Bieli C, Loeliger S, et al. Prenatal animal contact and gene expression of innate immunity receptors at birth are associated with atopic dermatitis. J Allergy Clin Immunol (2011) 127(1):179–85, 185.e1. doi: 10.1016/j.jaci.2010.10.010 PubMed Abstract | CrossRef Full Text | Google Scholar 9. Pfefferle PI, Büchele G, Blümer N, Roponen M, Ege MJ, Krauss-Etschmann S, et al. Cord blood cytokines are modulated by maternal farming activities and consumption of farm dairy products during pregnancy: The PASTURE Study. J Allergy Clin Immunol (2010) 125(1):108–15.e3. doi: 10.1016/j.jaci.2009.09.019 PubMed Abstract | CrossRef Full Text | Google Scholar 10. Ege MJ, Mayer M, Normand AC, Genuneit J, Cookson WO, Braun-Fahrländer C, et al. Exposure to environmental microorganisms and childhood asthma. N Engl J Med (2011) 364(8):701–9. doi: 10.1056/NEJMoa1007302 PubMed Abstract | CrossRef Full Text | Google Scholar 11. Stein MM, Hrusch CL, Gozdz J, Igartua C, Pivniouk V, Murray SE, et al. Innate Immunity and Asthma Risk in Amish and Hutterite Farm Children. N Engl J Med (2016) 375(5):411–21. doi: 10.1056/NEJMoa1508749 PubMed Abstract | CrossRef Full Text | Google Scholar 12. Schuijs MJ, Willart MA, Vergote K, Gras D, Deswarte K, Ege MJ, et al. Farm dust and endotoxin protect against allergy through A20 induction in lung epithelial cells. Science (2015) 349(6252):1106–10. doi: 10.1126/science.aac6623 PubMed Abstract | CrossRef Full Text | Google Scholar 13. Depner M, Taft DH, Kirjavainen PV, Kalanetra KM, Karvonen AM, Peschel S, et al. Maturation of the gut microbiome during the first year of life contributes to the protective farm effect on childhood asthma. 26(11):1766–75. doi: 10.1038/s41591-020-1095-x PubMed Abstract | CrossRef Full Text | Google Scholar 14. von Mutius E, Smits HH. Primary prevention of asthma: from risk and protective factors to targeted strategies for prevention. Lancet (2020) 396(10254):854–66. doi: 10.1016/S0140-6736(20)31861-4 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: asthma, children, allergy, immunology, epidemiology Citation: von Mutius E (2021) The “Hygiene Hypothesis” and the Lessons Learnt From Farm Studies. Front. Immunol. 12:635522. doi: 10.3389/fimmu.2021.635522 Received: 30 November 2020; Accepted: 02 March 2021; Published: 18 March 2021. Edited by: Reviewed by: Copyright © 2021 von Mutius. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Erika von Mutius, [email protected]
Published: 18 March 2021
Frontiers in Genetics, Volume 12; https://doi.org/10.3389/fgene.2021.635006

Abstract:
Editorial on the Research TopicDevelopment of Healthy and Nutritious Cereals: Recent Insights on Molecular Advances in Breeding Worldwide more than 2 billion people are affected by micronutrient deficiencies and most of them are residing in the developing countries of Asia, Africa and Latin America (Kennedy et al., 2002). Malnutrition is linked with heavy dependence on monotonous cereal staples without much dietary diversification or nutrient supplementation. Even though significant efforts have been made over the last six decades to improve production and productivity in most food crops, it lacked associated nutritional improvement (Bouis and Welch, 2010). So, the modern varieties do not have enough variability for several nutrients, making poor rural populations vulnerable to micronutrient deficiencies. More than two dozen mineral elements, vitamins, antioxidants, and health beneficial compounds must be supplied in optimal quantities daily for normal growth and development of humans. Biofortification of cereals with elevated levels of essential micronutrients, vitamins, and reduced levels of toxic elements help to address malnutrition and is a cost-effective approach in reaching target groups, especially rural populations (Bouis and Saltman, 2017). The sustainable development goals and the Lancet Commission Report have emphasized the need for promoting nutritious diets to eradicate malnutrition (Willet et al., 2019; https://sustainabledevelopment.un.org). Among these, deficiencies of iron (Fe), zinc (Zn), and vitamin A are major global health problems. As successful examples, one high Fe rice and several high Zn rice varieties have been successfully released for commercial cultivation (Palanog et al., 2019). Presently we have a better understanding of the genetic, physiological, and molecular basis, as well as the influence of environmental factors on nutrients accumulation in cereal grains (Swamy et al., 2016; Garcia-Oliveira et al., 2018; Ludwig and Slamet-Loedin, 2019). However, there is a need to integrate our understanding to achieve the goals of biofortification and review the current progress and the prospects for nutritious crops. In this Research Topic, we selected manuscripts on various aspects of nutritional improvement in cereals. Fourteen articles published in our special editorial topic, five of them provided updated review of cereals nutritional enhancement, and nine of them were original research articles on understanding the molecular basis of different grain nutrients and grain quality traits in cereals. Focusing on improving grain protein quality, Chandran et al. successfully pyramided Lysine, Tryptophan, and Provitamin A into Maize varieties based on opaque-2 and β-carotene through marker assisted selection (MAS). The improved lines possessed high lysine, tryptophan, and β-carotene content, but they had only slight yield reduction. Even though these lines can be used as genetic resources for maize improvement, they are not yet commercially viable, since successful biofortified crops should have similar or even higher yield along with the other desirable traits. Also aiming at improving protein content in rice, Jang et al. identified multiple genomic regions responsible for amino acid content (AAC) and protein content (PC). They identified two novel loci qAAC6.1 and qAAC7.1 and several transgressive segregants for both traits. These loci can be used for quantitative trait loci (QTL) pyramiding programs to develop rice lines with high protein content. It is quite interesting to note pleotropic effect of heading-date genes on protein content of rice. Xie et al. reported that in three nearly isogenic lines (NIL), the rice florigen genes RFT1 have a strong negative effect on the amino acids content governed by the Zhenshan97 allele with the genomic region consisting of 14 QTLs located in proximity to Hd3a. Bhuvaneswari et al. characterized 93 aromatic Chakhao rice germplasm from Manipur province of India. Wider variations were observed for the agro-morphological, grain quality and nutraceutical traits. The total anthocyanin content ranged from 29.8 to 275.8 mg.100g−1 DW, while total phenolics ranged from 66.5 to 700.3 mg GAE.100g−1 DW. The germplasm with higher levels of anthocyanin compounds such as cyanidin-3-O-glucoside (C3G) and peonidin-3-O-glucoside (P3G) are useful for improving the antioxidant properties in rice. Focusing on micronutrient biofortification, Ashokkumar et al. comprehensively reviewed recent advances in breeding for improved folate, provitamin A, and carotenoids content in rice, wheat, maize, and pearl millet. They discussed in detail the genetic variation, trait discovery, genes/QTL identification for nutritional traits and their introgressions into elite genetic backgrounds. Prasanna et al. carried out a detailed global analysis of molecular breeding for nutritional improvement in maize, a species where systematic efforts have been made to develop and deploy cultivars biofortified with quality protein maize (QPM), provitamin A, and kernel zinc. The limited germplasm characterization, lack of genetic variability, and diagnostic markers for some of the mineral elements is a constraint for breeding. Broadening the genetic base through exploitation of landraces and wild species, use of genomics technologies, market-driven breeding strategies, strengthening of seed systems, and collaborative interdisciplinary efforts were emphasized. Genetic Engineering (GE) and Genome Editing (GEd) technologies are the way forward for improving the traits with no variability and to achieve the target levels of multiple nutrients in cereals. Babu et al. characterized 40 rice genotypes for agronomic, yield and micronutrient traits. They identified stable high Zn donor lines and genome wide association analysis resulted in identification three loci on chromosomes 3 and 7, which were linked to new, uncharacterized putative candidate genes. Bollinedi et al. identified 18 novel marker-trait associations (MTAs) for grain Fe and Zn in brown and milled rice using 192 Indian rice germplasm accessions and found strong association between Zn concentrations in brown and milled rice. Fe concentration in brown rice, however, was not associated with Fe concentration in milled rice, highlighting the need for enriching the Fe concentration of rice endosperm. They have also identified four accessions with grain Zn concentration in milled rice with >28 mg/kg and one accession (IC-2127) with >12 mg/kg Fe, a target set by the HarvestPlus program for rice biofortification which will be used to develop high Fe and Zn varieties. Focusing on biofortification of not-so-typical grains, Bekkering and Tian provide an overview of how other cereals and non-grass pseudo-cereals can contribute to biofortification. The monocots species, broomcorn millet (Panicum miliaceum L.), canary seed (Phalaris canariensis L.), teff [Eragrostis tef (Zuccagni) Trotter], and the pseudo-cereals (i.e., seeds similar to cereals, but not from the Poaceae family), amaranth (Amaranthus spp.), buckwheat (Fagopyrum esculentum Moench.), chia (Salvia hispanica L.), and quinoa (Chenopodium quinoa Willd), are richer in protein and lipids, and have lower starch compared to the major staples. Other characteristics include a more balanced protein composition, higher levels of micronutrients and vitamins. Authors delineate possible avenues in which we should invest to improve and fully utilize these species to provide nutritious grains for consumption, from marketing to breeding to genomics/functional genetics. In the same line, Rodríguez et al., provided an extensive review on finger millet (Eleusine coracana) and foxtail millet (Setaria italica) and the pseudocereals quinoa, amaranth and buckwheat. These species can contribute to produce healthy grains, especially in harsh, stressful environments, for which these plants tend to be more resilient. Authors compare nutritional profiles, processes to increase bioavailability of nutrients and decrease anti-nutrients, and thoroughly review the current status of genome resources and molecular markers available to drive the efforts to improve these species, allowing their use in marginal lands to produce nutritious food to combat hidden hunger. Renganathan et al. focus on barnyard millet from the genus Echinochloa, including the cultivated Indian barnyard millet (Echinochloa frumentacea), Japanese barnyard millet (Echinochloa esculenta) and other wild species, which are used for human consumption and livestock feed, and also show tolerance to multiple stresses. The taxonomy, morphological and genetic diversity, genomic and genetic resources, and the potential of these species to contribute for food security and human nutrition are discussed. Altogether, these reviews are an excellent starting point for the biofortification community to launch efforts into including new species in our toolbox to increase human access to sufficient nutrients. From a genomic perspective, Butardo et al. addressed the structural, regulatory and nutrition roles of Starch Synthase IIa in O. sativa ssp. japonica rice endosperm, highlighting the importance of this key enzyme in seed morphology, starch granules size and distribution, amylopectin structure, amylose content and glycemic index. Using a different approach, Kishor et al. used whole-genome next generation sequencing to characterize traditional varieties of basmati rice and identified millions of SNP markers useful for genetic analysis. Finally, Bhuvaneswari et al., combined genomics and metabolomics to bring forward the nutraceutical importance of aromatic glutinous rice through the characterization of a set of 93 landraces for their agro-morphological traits, grain pigmentation, antioxidant properties, and molecular genetic variation. Altogether these three papers provide a solid platform of molecular markers for taxonomy, breeding and conservation programs. Our Research Topic combines different approaches on biofortification and provides a comprehensive collection of the efforts to improve grain nutrient quality and to increase human nutrition. All the authors equally contributed to prepare this editorial manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors would like to thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Rio Grande do Sul (FAPERGS) in Brazil; Fundação para a Ciência e a Tecnologia from Portugal, through the research unit UIDB/00239/2020 (CEF). Bill and Melinda Gates Foundation (BMGF) for funding Healthier Rice Project at International Rice Research Institute (IRRI). Bouis, H. E., and Saltman, A. (2017). Improving nutrition through biofortification: a review of evidence from HarvestPlus, 2003 through 2016. Global Food Security 12, 49–58. doi: 10.1016/j.gfs.2017.01.009 PubMed Abstract | CrossRef Full Text | Google Scholar Bouis, H. E., and Welch, R. M. (2010). Biofortification—a sustainable agricultural strategy for reducing micronutrient malnutrition in the global South. Crop Sci. 50, 20–32. doi: 10.2135/cropsci2009.09.0531 CrossRef Full Text | Google Scholar Garcia-Oliveira, A. L., Subhash, C., Rodomiro, O., Abebe, M., and Melaku, G. (2018). Genetic basis and breeding perspectives of grain iron and zinc enrichment in cereals. Front. Plant Sci. 9:937. doi: 10.3389/fpls.2018.00937 PubMed Abstract | CrossRef Full Text | Google Scholar Kennedy, G., Burlingame, B., and Nguyen, V. N. (2002). “Nutritional contribution of rice and impact of biotechnology and biodiversity in rice-consuming countries,” 20th International Rice Commission Bangkok Thailand 23–26 July 2002 (Bangkok). Google Scholar Ludwig, Y., and Slamet-Loedin, I. H. (2019). Genetic biofortification to enrich rice and wheat grain iron: from genes to product. Front. Plant Sci. 10:833. doi: 10.3389/fpls.2019.00833 PubMed Abstract | CrossRef Full Text | Google Scholar Palanog, A. D., Calayugan, M. I. C., Descalsota-Empleo, G. I., Amparado, A., Inabangan-Asilo, M. A., Arocena, E. C., et al. (2019). Zinc and iron nutrition status in the Philippines population and local soils: a review. Front. Nutr. 6:81. doi: 10.3389/fnut.2019.00081 CrossRef Full Text | Google Scholar Swamy, B. P. M., Rahman, M. A., Inabangan-Asilo, M. A., Amparado, A., Manito, C., Chadha-Mohanty, P., et al. (2016). Advances in breeding for high Zinc in Rice. Rice 9, 49–65. doi: 10.1186/s12284-016-0122-5 PubMed Abstract | CrossRef Full Text | Google Scholar Willet, W., Rockstrom, J., Loken, B., Springman, M., Lang, T., Vermeulen, S., et al. (2019). Food in the Aantropocene: the EAT-Lancet commission on healthy diets from sustainable food systems. Lancet 393, 447–492. doi: 10.1016/S0140-6736(18)31788-4 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: cereals, nutrition, breeding, omics, genome editing, biofortification Citation: Swamy BPM, Marathi B, Ribeiro-Barros AIF and Ricachenevsky FK (2021) Editorial: Development of Healthy and Nutritious Cereals: Recent Insights on Molecular Advances in Breeding. Front. Genet. 12:635006. doi: 10.3389/fgene.2021.635006 Received: 29 November 2020; Accepted: 23 February 2021; Published: 18 March 2021. Edited and reviewed by: Ahmed El-Sohemy, University of Toronto, Canada Copyright © 2021 Swamy, Marathi, Ribeiro-Barros and Ricachenevsky. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: B. P. Mallikarjuna Swamy, [email protected]; Balram Marathi, [email protected]; Ana I. F. Ribeiro-Barros, [email protected]; Felipe Klein Ricachenevsky, [email protected]
Published: 11 March 2021
Frontiers in Immunology, Volume 12; https://doi.org/10.3389/fimmu.2021.635371

Abstract:
The magnitude of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prompted the repurposing of several drugs to quickly stop the morbidity, mortality, and spread of this new disease. Repurposed drugs tested to fight COVID-19 have been chosen mainly on the basis of promising in vitro efficacy against SARS-CoV-2 or on previous therapeutic results with other human coronavirus diseases, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) (1). Numerous clinical trials have already been completed, but no repurposed drug evaluated to date has been found that could significantly impact the course of COVID-19 pandemic (2). Our experience with previous viral pandemics, such as human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV), has taught us that precise design and target specificity will be essential to obtaining potent and successful antivirals against SARS-CoV-2. Repurposed drugs that have been explored more thoroughly since the beginning of the COVID-19 pandemic include remdesivir, favipiravir, lopinavir-ritonavir, ribavirin, interferon, and hydroxychloroquine (1). Favipiravir, a purine nucleoside analog broad-spectrum inhibitor of viral RNA-dependent RNA polymerase (RdRp), approved for treatment of influenza virus infection in Japan, was chosen due to its in vitro activity against SARS-CoV-2, nevertheless, there is no evidence of its clinical efficacy. A prospective, randomized, open-label trial of early vs. late favipiravir in hospitalized patients with COVID-19 has shown that favipiravir did not significantly improve viral clearance (3). Lopinavir-ritonavir, a HIV-1 protease inhibitor, was investigated due to its SARS-CoV antiviral activity in tissue culture and infected patients. However, the lopinavir-ritonavir combination exhibited no clinical benefit against SARS-CoV-2 (4). From the very beginning of the COVID-19 pandemic, remdesivir has been the most promising drug against SARS-CoV-2. This adenosine nucleotide analog prodrug, a potentially inhibitor of RdRp, was initially developed by Gilead Sciences to treat filoviruses, such as the Ebola virus, and was explored due to its broad-spectrum antiviral activity in tissue culture and animal models against filoviruses, paramyxoviruses, pneumoviruses, and pathogenic coronaviruses, including SARS-CoV and MERS-CoV. Randomized controlled trials (RCTs) have found no effect of remdesivir on mortality (5). This drug has been approved to treat COVID-19 in the USA and Europe, but conclusive results to support the use of remdesivir are lacking (6, 7). The use of aminoquinoline drugs chloroquine and hydroxychloroquine is paradigmatic of the failure of repurposed drugs to treat COVID-19. These cheap drugs are generic antimalarials used to treat amoebic liver abscess and rheumatic disease. The early promising results with these two drugs showing antiviral activity against SARS-CoV-2 at micromolar concentrations in tissue culture and their clinical benefit in dubious observational trials of a few patients positioned them at the forefront of possible treatments for COVID-19. However, large observational clinical trials and RCTs have shown no effect of hydroxychloroquine in reducing mortality and/or mobility (2). Moreover, in some studies, a worse infection course was observed in hospitalized patients treated with these aminoquinolines (8). Importantly, recent studies have demonstrated that chloroquine does not inhibit infection of human lung cells with SARS-CoV-2 (9). Previous studies have shown that chloroquine and hydroxychloroquine inhibit the ability of SARS-CoV-2 to infect African green monkey kidney-derived Vero cells. However, when Vero cells were engineered to express TMPRSS2, a cellular protease that activates SARS-CoV-2 for entry into lung cells rendered SARS-CoV-2-infected Vero cells insensitive to chloroquine (9). Furthermore, chloroquine does not block infection with SARS-CoV-2 in TMPRSS2-expressing human lung Calu-3 cells, indicating that chloroquine targets a pathway for viral activation that is not active in lung cells and is unlikely to protect against the spread of SARS-CoV-2. These results emphasize the necessity of being cautious with observed drug inhibition of viral replication in tissue culture. Ivermectin, another cheap antiparasitic drug with in vitro efficacy against SARS-CoV-2, is being prescribed as a preventative against COVID-19. However, the evidence that ivermectin protects people from COVID-19 is limited (10). We should be prudent using ivermectin, or other potential drugs, outside clinical trials. In some countries, ivermectin is being also administered to SARS-CoV-2 infected patients. Different doses and posology have been used and confounding results have been reported. A recent pilot clinical trial found no significant differences in detection of SARS-CoV-2 RNA from nasopharyngeal swabs at days four and seven after treating with a single oral dose of 400 mcrg/Kg of ivermectin (11). Virus target specificity (e.g., isolation or drug-resistant viruses) should be tested and demonstrated before initiating treatments in virus-infected patients. As hydroxychloroquine showed no effect in SARS-CoV-2 infection in non-human primates (12), testing animal models will be preferable before translating these drugs to humans. In addition to drugs specifically aimed to inhibit SARS-CoV-2 replication, therapeutics that modulate inflammation have also been tested and, in this case, they seem to be a more effective therapeutic strategy for treating COVID-19 morbidity and mortality. Immunomodulators are being tested in several clinical trials for the treatment of SARS-CoV-2-generated cytokine storm. However, data to support the use of one of the most explored compounds to modulate inflammation, tocilizumab, a monoclonal antibody against interleukin-6 receptors, come largely from observational studies (13). Large RCTs with tocilizumab should provide answers regarding its clinical benefit. Immunomodulators that appear to work are corticosteroids. A recent RTC performed with dexamethasone showed that, in patients with moderate or severe COVID-19, dexamethasone plus standard care significantly increases survival and reduces morbidity (14, 15). Other drugs that could offer clinical effects despite the lack of specific evidence for COVID-19 include anti-androgens, statins, N-acetyl cysteine, ACE2 inhibitors, angiotensin receptor blockers, and direct TMPRSS-2 inhibitors (16). Although immunomodulators may be an excellent clinical tool, it is desirable to potently and specifically stop SARS-CoV-2 replication after the onset of the first COVID-19 symptoms to avoid the pathogenic course of the disease. Ideally, we should stop SARS-CoV-2 in the first days of the infection. For example, neuraminidase inhibitors may not produce any detectable effect in a patient hospitalized with severe influenza virus infection, but can be useful in preventing the development of severe disease. The most appropriate therapy goal of an acute viral infection is therefore not to cure severe disease, but to keep the disease from becoming severe, and prevent hospitalization. Treatment early in the course of illness could also limit person-to-person transmission. A way to stop the early spread of SARS-CoV-2 will be through a sterilizing vaccine. SARS-CoV-2-neutralizing antibodies have been associated with protection (17). This is not surprising as natural infection induces both mucosal antibody responses (secretory IgA) and systemic antibody responses (IgG). The upper respiratory tract is mostly protected by secretory IgA, whereas the lower respiratory tract is mostly protected by IgG. Because most vaccines currently in development will be administered intramuscularly or intradermally, they will induce mostly IgG, but no secretory IgA (18). Therefore, these vaccines would probably prevent disease but not generate sterilizing immunity; that is, they may still allow for transmission of the virus (18). In this scenario, the current pandemic will require different strategies utilized in concert, including an effective vaccine and competent antivirals. HIV therapy is arguably among the most successful in treating any single human disease. The success of HIV therapeutics is illustrated by the number of antiretroviral agents and unique drug classes available (19). To date, the Food and Drug Administration (FDA) has approved 23 drugs to treat HIV infection. Based on their molecular mechanism and drug-resistance profile, antiretrovirals are classified into eight different classes: nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, fusion inhibitors that block HIV entering CD4+ cells, CCR5 antagonists that block the CCR5 co-receptor that HIV needs to enter the cells, integrase inhibitors, attachment inhibitors that bind HIV glycoprotein 120, and post-attachment inhibitors that block cellular CD4 receptor. In addition to antiretrovirals, one pharmacokinetic enhancer has been approved to increase antiretroviral effectiveness. In contrast to the diffuse viral targets of most of the repurposed drugs mentioned above, antiretroviral target specificity was defined through the in vitro or in vivo selection of HIV-resistant variants for the different drugs. No antiretroviral has been approved in the absence of a specific viral target. Although the virus was discovered in 1983, few antiretroviral treatment options existed for HIV infection before 1996. HIV therapeutics consisted mainly of prophylaxis against common opportunistic pathogens and managing AIDS-related illnesses. The development of HIV reverse transcriptase and protease inhibitors in the mid-1990s, and the introduction of drug regimens that combined these two classes of inhibitors to increase their efficacy, completely revolutionized the clinical approach to HIV. These combination treatments transformed HIV infection from a life-threatening disease to a manageable chronic disease. The success of antiretroviral therapies has strongly impacted the development of therapies against other viral infections. The best example is HCV, another pandemic, life-threatening, human viral infection discovered in 1989. The first generation of FDA-approved HCV drugs included interferon alfacon-1 (approval year: 1997, discontinued in 2013 due to severe adverse events), ribavirin (1998), pegylated interferon alfa-2b (2001), and pegylated interferon alfa-2a (2002) (20). Although these drugs had low cure rates, a treatment duration of 48 weeks, and may cause severe adverse events, they were the only standard-of-care treatments over a decade. Interferons and ribavirin were chosen because they exhibited a certain inhibitory capacity against other viral infections, and their low effectivity is largely due to their low specificity against HCV. Fortunately, the development of direct-acting antivirals (DAAs) targeting the two main HCV enzymes, NS3 protease and RdRp (20), has decreased treatment duration to 8 weeks and increased the cure rate to nearly 100%. DAA therapy is among the best examples of success in the fight against viral infections. DAAs have transformed HCV management and opened the door to the global eradication of HCV. Patients infected with HIV or HCV have a prolonged course of infection measured in months or years, during which they are asymptomatic or only mildly ill, providing ample opportunity to intervene with an antiviral drug. Because viremia is prolonged and relatively steady, a patient can serve as his own control to measure a drug effect. Although the situation is quite different for patients who have developed COVID-19, a rapidly progressive disease in whom might be more difficult to expect an antiviral to provide detectable benefit and more difficult to diagnose in its earlier stages when antiviral approaches would be more likely to be effective, HIV-1 and HCV examples should be mirrors in which we should look for antiviral solutions. It can be argue that a repurposed drug that has not shown any benefit in hospitalized COVID-19 patients might still be useful in slowing the development of illness, preventing severe disease and making hospitalization unnecessary. In the absence of vaccines, a repurposed drug with limited antiviral activity might be given to persons who have been exposed to an infected individual, or are in a situation in which exposure is likely to occur (post- or pre-exposure prophylaxis). However, such benefit would be more difficult to demonstrate than a standard RCT, but could still be tested. A combinatorial approach of repurposing drugs targeting both the virus and host target mechanisms has been also proposed for the management of COVID-19 severity (21). The recent resolution of the crystal structures of the three most likely SARS-CoV-2 targetable proteins (spike, RdRp, and the main protease) is allowing the identification of first-generation SARS-CoV-2-specific antivirals (22–24). Even if the benefits of SARS-CoV-2-specific antivirals remain to be elucidated, we should quickly move these first-generation specific and potent antivirals to the clinic. Antiviral drugs approved for the treatment of human virus infectious diseases have saved tens of millions of human beings over the last decades. It is a challenge to pursue effective, low-toxicity, and well-tolerated drugs that enhance patient compliance and drug administration. Nevertheless, effective antivirals will positively impact COVID-19 therapy, and SARS-CoV-2 transmission and eradication. The success of antiretroviral and antiviral therapies developed against HIV and HCV should provide a point of reference for SARS-CoV-2 drug development and a roadmap for the development of novel COVOD-19 prevention strategies. MM designed and wrote this manuscript. This work was supported by the Spanish Ministry of Science and Innovation (PID2019-103955RB-100). The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 1. Martinez MA. Compounds with therapeutic potential against novel respiratory 2019 coronavirus. Antimicrob Agents Chemother. (2020) 64:e00399–20. doi: 10.1128/AAC.00399-20 PubMed Abstract | CrossRef Full Text | Google Scholar 2. Martinez MA. Clinical trials of repurposed antivirals for SARS-CoV-2. Antimicrob Agents Chemother. (2020) 64:e01101–20. doi: 10.1128/AAC.01101-20 PubMed Abstract | CrossRef Full Text | Google Scholar 3. Doi Y, Hibino M, Hase R, Yamamoto M, Kasamatsu Y, Hirose M, et al. A prospective, randomized, open-label trial of early versus late favipiravir in hospitalized patients with COVID-19. Antimicrob Agents Chemother. (2020) 64:e01897–20. doi: 10.1128/AAC.01897-20 PubMed Abstract | CrossRef Full Text | Google Scholar 4. Cao B, Wang Y, Wen D, Liu W, Wang J, Fan G, et al. A Trial of Lopinavir–Ritonavir in adults hospitalized with severe Covid-19. N Engl J Med. (2020) 382:1787–99. doi: 10.1056/NEJMoa2001282 PubMed Abstract | CrossRef Full Text | Google Scholar 5. Wang Y, Zhang D, Du G, Du R, Zhao J, Jin Y, et al. Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trial. Lancet. (2020) 395:1569–78. doi: 10.1016/S0140-6736(20)31022-9 PubMed Abstract | CrossRef Full Text | Google Scholar 6. Beigel JH, Tomashek KM, Dodd LE, Mehta AK, Zingman BS, Kalil AC, et al. Remdesivir for the treatment of Covid-19 — final report. N Engl J Med. (2020) 383:1813–26. doi: 10.1056/NEJMoa2007764 PubMed Abstract | CrossRef Full Text | Google Scholar 7. Goldman JD, Lye DCB, Hui DS, Marks KM, Bruno R, Montejano R, et al. Remdesivir for 5 or 10 days in patients with severe Covid-19. N Engl J Med. (2020) 238:1827–37. doi: 10.1056/NEJMoa2015301 PubMed Abstract | CrossRef Full Text | Google Scholar 8. Magagnoli J, Narendran S, Pereira F, Cummings TH, Hardin JW, Sutton SS, et al. Outcomes of Hydroxychloroquine Usage in United States Veterans Hospitalized with COVID-19. Med. (2020) 1:114–27. doi: 10.1016/j.medj.2020.06.001 PubMed Abstract | CrossRef Full Text | Google Scholar 9. Hoffmann M, Mösbauer K, Hofmann-Winkler H, Kaul A, Kleine-Weber H, Krüger N, et al. Chloroquine does not inhibit infection of human lung cells with SARS-CoV-2. Nature. (2020) 585:588–90. doi: 10.1038/s41586-020-2575-3 PubMed Abstract | CrossRef Full Text | Google Scholar 10. Mega ER. Latin America's embrace of an unproven COVID treatment is hindering drug trials. Nature. (2020) 586:481–2. doi: 10.1038/d41586-020-02958-2 PubMed Abstract | CrossRef Full Text | Google Scholar 11. Chaccour C, Casellas A, Blanco-Di Matteo A, Pineda I, Fernandez-Montero A, Ruiz-Castillo P, et al. The effect of early treatment with ivermectin on viral load, symptoms and humoral response in patients with non-severe COVID-19: a pilot, double-blind, placebo-controlled, randomized clinical trial. EClinicalMedicine. (2021) 100720. doi: 10.1016/j.eclinm.2020.100720 PubMed Abstract | CrossRef Full Text | Google Scholar 12. Maisonnasse P, Guedj J, Contreras V, Behillil S, Solas C, Marlin R, et al. Hydroxychloroquine use against SARS-CoV-2 infection in non-human primates. Nature. (2020) 585:584–7. doi: 10.1038/s41586-020-2558-4 PubMed Abstract | CrossRef Full Text | Google Scholar 13. Stone JH, Frigault MJ, Serling-Boyd NJ, Fernandes AD, Harvey L, Foulkes AS, et al. Efficacy of Tocilizumab in Patients Hospitalized with Covid-19. N Engl J Med. (2020) 383:2333–44. doi: 10.1056/NEJMoa2028836 PubMed Abstract | CrossRef Full Text | Google Scholar 14. Horby PW, Mafham M, Bell JL, Linsell L, Staplin N, Emberson J. Dexamethasone in hospitalized patients with Covid-19 — Preliminary Report. N Engl J Med. (2020) 396:1345–52. doi: 10.1056/NEJMoa2021436 CrossRef Full Text | Google Scholar 15. Tomazini BM, Maia IS, Cavalcanti AB, Berwanger O, Rosa RG, Veiga VC, et al. Effect of dexamethasone on days alive and ventilator-free in patients with moderate or severe acute respiratory distress syndrome and COVID-19: The CoDEX randomized clinical trial. J Am Med Assoc. (2020) 324:1307–16. doi: 10.1001/jama.2020.17021 CrossRef Full Text | Google Scholar 16. Cadegiani FA. Repurposing existing drugs for COVID-19: an endocrinology perspective. BMC Endocr Disord. (2020) 20:149. doi: 10.1186/s12902-020-00626-0 CrossRef Full Text | Google Scholar 17. Addetia A, Crawford KHD, Dingens A, Zhu H, Roychoudhury P, Huang M-L, et al. Neutralizing antibodies correlate with protection from SARS-CoV-2 in humans during a fishery vessel outbreak with high attack rate. J Clin Microbiol. (2020) 58:e02107–20. doi: 10.1128/JCM.02107-20 PubMed Abstract | CrossRef Full Text | Google Scholar 18. Krammer F. SARS-CoV-2 vaccines in development. Nature. (2020) 586:516–27. doi: 10.1038/s41586-020-2798-3 CrossRef Full Text | Google Scholar 19. Arts EJ, Hazuda DJ. HIV-1 antiretroviral drug therapy. Cold Spring Harb Perspect Med. (2012) 2:a007161. doi: 10.1101/cshperspect.a007161 CrossRef Full Text | Google Scholar 20. Li G, De Clercq E. Current therapy for chronic hepatitis C: the role of direct-acting antivirals. Antiviral Res. (2017) 142:83–122. doi: 10.1016/j.antiviral.2017.02.014 PubMed Abstract | CrossRef Full Text | Google Scholar 21. Sharma D, Kunamneni A. Recent progress in the repurposing of drugs/molecules for the management of COVID-19. Expert Rev Anti Infect Ther. (2020) 1–9. doi: 10.1080/14787210.2021.1860020 PubMed Abstract | CrossRef Full Text | Google Scholar 22. Gao Y, Yan L, Huang Y, Liu F, Zhao Y, Cao L, et al. Structure of the RNA-dependent RNA polymerase from COVID-19 virus. Science. (2020) 368:779–82. doi: 10.1126/science.abb7498 PubMed Abstract | CrossRef Full Text | Google Scholar 23. Lan J, Ge J, Yu J, Shan S, Zhou H, Fan S, et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature. (2020) 581:215–20. doi: 10.1038/s41586-020-2180-5 PubMed Abstract | CrossRef Full Text | Google Scholar 24. Zhang L, Lin D, Sun X, Curth U, Drosten C, Sauerhering L, et al. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved a-ketoamide inhibitors. Science. (2020) 368:409–12. doi: 10.1126/science.abb3405 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: COVID-19, repurposed drugs, treatment efficacy, HIV, HCV Citation: Martinez MA (2021) Lack of Effectiveness of Repurposed Drugs for COVID-19 Treatment. Front. Immunol. 12:635371. doi: 10.3389/fimmu.2021.635371 Received: 01 December 2020; Accepted: 19 February 2021; Published: 11 March 2021. Edited by: Reviewed by: Copyright © 2021 Martinez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Miguel Angel Martinez, [email protected]
Le Anh Tuan, Bui Son Nhat, Nguyen Hong Long, Nguyen Thi Ngan, Nguyen Thi Lien Huong, Le Thi Luyen
VNU Journal of Science: Medical and Pharmaceutical Sciences, Volume 37; https://doi.org/10.25073/2588-1132/vnumps.4278

Abstract:
The aims of this systematic review are to provide knowledge concerning population pharmacokinetics of isoniazid (INH) and to identify factors influencing INH pharmacokinetic variability. Pubmed and Embase databases were systematically searched from inception to July, 2017. Relevant articles from reference lists were also included. All population pharmacokinetic studies of INH written in English, conducted in human (either healthy subjects or pulmonary tuberculosis patients) were included in this review. Ten studies were included in this review. Most studies characterized a two-compartment model with first-order kinetics for INH with a transit-compartment model for absorption suggested. Frequently reported significant predictors for INH clearance is NAT2 acetylator types (slow/intermediate/fast), while weight is a significant covariate for INH volume of distribution (both central and peripheral). In children, enzyme maturation had a profound affect on INH clearance. Keywords: Population pharmacokinetics, Isoniazid. References [1] World Health Organization, Global Tuberculosis Report 2019. https://apps.who.int/iris/bitstream/handle/10665/329368/9789241565714-eng.pdf (accessed 18 December 2019).[2] United Nations, Transforming our world: The 2030 agenda for sustainable development, New York, USA, 2015.[3] K. Takayama, L. Wang, H.L. David, Effect of isoniazid on the in vivo mycolic acid synthesis, cell growth, and viability of Mycobacterium tuberculosis, Antimicrob Agents Chemother 2.1 (1972) 29-35. https://doi.org/10.1128/aac.2.1.29 [4] A. Jindani, V.R. Aber, E. A. Edwards, D. A. Mitchison, The early bactericidal activity of drugs in patients with pulmonary tuberculosis. Am Rev Respir Dis 121(6) (1980) 939-49. https://doi.org/10.1164/arrd.1980.121.6.939 [5] P.R. Donald, The influence of human N-acetyltransferase genotype on the early bactericidal activity of isoniazid. Clin Infect Dis 39(10) (2004) 1425-30. https://doi.org/10.1086/424999 [6] D.A. Mitchison, Basic mechanisms of chemotherapy, Chest 76(6 Suppl) (1979) 771-81. https://doi.org/10.1378/chest.76.6_supplement.771 [7] H. McIlleron et al., Determinants of rifampin, isoniazid, pyrazinamide, and ethambutol pharmacokinetics in a cohort of tuberculosis patients, Antimicrob Agents Chemother 50(4) (2006) 1170-7. https://doi.org/10.1128/aac.50.4.1170-1177.2006 [8] S. Chideya et al., Isoniazid, rifampin, ethambutol, and pyrazinamide pharmacokinetics and treatment outcomes among a predominantly HIV-infected cohort of adults with tuberculosis from Botswana, Clin Infect Dis 48(12) (2009) 1685-94. https://doi.org/10.1086/599040 [9] N. Singh et al., Study of NAT2 gene polymorphisms in an Indian population: association with plasma isoniazid concentration in a cohort of tuberculosis patients. Mol Diagn Ther 13(1) (2009) 49-58. https://doi.org/10.1007/bf03256314 [10] N. Buchanan, C. Eyberg, M.D. Davis, Isoniazid pharmacokinetics in kwashiorkor. S Afr Med J 56(8) (1979) 299-300.[11] U.S. Food and Drug Administration (1999), "Guidance for Industry. Populationpharmacokinetics",Retrieved from http://www.fda.gov/downloads/Drugs/.../Guidances/UCM072137.pdf[12] D. R Mould, R. N. Upton, Basic concepts in population modeling, simulation, and model‐based drug development, CPT: pharmacometrics & systems pharmacology 1(9) (2012) 1-14. https://doi.org/10.1038/psp.2012.4 [13] P. Denti et al., Pharmacokinetics of isoniazid, pyrazinamide, and ethambutol in newly diagnosed pulmonary TB patients in Tanzania, PLoS ONE 10(10) (2015), e0141002. https://doi.org/10.1371/journal.pone.0141002 [14] B. Guiastrennec et al., Suboptimal Antituberculosis Drug Concentrations and Outcomes in Small and HIV-Coinfected Children in India: Recommendations for Dose Modifications, Clin Pharmacol Ther 104(4) (2017), 733-741. https://doi.org/10.1002/cpt.987 [15] M. Kinzig-Schippers et al., Should we use N-acetyltransferase type 2 genotyping to personalize isoniazid doses?, Antimicrobial Agents and Chemotherapy 49(5) (2005), 1733-1738. https://doi.org/10.1128/aac.49.5.1733-1738.2005 [16] J.J. Kiser et al., Isoniazid pharmacokinetics, pharmacodynamics, and dosing in South African infants, Therapeutic Drug Monitoring 34(4) (2012) 446-451. https://doi.org/10.1097/ftd.0b013e31825c4bc3 [17] L. Lalande, Population modeling and simulation study of the pharmacokinetics and antituberculosis pharmacodynamics of isoniazid in lungs, Antimicrobial Agents and Chemotherapy 59(9) (2015) 5181-5189. https://doi.org/10.1128/aac.00462-15 [18] C. Magis-Escurra et al., Population pharmacokinetics and limited sampling strategy for first-line tuberculosis drugs and moxifloxacin, International Journal of Antimicrobial Agents 44(3) (2014) 229-234. https://doi.org/10.1016/j.ijantimicag.2014.04.019 [19] C.A. Peloquin et al., Population pharmacokinetic modeling of isoniazid, rifampin, and pyrazinamide, Antimicrobial Agents and Chemotherapy 41(12) (1997) 2670-2679. https://doi.org/10.1128/aac.41.12.2670 [20] K.Y. Seng et al., Population pharmacokinetic analysis of isoniazid, acetylisoniazid, and isonicotinic acid in healthy volunteers, Antimicrobial Agents and Chemotherapy 59(11) (2015) 6791-6799. https://doi.org/10.1128/aac.01244-15 [21] J.J. Wilkins et al., Variability in the population pharmacokinetics of isoniazid in South African tuberculosis patients, British Journal of Clinical Pharmacology 72(1) (2011) 51-62. https://doi.org/10.1111/j.1365-2125.2011.03940.x [22] S.P. Zvada et al., Population pharmacokinetics of rifampicin, pyrazinamide and isoniazid in children with tuberculosis: In silico evaluation of currently recommended doses, Journal of Antimicrobial Chemotherapy 69(5) (2014) 1339-1349. https://doi.org/10.1093/jac/dkt524 [23] World Health Organization, Guidance for national tuberculosis programmes on the management of tuberculosis in children (No. WHO/HTM/TB/2014.03). World Health Organization, 2014.[24] World Health Organization, & Stop TB Initiative (World Health Organization), Treatment of...
, George M. Eliopoulos
Antimicrobial Agents and Chemotherapy, Volume 65; https://doi.org/10.1128/aac.02295-20

Abstract:
Since its inaugural issue nearly half a century ago, Antimicrobial Agents and Chemotherapy has served as a premier source for reports on scientific and clinical advances in the field of antimicrobial chemotherapy. As a follow-up to the previous “History of Antimicrobial Agents and Chemotherapy from 1972 to 1998” written by George A. Jacoby (Antimicrob Agents Chemother 43:999–1002, 1999, https://doi.org/10.1128/AAC.43.5.999 ), we herein highlight the further evolution of this comprehensive and authoritative journal in response to changing science, demographics, and information technology.
Published: 11 February 2021
Frontiers in Animal Science, Volume 2; https://doi.org/10.3389/fanim.2021.650324

Abstract:
Animal agriculture is a vital social and economic component of human communities, supplying them with food, labor, companionship, and raw material for a myriad of goods. The demand for animal protein and animal products is rapidly growing, given the continued world population growth as well as rising incomes and urbanization, more notably in developing countries (FAO, 2018). However, meeting such an upward demand should comply with moral and economic constraints related to animal welfare and the sustainability of agricultural enterprises while also reducing the environmental impact of animal production systems (FAO, 2017). Precision livestock farming (PLF) proposes to address this challenge by applying technology within the animal space for automated and real-time decision making at the individual animal and group level in livestock production (Berckmans, 2017; Benjamin and Yik, 2019). Data collected by sensors (such as cameras, microphones, accelerometers, gas analyzers, and spectrometers) on animals or on their environment, coupled with advanced analytical techniques, provide efficient tools to monitor animals to improve their welfare and optimize resource use, such as feed, water, land, and human labor. Traditionally, in livestock production, the management unit is generally a herd or flock, e.g., a group of animals in a paddock or housed together within a facility. Typical examples are large beef cattle production in either feedlots or grazing systems and modern production of pigs and poultry. Animal caretakers still try to monitor the condition of individual animals in terms of their overall health, well-being, and performance, but this is quite often a difficult task when working with large groups of animals and with limited time. Nonetheless, ideally, management practices should be efficient enough for early detection of diseases, even if subclinical, and tailored for individual animal needs to maximize animal welfare, reproductive, and production performance, as well as homogeneity within groups of pen mate animals. Precision livestock farming technologies can alert the animal caretakers in real-time to deliver individualized care to an animal showing altered behavior as a result of disease, injury, or a stressor. PLF is also useful for many other applications to improve the efficiency of livestock operations, for e.g., to detect estrus in beef and dairy cattle for optimal reproductive management of herds, precision feeding by monitoring daily feed intake and weight gain, etc. In addition, PLF systems can measure novel phenotypes or indicator traits to be used in advanced breeding programs (Rosa, 2011). Phenotypes of economic or societal importance that are generally difficult to measure without digital technologies include, for e.g., individual feed consumption and feed efficiency, heat tolerance, mothering ability, aggressive behavior, disease resistance, among many others. Moreover, the collection of individual animal data in commercial farms provides an in-depth look at the interaction between environment (stocking density, lighting, temperature, etc.) and behavior and performance of individual animals within the space. This can help the genetic improvement of adapted animals for different environments and the development of more efficient animal facilities and equipment for enhanced animal welfare and performance. Despite the potential of PLF to increase the efficiency and sustainability of livestock production systems, on-farm adoption of PLF is still incipient. Nonetheless, the trend is that PLF will be more broadly adopted as the price of sensor technologies decreases, and the animal science community develops groundbreaking basic and translational research for efficient use of PLF. The key to PLF is real-time data collection on either individual animals or groups of animals with little or no disruption of animal activities. Monitoring groups of animals or their environment requires sensors strategically positioned within their spaces (e.g., barns and paddocks), while monitoring individuals animals involves small wearable sensors integrated with animal identification and video or image analysis of the animals. Gathered data need to be processed through sophisticated machine learning and statistical analyses, which also involves efficient data management, including data transfer, storage, and editing. Lastly, independently of how modern, sophisticated, and efficient any specific PLF tool is, and its potential to improve lives of animals and animal caretakers, there is also another fundamental component for the success of PLF applications, which is the economic viability of each specific tool, and its accessibility and acceptability by farmers and farmworkers. In this context, the development and successful application of PLF tools should involve a truly multidisciplinary approach for research and development as well as extension and outreach activities at universities, research institutions, and agriculture industries. To deal with such complexities and challenges posed by PLF, a joint effort is required by individuals from various disciplines and expertise, including animal scientists and veterinarians, electrical engineers and computer scientists, data scientists, agricultural and biological systems engineers, economists, and sociologists, among others. Certainly, there are still many challenges that must be tackled so that PLF can be more widely used for different livestock species, and different production systems. First, PLF tools should be designed according to the reality of each country or global region, not only in terms of the specific climate and weather conditions, but also farm sizes and technological level, labor availability and labor training, market requirements, etc. For example, while electricity, Internet, and cell phone signal may be more broadly available in rural areas of developed countries, this is certainly not the case in many developing regions of the world. Also, countries can have vastly different labor force participation rates in agriculture, so that PLF and farm automation can affect them differently. Another challenge for PLF is the continuous development of efficient and reliable sensors (in terms of sensitivity to gather the intended signal, durability, cost, miniaturization, and efficiency of power utilization) as well as Internet of Things (IoT) systems to integrate PLF tools and to provide efficient data transmission and data storage, including data security and privacy. All this requires dedicated professionals in the areas of electrical engineering and computer science, biological systems engineering, among others, with a good understanding of farm production systems and livestock biology. The potential amount of data generated through the use of sensors and camera technologies is unprecedented. Sensor technologies can generate multiple data points per animal daily. For example, consider a PLF device based on a camera sensor capturing a single image of 5 megabytes every second from a pen or individual animal. In a single day, there would be 86,400 images for a total of 432 gigabytes, or about 13 terabytes per month. This definitely poses a tremendous challenge from a data transmission and data storage standpoint, and so computer science expertise is essential to optimize such systems. In terms of data analysis, careful data editing and quality control protocols, and sophisticated machine learning algorithms and statistical models are required to deal with such multidimensional datasets, involving complex interdependencies between interacting variables measured over time, including missing data and potential recording errors, confounding effects, and redundancies. Most applications of sensor technologies involve a prediction task, so techniques of machine learning and pattern recognition, time series, and cross-validation are extremely important. The power of information in more complex data streams, as in computer vision systems, has been harvested through deep learning algorithms, which have been reported as extremely useful analytical tools (Fernandes et al., 2020). Lastly, another critical component of PLF is how to best utilize the additional information and insight provided by sensors and related predictive algorithms for optimal data-driven decisions. Some PLF applications are straightforward in this regard, for e.g., sensors used for animal identification or heat detection in cattle for insemination decisions. Other applications, however, will provide an opportunity for a complete paradigm shift in terms of animal care. For example, years of research in veterinary medicine allowed the development of efficient protocols for treatment of animals diagnosed with specific diseases, including for example the choice of antibiotics, the appropriate dose and treatment period. Suppose now that a PLF tool allows the detection of a particular disease a few days in advance, potentially at its very early stages of development. This may provide a huge opportunity to develop new protocols of preventive medicine and treatment, to minimize the impact on the individual and to prevent or slow the spread of the disease through the herd/flock. More importantly, the earlier detection of diseases may reduce the antibiotic doses or even motivate the development of antibiotic-free treatments. Another example is in the context of feeding. Nowadays, most commercial livestock operations manage animals on a group (or pen) basis only, with diet composition and feed delivery tailored for the group average. However, due to animal-to-animal variation, some individuals may be underfed while others may be overfed. Nonetheless, with the advent of PLF technology that allows monitoring of individual animal feed intake as well as body weight gain and development, diets might be mixed in real-time and delivered precisely according to each animal's need, maximizing animal performance, and minimizing feed losses (Pomar and Remus, 2019). PLF advancements will then contribute decisively to the implementation of optimal “individualized” treatment of animals, improving upon the traditional “pen-alyzed” approach based on the group average. Surely, PLF tools will open new avenues for a myriad of opportunities for advances in livestock production and animal care, which will only be fully accomplished with dedicated and innovative research. This is precisely the main goal of Frontiers in Animal Science | Precision Livestock Farming (PFL), i.e., to provide an open forum for dissemination and discussion of scientific research findings on the development and application of PFL tools to advance the efficiency of livestock systems while improving animal welfare and reducing the environmental footprint of animal agriculture. Manuscripts contributing to the advance of any aspect of PLF are welcome, including hypothesis-driven research in precision feeding, reproduction, and overall management of animals and animal systems, animal behavior and well-being, as well as research on social and economic implications of PLF adoption, the development of innovative technologies and novel statistical and computational algorithms for implementing PLF tools. The author confirms being the sole contributor of this work and has approved it for publication. The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author acknowledges support from the Wisconsin Agriculture Experiment Station Hatch grant (142-AAC7939) and the United States Department of Agriculture (USDA). Benjamin, M., and Yik, S. (2019). Precision livestock farming in swine welfare: a review for swine practitioners. Animals 9:133. doi: 10.3390/ani9040133 PubMed Abstract | CrossRef Full Text | Google Scholar Berckmans, D. (2017). General introduction to precision livestock farming. Anim. Front. 7:6. doi: 10.2527/af.2017.0102 CrossRef Full Text | Google Scholar FAO (2017). Livestock Solutions for Climate Change. FAO. Available online at: http://www.fao.org/3/a-i8098e.pdf FAO (2018). “Shaping the future of livestock sustainably, responsibly, efficiently,” in The 10th Global Forum for Food and Agriculture. Berlin: FAO. Available online at: http://www.fao.org/3/i8384en/I8384EN.pdf Fernandes, A. F. A., Dórea, J. R. R., and Rosa, G. J. M. (2020). Image analysis and computer vision applications in animal sciences: an overview. Front. Vet. Sci. 7:551269. doi: 10.3389/fvets.2020.551269 PubMed Abstract | CrossRef Full Text | Google Scholar Pomar, C., and Remus, A. (2019). Precision pig feeding: a breakthrough toward sustainability. Anim. Front. 9, 52–55. doi: 10.1093/af/vfz006 PubMed Abstract | CrossRef Full Text | Google Scholar Rosa, G. J. M. (2011). Grand challenge in livestock genomics: for food, for medicine, for the environment, for knowledge. Front. Gene. 2:34. doi: 10.3389/fgene.2011.00034 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: digital agriculture, sensor technology, computer vision, animal welfare, sustainability Citation: Rosa GJM (2021) Grand Challenge in Precision Livestock Farming. Front. Anim. Sci. 2:650324. doi: 10.3389/fanim.2021.650324 Received: 06 January 2021; Accepted: 21 January 2021; Published: 11 February 2021. Edited by: Reviewed by: Copyright © 2021 Rosa. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Guilherme J. M. Rosa, [email protected]
, Franca Marino, Isabel Varela-Nieto,
Published: 9 February 2021
Frontiers in Neurology, Volume 12; https://doi.org/10.3389/fneur.2021.635359

Abstract:
Editorial on the Research TopicNeuroimmunology of the Inner Ear Although the term was first officially used in 1982 (1), neuroimmunology is now a mature field that has gained immense traction in the past decade. Thanks to novel technological advances, the cellular and molecular mechanisms that mediate the crosstalk between the immune and nervous systems are increasingly appreciated in both physiological and pathological states (1). Similar to the brain, the inner ear has long been considered an “isolated” system devoted to auditory and vestibular signal processing and protected by a blood-labyrinth barrier (BLB) (2–5), and the early neuroimmunology of the inner ear was mainly focused on autoimmunity (6, 7), and on the role of macrophages in cochlear damage (8, 9). In parallel to the brain, awareness about non-neural cells and molecules affecting inner ear functions has been steadily growing1, and neuro-immunological studies of the inner ear face multiple challenges, including an overwhelming number of cellular and molecular interactions, which will require a systems biology approach to grasp their full functionality. In addition, the inner ear poses unique difficulties due to its tight bone encasing and complex fluid regulation. Like most organs, including the brain, the inner ear immune cells are dominated by several populations of macrophages [reviewed in (10, 11)], which largely contribute to both inflammatory/phagocytic and regenerative/protective responses. However, several questions are still open, such as: • What are the signals exchanged between immune cells and inner ear cells in healthy and pathological settings? • How much communication is there between the inner ear and surrounding tissues and fluids? • What is the neuroimmune role of the endolymphatic sac? • What are the roles, nature, and location of several immune cell populations and subpopulations, e.g., mast cells (12), lymphocytes (13), or other leukocytes (14)? • How are local and systemic immune responses regulated—and especially dysregulated- in various kinds of damage (e.g., infection, noise trauma, and ototoxicity)? • How do neuroimmune interactions translate in the modulation of inner ear functions? The articles collected in this Research Topic reflect this increasingly diverse field in several main threads, broadly divided into immune cell characterization, responses to diseases or damage, biomarkers, and immune molecular targets (Figure 1). Both human and animal studies are included in this Special Topic. The system's complexity at both cellular and functional levels requires the integration of invasive approaches only possible in animals and testing of functions and markers that may be incompletely overlapping between humans and other animals. Table 1 summarizes the main points that were contributed by human and animal studies within this Research Topic. Figure 1. Neuroimmunology of the inner ear. Visual abstract of this Research Topic: each paper within this Topic explores molecules, cells and/or functions related to inner ear neuroimmune interactions. HL, hearing loss; ES, endolymphatic sac; SCC, semicircular canals. Created with Biorender.com. Table 1. A summary of data published in the Research Topic “Neuroimmunology of the Inner Ear” based on the type of manuscript and the research model used. Five papers published in this Research Topic focus on the description of the immune cells of the inner ear, with a predominance of studies regarding cochlear macrophages, consistent with their role as leading players both by number and function, as reviewed in (10, 11). The paper “Early Development of Resident Macrophages in the Mouse Cochlea Depends on Yolk Sac Hematopoiesis” describes the presence of two resident macrophage populations in the mouse cochlea, derived respectively from the yolk sac and fetal liver (Kishimoto et al.). These populations are different in their Csf1-dependence and final cochlear localization. The origin of macrophages may be reflected in their various shapes in the adult cochlea, as seen in human samples in the following two papers. The first, “Age-Related Changes in Immune Cells of the Human Cochlea,” describes macrophage populations associated with various parts of the human cochlea and the aging-dependent changes occurring in these cells (Noble et al.). Similarly, the paper “Human Inner Ear Immune Activity: A Super-Resolution Immunohistochemistry Study,” describes immune cells (mainly macrophages and to a lesser extent T lymphocytes) associated with the human cochlea and endolymphatic sac (Liu et al.). Both papers observed an association between cell shapes and their localization in the inner ear. Finally, two groups studied macrophage-related gene knock-out effects on the inner ear function and anatomy using the mouse model. “Csf1 Signaling Regulates Maintenance of Resident Macrophages and Bone Formation in the Mouse Cochlea” underlines the impact of the Csf1op/op genotype (where no Csf1 is produced) on cochlear bone remodeling and cochlear macrophages, therefore pointing to a possible connection between immune responses and bony capsule metabolism (Okano and Kishimoto). On the other hand, the paper “Lack of Fractalkine Receptor on Macrophages Impairs Spontaneous Recovery of Ribbon Synapses After Moderate Noise Trauma in C57BL/6 Mice” demonstrates the negative impact of knocking-out the fractalkine receptor on ribbon synapse recovery after noise trauma, suggesting a protective role for auditory nerve-associated macrophages (Kaur et al.). Eight other papers in this Research Topic studied immune responses in the inner ear in a pathological context, three of which (two in humans and one in a rat model) focus on infectious diseases. Passive barriers strongly protect the inner ear, but pathogens may enter it through its connections with CSF and middle ear, plus vascular and neural routes additionally available for viruses (15). Moreover, even systemic responses to pathogens may affect the ear indirectly, due to immune crossreactivity (as suggested after viral or fungal infection, reviewed in (16) or BLB impairment opening the way to ototoxic factors into the inner ear (17–19). The review “Main Aspects of Peripheral and Central Hearing System Involvement in Unexplained HIV-Related Hearing Complaints” presents an interesting study on the auditory consequences of HIV viral infection (which show similarities to age-induced hearing impairment), an aspect not well-understood and of enormous importance (de Jong et al.). The paper “Vertigo and Severe Balance Instability as Symptoms of Lyme Disease—Literature Review and Case Report” describes the effects of neuroborreliosis, which can target the 8th nerve, on the human vestibular system from a clinical point of view (Jozefowicz-Korczynska et al.). The work described in “Anti-inflammatory and Oto-Protective Effect of the Small Heat Shock Protein Alpha B-Crystallin (HspB5) in Experimental Pneumococcal Meningitis” demonstrates in a rat model that inner ear damage due to meningococcal infection goes together with an increase of proinflammatory cytokines in CSF, and rise in the numbers of cochlear neutrophils and macrophages (Erni et al.). The cytokine (but not the cellular) response could be reduced by intracisternal injection with the small heat shock protein alpha B-crystallin (HspB5), which also ameliorated hearing loss. Besides infections, the inner ear can be affected by exposure to stress factors (including noise, drugs, and surgery), aging, genetic defects, or pathologies of unclear etiology, such as Menière's disease (MD). Many of these settings are accompanied by inflammation—a classical response to damage that is beneficial per se but may become detrimental if dysregulated, further damaging the inner ear [reviewed in (20, 21)]. Moreover, inflammation related to invasive inner ear intervention, such as cochlear implantation (CI), may lead to fibrosis and bone neoformation (22, 23), which degrade residual hearing. An effective otoprotective strategy in CI is the use of dexamethasone-eluting electrodes (24–26). However, understanding the otoprotective mechanisms of dexamethasone in CI is difficult since steroids can block all inflammatory response phases (20). Interestingly, CI influences the composition of macrophages subsets in animals (27, 28) and humans (29, 30). In the animal model, where the entire inflammatory process can be followed, macrophages have been found in the inflammatory, cytotoxic phenotype (27), and in the reparative phenotype, which is associated to matrix deposition and remodeling, and therefore to fibrosis (28). Moreover, a protective macrophage subpopulation has been observed in rodents' spiral ganglion (31) and suggested to exist in humans (29). The foreign-body responses may also induce the macrophages to form giant multinucleated cells with osteoblast-like properties (32). Finally, CI may also recruit B and T lymphocytes (33). The manuscript “Immune Response After Cochlear Implantation” shows that dexamethasone-eluting electrodes reduce both the cellular and cytokine signature of acute inflammatory response and fibrosis associated with implantation in a guinea pig, a well-established animal model for CI (Simoni et al.). The paper “Genetic Hearing Loss Associated With Autoinflammation” describes deafness correlated with mutations affecting the NLRP3 inflammasome and other genes that influence macrophages (Nakanishi et al.). Inflammation of strial blood vessels was suggested, among other possible causes, in ototoxicity secondary to methadone treatment in humans in the paper “Early Diagnosis of Hearing Loss in Patients Under Methadone Maintenance Treatment” by Bayat et al.. Finally, two papers focus on the immune-inflammatory response in Menière's disease. As of today, MD diagnosis and monitoring are based on clinical symptoms, and no selective biomarker is available (34, 35). Endolymphatic hydrops has been long associated (although not exclusively) with MD, and endolymph-producing and reabsorbing structures in the ear are being targeted in treatment options [reviewed in (34, 35)]. Also, recent studies show a breakdown of the human utricular BLB in MD due to increased vesicular transport in endothelial cells and pericytes (36). Therefore, perilymph production appears to be affected, as well. In fact, a diagnostic tool based on gadolinium-enhanced MRI is being considered for MD (37, 38). MD etiology is still debated and includes a combination of genetic, immune, inflammatory, environmental, hemodynamic, and hormonal factors (39). Immune-inflammatory responses seem however central, given that several genes linked to MD belong to immune or inflammation pathways (40), and that a significant percentage of patients with MD is also affected by other autoimmune diseases (41), or displays enhanced and anomalous inflammatory responses (42). Moreover, the endolymphatic sac, which is involved in endolymph reabsorption and displays morphological changes in MD (43), is the primary immune structure of the inner ear (44), and inner ear vasculature permeability is strongly affected by inflammation (45). Finally, MD immune-inflammatory model explains the effectiveness of steroid treatment (46). However, as for other immune diseases, treatment of subjects non-responsive to steroids requires personalized approaches that depend on the particular pathway being deranged (40). The report “Subcellular Abnormalities of Vestibular Nerve Morphology in Patients with Intractable Menière's Disease” found the presence of structures associated to neurodegeneration (corpora amylacea, lipofuscin, microglia) in the vestibular nerve of MD patients (Wang et al.). On the other hand, the paper “Differential Proinflammatory Signature in Vestibular Migraine and Meniere Disease” describes a cytokine subset whose expression level in peripheral blood mononuclear cells can discriminate between healthy controls, vestibular migraine patients, and two subgroups of MD patients, thus yielding the basis for a blood test helping MD diagnosis (Flook et al.). Two papers focused on a hot spot in auditory research: biomarkers for various types of inner ear diseases. “Cytokine Levels in Inner Ear Fluid of Young and Aged Mice as Molecular Biomarkers of Noise-Induced Hearing Loss” approaches the connection between noise-induced and age-related hearing loss using the mouse model (Landegger et al.). The second paper, “Biomarkers in Vestibular Schwannoma–Associated Hearing Loss,” reviews the proteins and genes that could potentially be included in the clinical evaluation panel of vestibular schwannoma (Lassaletta et al.). The paper “Defining the Inflammatory Microenvironment in the Human Cochlea by Liquid Biopsy and Perilymph Analysis” studied human perilymph from patients assigned for CI, observing the expression of a panel of immune-related and growth factor-related proteins (Warnecke et al.). Finally, in the paper “Correlations Between Vestibular Function and Imaging of the Semicircular Canals in DFNA9 Patients,” MRI was used to visualize abnormalities of the semicircular canals that were attributed to cochlin deposits and fibrosis in the light of functional deficiencies (Ihtijarevic et al.). Regarding molecular targets for neuroimmune signaling in the ear, most papers in this Research Topic confirmed an essential role for TNF signaling [see discussion in (47)]. Two papers focused on that issue. “Intracochlear Perfusion of Tumor Necrosis Factor-Alpha Induces Sensorineural Hearing Loss and Synaptic Degeneration in Guinea Pigs” found that TNF-α perfusion rapidly induced synaptic loss and CAP reduction, which resembled primary cochlear neuropathy (Katsumi et al.), whereas “Nfatc4 Deficiency Attenuates Ototoxicity by Suppressing Tnf-Mediated Hair Cell Apoptosis in the Mouse Cochlea” describes the expression of Nfatc4 in the auditory hair cells being linked to TNF-dependent apoptosis (Zhang et al.). The last paper, “Cannabinoids, inner ear, hearing and tinnitus: a neuroimmunological perspective” reviews cannabinoid effects on the immune system and their possible roles in tinnitus and hearing loss, for which only neuronal effects have been considered so far (Perin et al.). By exploring several consolidated or novel mechanisms and effects of neuroimmune interactions in the inner ear, this Research Topic yields a broad perspective on possible innovative therapeutic and diagnostic approaches to audiovestibular diseases and contributes to increasing visibility of this fascinating subject. We hope that in the next decade, the current research will elucidate biochemical pathways connecting immune responses in sensory circuits to functional changes at the cellular and systems levels. Further exploration of the inner ear's neuro-immuno-sensory axis might impact future therapy and monitoring of some otological diseases. PP and AS drafted the manuscript. PP drew the figure. PP, FM, IV-N, and AS revised and approved the final version of this manuscript. All authors contributed to the article and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 1. ^PUBMED search with the query “(inner ear OR cochlea OR vestibular) AND (neuroimmune OR neuroimmunology)” only retrieves 14 results, but this reflects the still sparse use of “neuroimmune”, since the related queries “(inner ear OR cochlea OR vestibular) AND (immune OR macrophage OR cytokine)” and “(inner ear OR cochlea OR vestibular) AND (inflammatory OR inflammation)” retrieve 1,734 and 2,043 results, respectively. 1. Nutma E, Willison H, Martino G, Amor S. Neuroimmunology - the past, present and future. Clin Exp Immunol. (2019) 197:278–93. doi: 10.1111/cei.13279 CrossRef Full Text | Google Scholar 2. Jahnke K. The blood-perilymph barrier. Arch Otorhinolaryngol. (1980) 228:29–34. doi: 10.1007/BF00455891 CrossRef Full Text | Google Scholar 3. Juhn SK, Rybak LP, Fowlks WL. Transport characteristics of the blood–perilymph barrier. Am J Otolaryngol. (1982) 3:392–6. doi: 10.1016/S0196-0709(82)80016-1 CrossRef Full Text | Google Scholar 4. Cohen-Salmon M, Regnault B, Cayet N, Caille D, Demuth K, Hardelin JP, et al. Connexin30 deficiency causes instrastrial fluid-blood barrier disruption within the cochlear stria vascularis. Proc Natl Acad Sci USA. (2007) 104:6229–34. doi: 10.1073/pnas.0605108104 PubMed Abstract | CrossRef Full Text | Google Scholar 5. Shi X. Pathophysiology of the cochlear intrastrial fluid-blood barrier (review). Hear Res. (2016) 338:52–63. doi: 10.1016/j.heares.2016.01.010 PubMed Abstract | CrossRef Full Text | Google Scholar 6. Quick CA. Antigenic causes of hearing loss. Otolaryngol Clin North Am. (1975) 8:385–94. doi: 10.1016/S0030-6665(20)32776-6 CrossRef Full Text | Google Scholar 7. Tomiyama S, Harris JP. The endolymphatic sac: its importance in inner ear immune responses. Laryngoscope. (1986) 96:685–91. doi: 10.1288/00005537-198606000-00018 PubMed Abstract | CrossRef Full Text | Google Scholar 8. Hirose K, Discolo CM, Keasler JR, Ransohoff R. Mononuclear phagocytes migrate into the murine cochlea after acoustic trauma. J Comp Neurol. (2005) 489:180–94. doi: 10.1002/cne.20619 PubMed Abstract | CrossRef Full Text | Google Scholar 9. Sato E, Shick HE, Ransohoff RM, Hirose K. Repopulation of cochlear macrophages in murine hematopoietic progenitor cell chimeras: the role of CX3CR1. J Comp Neurol. (2008) 506:930–42. doi: 10.1002/cne.21583 PubMed Abstract | CrossRef Full Text | Google Scholar 10. Hirose K, Rutherford MA, Warchol ME. Two cell populations participate in clearance of damaged hair cells from the sensory epithelia of the inner ear. Hear Res. (2017) 352:70–81. doi: 10.1016/j.heares.2017.04.006 PubMed Abstract | CrossRef Full Text | Google Scholar 11. Warchol ME. Interactions between macrophages and the sensory cells of the inner ear. Cold Spring Harb Perspect Med. 9:a033555. doi: 10.1101/cshperspect.a033555 PubMed Abstract | CrossRef Full Text | Google Scholar 12. Szczepek AJ, Dudnik T, Karayay B, Sergeeva V, Olze H, Smorodchenko A. Mast cells in the auditory periphery of rodents. Brain Sci. (2020) 10:697. doi: 10.3390/brainsci10100697 PubMed Abstract | CrossRef Full Text | Google Scholar 13. Rai V, Wood MB, Feng H, Schabla NM, Tu S, Zuo J. The immune response after noise damage in the cochlea is characterized by a heterogeneous mix of adaptive and innate immune cells. Sci Rep. (2020) 10:15167. doi: 10.1038/s41598-020-72181-6 PubMed Abstract | CrossRef Full Text | Google Scholar 14. Hu BH, Zhang C, Frye MD. Immune cells and non-immune cells with immune function in mammalian cochleae. Hear Res. (2018) 362:14–24. doi: 10.1016/j.heares.2017.12.009 PubMed Abstract | CrossRef Full Text | Google Scholar 15. Dewyer NA, Kiringoda R, Mckenna MJ. Inner Ear Infections (Labyrinthitis). In: Durand M, Deschler D, editors. Infections of the Ears, Nose, Throat, and Sinuses. Cham: Springer (2018). p. 79–88. doi: 10.1007/978-3-319-74835-1_7 CrossRef Full Text | Google Scholar 16. Buki B, Junger H, Zhang Y, Lundberg YW. The price of immune responses and the role of vitamin D in the inner ear. Otol Neurotol. (2019) 40:701–9. doi: 10.1097/MAO.0000000000002258 PubMed Abstract | CrossRef Full Text | Google Scholar 17. Oh GS, Kim HJ, Choi JH, Shen A, Kim CH, Kim SJ, et al. Activation of lipopolysaccharide-TLR4 signaling accelerates the ototoxic potential of cisplatin in mice. J Immunol. (2011) 186:1140–50. doi: 10.4049/jimmunol.1002183 PubMed Abstract | CrossRef Full Text | Google Scholar 18. Hirose K, Li SZ, Ohlemiller KK, Ransohoff RM. Systemic lipopolysaccharide induces cochlear inflammation and exacerbates the synergistic ototoxicity of kanamycin and furosemide. J Assoc Res Otolaryngol. (2014) 15:555–70. doi: 10.1007/s10162-014-0458-8 PubMed Abstract | CrossRef Full Text | Google Scholar 19. Koo JW, Quintanilla-Dieck L, Jiang M, Liu J, Urdang ZD, Allensworth JJ, et al. Endotoxemia-mediated inflammation potentiates aminoglycoside-induced ototoxicity. Sci Transl Med. (2015) 7:298ra118. doi: 10.1126/scitranslmed.aac5546 PubMed Abstract | CrossRef Full Text | Google Scholar 20. Kalinec GM, Lomberk G, Urrutia RA, Kalinec F. Resolution of cochlear inflammation: novel target for preventing or ameliorating drug-, noise- and age-related hearing loss. Front Cell Neurosci. (2017) 11:192. doi: 10.3389/fncel.2017.00192 PubMed Abstract | CrossRef Full Text | Google Scholar 21. Wood MB, Zuo J. The contribution of immune infiltrates to ototoxicity and cochlear hair cell loss. Front Cell Neurosci. (2017) 11:106. doi: 10.3389/fncel.2017.00106 PubMed Abstract | CrossRef Full Text | Google Scholar 22. Fayad JN, Makarem AO, Linthicum FHJr. Histopathologic assessment of fibrosis and new bone formation in implanted human temporal bones using 3D reconstruction. Otolaryngol Head Neck Surg. (2009) 141:247–52. doi: 10.1016/j.otohns.2009.03.031 PubMed Abstract | CrossRef Full Text | Google Scholar 23. Jia H, Wang J, Francois F, Uziel A, Puel JL, Venail F. Molecular and cellular mechanisms of loss of residual hearing after cochlear implantation. Ann Otol Rhinol Laryngol. (2013) 122:33–9. doi: 10.1177/000348941312200107 PubMed Abstract | CrossRef Full Text | Google Scholar 24. Astolfi L, Simoni E, Giarbini N, Giordano P, Pannella M, Hatzopoulos S, et al. Cochlear implant and inflammation reaction: Safety study of a new steroid-eluting electrode. Hear Res. (2016) 336:44–52. doi: 10.1016/j.heares.2016.04.005 PubMed Abstract | CrossRef Full Text | Google Scholar 25. Bas E, Bohorquez J, Goncalves S, Perez E, Dinh CT, Garnham C, et al. Electrode array-eluted dexamethasone protects against electrode insertion trauma induced hearing and hair cell losses, damage to neural elements, increases in impedance and fibrosis: a dose response study. Hear Res. (2016) 337:12–24. doi: 10.1016/j.heares.2016.02.003 PubMed Abstract | CrossRef Full Text | Google Scholar 26. Wilk M, Hessler R, Mugridge K, Jolly C, Fehr M, Lenarz T, et al. Impedance changes and fibrous tissue growth after cochlear implantation are correlated and can be reduced using a dexamethasone eluting electrode. PLoS ONE. (2016) 11:e0147552. doi: 10.1371/journal.pone.0147552 PubMed Abstract | CrossRef Full Text | Google Scholar 27. Bas E, Gupta C, Van De Water TR. A novel organ of corti explant model for the study of cochlear implantation trauma. Anat Rec. (2012) 295:1944–56. doi: 10.1002/ar.22585 PubMed Abstract | CrossRef Full Text | Google Scholar 28. Bas E, Goncalves S, Adams M, Dinh CT, Bas JM, Van De Water TR, et al. Spiral ganglion cells and macrophages initiate neuro-inflammation and scarring following cochlear implantation. Front Cell Neurosci. (2015) 9:303. doi: 10.3389/fncel.2015.00303 PubMed Abstract | CrossRef Full Text | Google Scholar 29. Okayasu T, O'Malley JT, Nadol JB Jr. Density of macrophages immunostained with anti-iba1 antibody in the vestibular endorgans after cochlear implantation in the human. Otol Neurotol. (2019) 40:e774–81. doi: 10.1097/MAO.0000000000002313 PubMed Abstract | CrossRef Full Text | Google Scholar 30. Noonan KY, Lopez IA, Ishiyama G, Ishiyama A. Immune response of macrophage population to cochlear implantation: cochlea immune cells. Otol Neurotol. (2020) 41:1288–95. doi: 10.1097/MAO.0000000000002764 PubMed Abstract | CrossRef Full Text | Google Scholar 31. Kaur T, Zamani D, Tong L, Rubel EW, Ohlemiller KK, Hirose K, et al. Fractalkine signaling regulates macrophage recruitment into the cochlea and promotes the survival of spiral ganglion neurons after selective hair cell lesion. J Neurosci. (2015) 35:15050–61. doi: 10.1523/JNEUROSCI.2325-15.2015 PubMed Abstract | CrossRef Full Text | Google Scholar 32. Barbeck M, Booms P, Unger R, Hoffmann V, Sader R, Kirkpatrick CJ, et al. Multinucleated giant cells in the implant bed of bone substitutes are foreign body giant cells-New insights into the material-mediated healing process. J Biomed Mater Res A. (2017) 105:1105–11. doi: 10.1002/jbm.a.36006 PubMed Abstract | CrossRef Full Text | Google Scholar 33. Nadol JB, O'Malley JT, Burgess BJ, Galler D. Cellular immunologic responses to cochlear implantation in the human. Hear Res. (2014) 318:11–17. doi: 10.1016/j.heares.2014.09.007 PubMed Abstract | CrossRef Full Text | Google Scholar 34. Magnan J, Ozgirgin ON, Trabalzini F, Lacour M, Escamez AL, Magnusson M, et al. European position statement on diagnosis, and treatment of Meniere's disease. J Int Adv Otol. (2018) 14:317–21. doi: 10.5152/iao.2018.140818 PubMed Abstract | CrossRef Full Text | Google Scholar 35. Basura GJ, Adams ME, Monfared A, Schwartz SR, Antonelli PJ, Burkard R, et al. Clinical Practice guideline: Meniere's disease. Otolaryngol Head Neck Surg. (2020) 162:S1–55. doi: 10.1177/0194599820909438 PubMed Abstract | CrossRef Full Text | Google Scholar 36. Ishiyama G, Lopez IA, Ishiyama P, Vinters HV, Ishiyama A. The blood labyrinthine barrier in the human normal and Meniere's disease macula utricle. Sci Rep. (2017) 7:253. doi: 10.1038/s41598-017-00330-5 PubMed Abstract | CrossRef Full Text | Google Scholar 37. Nakashima T, Naganawa S, Sugiura M, Teranishi M, Sone M, Hayashi H, et al. Visualization of endolymphatic hydrops in patients with Meniere's disease. Laryngoscope. (2007) 117:415–20. doi: 10.1097/MLG.0b013e31802c300c CrossRef Full Text | Google Scholar 38. Van Steekelenburg JM, Van Weijnen A, De Pont LMH, Vijlbrief OD, Bommeljé CC, Koopman JP, et al. Value of endolymphatic hydrops and perilymph signal intensity in suspected Ménière disease. AJNR Am J Neuroradiol. (2020) 41:529–34. doi: 10.3174/ajnr.A6410 PubMed Abstract | CrossRef Full Text | Google Scholar 39. Oberman BS, Patel VA, Cureoglu S, Isildak H. The aetiopathologies of Meniere's disease: a contemporary review. Acta Otorhinolaryngol Ital. (2017) 37:250–63. doi: 10.14639/0392-100X-793 PubMed Abstract | CrossRef Full Text | Google Scholar 40. Lopez-Escamez JA, Batuecas-Caletrio A, Bisdorff A. Towards personalized medicine in Meniere's disease. F1000Res. (2018) 7:F1000 Faculty Rev-1295. doi: 10.12688/f1000research.14417.1 PubMed Abstract | CrossRef Full Text | Google Scholar 41. Greco A, Gallo A, Fusconi M, Marinelli C, Macri G, De Vincentiis M. Meniere's disease might be an autoimmune condition? Autoimmun Rev. (2012) 11:731–8. doi: 10.1016/j.autrev.2012.01.004 CrossRef Full Text | Google Scholar 42. Frejo L, Gallego-Martinez A, Requena T, Martin-Sanz E, Amor-Dorado JC, Soto-Varela A, et al. Proinflammatory cytokines and response to molds in mononuclear cells of patients with Meniere disease. Sci Rep. (2018) 8:5974. doi: 10.1038/s41598-018-23911-4 PubMed Abstract | CrossRef Full Text | Google Scholar 43. Bachinger D, Luu NN, Kempfle JS, Barber S, Zurrer D, Lee DJ, et al. Vestibular aqueduct morphology correlates with endolymphatic sac pathologies in meniere's disease-a correlative histology and computed tomography study. Otol Neurotol. (2019) 40:e548–55. doi: 10.1097/MAO.0000000000002198 PubMed Abstract | CrossRef Full Text | Google Scholar 44. Kampfe Nordstrom C, Danckwardt-Lilliestrom N, Laurell G, Liu W, Rask-Andersen H. The human endolymphatic sac and inner ear immunity: macrophage interaction and molecular expression. Front Immunol. (2018) 9:3181. doi: 10.3389/fimmu.2018.03181 PubMed Abstract | CrossRef Full Text | Google Scholar 45. Trune DR, Nguyen-Huynh A. Vascular pathophysiology in hearing disorders. Semin Hear. (2012) 33:242–50. doi: 10.1055/s-0032-1315723 CrossRef Full Text | Google Scholar 46. Hao W, Yu H, Li H. Effects of intratympanic gentamicin and intratympanic glucocorticoids in Meniere's disease: a network meta-analysis. J Neurol. (2021). doi: 10.1007/s00415-020-10320-9. [Epub ahead of print]. PubMed Abstract | CrossRef Full Text | Google Scholar 47. Ren Y, Stankovic KM. The Role of Tumor Necrosis Factor Alpha (TNFalpha)in hearing loss and vestibular schwannomas. Curr Otorhinolaryngol Rep. (2018) 6:15–23. doi: 10.1007/s40136-018-0186-4 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: inner ear, immunology, neuroimmunology, balance, hearing, disorders Citation: Perin P, Marino F, Varela-Nieto I and Szczepek AJ (2021) Editorial: Neuroimmunology of the Inner Ear. Front. Neurol. 12:635359. doi: 10.3389/fneur.2021.635359 Received: 30 November 2020; Accepted: 07 January 2021; Published: 09 February 2021. Edited by: Reviewed by: Copyright © 2021 Perin, Marino, Varela-Nieto and Szczepek. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Paola Perin, [email protected]; Agnieszka J. Szczepek, [email protected]
Jiao Liu, Yue-Tian Yu, Chun-Hui Xu, De-Chang Chen
Published: 4 February 2021
Frontiers in Medicine, Volume 7; https://doi.org/10.3389/fmed.2020.598037

Abstract:
Candida spp. is one of the most important components of human microecology. Among hospitalized patients, the isolation rate of Candida spp. by active screening is about 15%, while in critically ill patients, the rate can be as high as 25% (1). Although microbial colonization plays an important role in secondary infections, Candida pneumonia is seldom documented even in the intensive care unit (ICU). Thus, the common consensus is that anti-Candida therapy is rarely necessary in most cases and it should be considered as colonization in which Candida spp. are isolated from the respiratory tract (RT) (2). The co-existence of bacteria and fungi has raised great concern in the last decade. It has been indicated by some studies that Candida colonization in the RT might be an independent risk factor that could promote ventilator-associated pneumonia (VAP) and even change the antibiotic resistance patterns of pathogenic bacteria by polymicrobial biofilm formation (3, 4). Therefore, the significance of Candida colonization in RT remains controversial, and many clinical problems need to be reinterpreted. The rate of Candida spp. isolation in the RT is relatively high, especially in those with mechanical ventilation (MV) (3). However, whether VAP can be caused by Candida spp. remains controversial and the main reasons for this are listed as follows: (1) No matter what the pathogenic microorganism is, the diagnosis of VAP is still difficult due to the lack of pathological evidence. The clinical diagnostic criteria for suspected VAP are not specific, and it is difficult to distinguish between colonization and infection (5). (2) The understanding of the importance of bacterial and fungal co-existence is not deep enough. Some microbiological laboratories have not conducted further analysis when fast-growing Candida spp. are isolated from RT samples. What's more, only filamentous fungi isolation were reported in some institutions (6). (3) It is widely accepted that the cutoff value for the number of pathogenic bacteria for VAP diagnosis is 103 cfu/mL (protected specimen brush sample) or 104 cfu/mL (bronchoalveolar lavage fluid sample), but such a threshold has not yet been established for Candida (5). Therefore, Candida pneumonia must be diagnosed by histopathology. Hence, it is generally thought that Candida pneumonia is quite rare in the ICU, and the guidelines for the management of Candida spp. of both the IDSA and ESCMID do not recommend antifungal treatment unless there is clear histological evidence of infection (2, 7). Alveolar macrophages act as the first line of defense against Candida in critically ill patients. Toll-like receptor (TLR) induces a Th1 cytokine pattern to increase the levels of IFN-γ and TNF-α to facilitate the clearance of Candida spores from the alveoli. What is more, other researches have also indicated that IFN-γ favors the intracellular killing of the fungus after internalization in professional phagocytes (8). Thus, it can be inferred that Candida pneumonia may not exist in the ICU. An autopsy study with 135 patients who died of pneumonia showed that among them, 77 (57%) severely affected patients had Candida airway colonization during their hospital stay. However, none of these cases was pathologically confirmed as Candida pneumonia (9). Meanwhile, one controlled before-after study in a microbiology laboratory at Illinois University showed that limiting the identification of respiratory secretions (only filamentous fungi were reported) could reduce the prescription of antifungal drug treatment (21 vs. 39%) and shorten the length of hospital stay (10.1 vs. 12.1 days) compared with full identification (all rapidly growing yeasts were reported), p< 0.05 (6). What should ICU physicians do when they receive a microbial culture report which indicates that Candida spp. are growing fast in airway secretions? The practice guidelines recommend that antifungal therapy should not be routinely used in those with Candida airway colonization (2, 7). However, should Candida colonization in the airway of critically ill patients simply be ignored? Some in vitro experiments on the co-existence of bacteria and fungi came to different conclusions. The cell wall of Candida spp. is combined with polysaccharides and proteins. Among them, Beta-glucan (BG) is a proinflammatory factor that can cause dysfunction of macrophages and neutrophils in alveoli as well as reduce the production of reactive oxygen species (10). It is also reported that there is a strong interaction among Candida, Gram-positive and Gram-negative bacteria through quorum sensing (QS) molecules, and the extensive interaction of metabolic processes and intercellular communication among them are the basis of synergistic and antagonistic interactions (11). Through an observational study of rats injected with active Candida albicans, it was found that the increased production of cellular inflammatory factors, including interleukin-6, interferon-γ and tumor necrosis factor-α, inhibited phagocytosis by alveolar macrophages. This phenomenon led to changes in airway microecology, and an increase in the airway colonization rate of Pseudomonas aeruginosa was found (12). Moreover, this effect was not unique to Pseudomonas aeruginosa. Another study showed that Candida colonization was also beneficial for the colonization of Staphylococcus aureus and Enterobacteriaceae, which led to an increase in bacterial pneumonia (13). Candida biofilms show a reticular structure composed of Candida spores and hyphae and are easily found on the surfaces of artificial materials (such as endotracheal tubes). The biofilm matrix contains polysaccharides, proteins and other unknown components, which show strong adhesion and are difficult to remove (14) (Figure 1). Biofilms not only have a protective effect on Candida but also have a strong adsorption effect on co-existing bacteria. Animal experiments and electron microscopic studies show that bacteria and fungi can produce small molecules to interact with each other and change their morphology, function and growth environment, resulting in bacteria that are firmly adsorbed between Candida spores or biofilms. Such structures are difficult to remove. Even though the spore activity of some Candida spp. is decreased, the adsorption phenomenon is still observed (4, 15). Figure 1. Interaction of Candida spp. and bacteria in patients with mechanical ventilation. Candida biofilms are easily found on the respiratory tract or the surfaces of endotracheal tubes. Biofilms not only have a protective effect on Candida but also have a strong adsorption effect on co-existing bacteria. Multidrug-resistant bacteria could be isolated by the transmission of drug-resistant plasmid transmission and polymicrobial biofilm formation (Drawn by Chunhui Xu). Candida colonization can also change the virulence and/or host immune function of colonized bacteria. A series of animal experiments have shown that after the mixed inoculation of Candida and bacteria in the airway of mice, even if the number of inoculated Candida is very small, the bacterial load still occupies a high percentage of the alveoli. It has been suggested that the presence of Candida albicans protects the bacteria from clearance by normal alveolar macrophages (16). Acinetobacter baumannii can affect the morphology of Candida albicans through the QS molecule N-acyl homoserine lactone, whereas farnesol is the main QS molecule of Candida albicans (11). This can affect the movement ability and virulence factor expression of Acinetobacter baumannii. An animal experiment has also found that the degree of alveolar invasiveness of Acinetobacter baumannii in mice with Candida colonization during pneumonia is much higher than that of Acinetobacter baumannii during pulmonary infection (17). The existence of biofilms can also increase the resistance of bacteria to antibiotics. It is showed that Staphylococcus aureus could form a single biofilm (monoculture biofilm) in serum, but its integrity was poor, and it was easy to dissociate. If there is co-growth with Candida albicans, Staphylococcus aureus can form microcolonies on the fungal biofilm, which is closely connected to the bottom hyphae “scaffold,” to form a multi-bacterial biofilm (polymicrobial biofilm) (Supplementary Figure 1). Staphylococcus aureus matrix staining showed different phenotypes of multi-bacterial biofilms and single cell membranes (18), indicating that Staphylococcus aureus may be encapsulated in the matrix secreted by Candida albicans, resulting in an increase in its resistance to vancomycin. Further studies showed that in the environment of multi-bacterial biofilm formation, 27 Staphylococcus aureus-specific proteins were identified by gel electrophoresis, some of which could upregulate the expression of L-lactate dehydrogenase I, confer the ability to resist host-derived oxidative stress to bacteria and enhance resistance to antibiotics, while other proteins could downregulate the expression of the virulence factor CodY (19). These findings suggest that the occurrence of VAP caused by MRSA in patients with Candida albicans airway colonization is not only the result of the expression of QS molecules but can also be attributed to the differential regulation of specific drug resistance genes and virulence factors. Similar results have been obtained in other studies of Gram-negative bacteria (20, 21). In vitro studies suggest that there is mutual induction of the process of the co-existence of bacteria and fungi, so it is necessary to further describe and study the complex interactions between pathogens at the molecular level. The transition from basic research to clinical research may help to design new treatment or prevention and control strategies for bacterial and fungal superinfection. Clinical studies have pointed out that the isolation rate of Candida from the RT of ICU patients with MV could be as high as 50%, which prolonged the median hospital stay (59.9 vs. 38.6 days, p = 0.006) or even increased the hospital mortality (34.2 vs. 21.0%, p = 0.003) (22). Moreover, it might be associated with persistent immunosuppression and inflammation (23). Candida airway colonization and its concomitant secretion of inflammatory factors may affect host cellular immune function, especially in immunosuppressed hosts with severe monocyte and lymphocyte dysfunction, which results in a decrease in the effective clearance of bacteria and fungi and an increase in the incidence of VAP (24). However, the effect of Candida RT colonization on bacterial colonization and antibacterial resistance patterns has always been controversial in clinical research. It is still unclear whether Candida airway colonization could increase the incidence of VAP and whether patients with Candida airway colonization can benefit from antifungal therapy (Supplementary Table 1). One early prospective cohort study reported that Candida RT colonization could increase the incidence of VAP caused by Pseudomonas aeruginosa (9 vs. 4.8%, p = 0.048), and Candida RT colonization was proven to be an independent risk factor (18). Similarly, another single-center retrospective case-control study indicated that antifungal therapy in those with Candida albicans airway colonization could prevent the occurrence of Pseudomonas aeruginosa VAP (25). Some studies have also pointed out that Candida airway colonization is associated with the pathogenesis of Acinetobacter baumannii VAP. In addition, another cohort study showed that aerosol inhalation of amphotericin B in patients with MV significantly reduced the Candida load in the airway but did not change the morbidity due to VAP or mortality during the ICU stay (26, 27). The EMPIRICUS study is a randomized trial to evaluate the efficacy of micafungin for the treatment of patients with Candida colonization in multiple sites and sepsis with organ failure (28). The study noted that the incidence of VAP and the 28-days mortality during the ICU stay did not decrease in the micafungin group compared with those in the placebo group (32 vs. 39.8%, p > 0.05). Therefore, the above studies led to a change in the understanding of the co-existence of bacteria and fungi and their effects on immune function in clinical studies. FUNGIBACT, as a prospective cohort study, included 146 patients with MV for more than 96 h. After adjusting for the immune index mHLA-DR, it was concluded that there was no correlation between airway Candida colonization and the incidence of VAP [HR: 0.98; 95% CI (0.59–1.65), p = 0.95] (29). Another retrospective study reviewed 269 systemic lupus erythematosus patients with hospital-acquired pneumonia. Among them, 186 (69.1%) were found to have airway Candida colonization. Compared with that in the non-colonized group, the detection rate of multidrug-resistant bacteria was higher (58.6 vs. 36.1%, p< 0.001), and the secreted IgA and IL-17 levels returned to normal range faster after anti-fungal treatment, but this had no effect on 28-days mortality (14.5 vs. 10.8, p > 0.05) (30). One meta-analysis about the influence of Candida spp. airway colonization on clinical outcomes in patients with VAP included four prospective studies, three retrospective studies, and one cross-sectional study (31). It revealed that those with airway Candida colonization had longer durations of MV. The most noteworthy feature of the meta-analysis is that patients with Candida colonization had higher 28-days mortality (RR: 1.64; 95% CI: 1.27–2.12) and ICU mortality (RR: 1.57; 95% CI: 1.26–1.94) than those without Candida colonization. Although it has included almost all the clinical research about airway Candida colonization with high quality, limitations still exist. First, attributable mortality rate could hardly find in these studies duo to the effects of confounding factors and the insufficient sample size. Second, a highly heterogeneity could be recognized in the baseline of the enrolled patients. Reasons for MV, severity of VAP, antibiotic exposures before the diagnosis of VAP and the immune state was probably diverse among studies. Although “Candida pneumonia” is rarely confirmed in critically ill patients, Candida airway colonization may affect bacterial colonization and antibacterial resistance patterns, playing an important role in the development of bacterial pneumonia. However, the conclusions of current clinical studies are not consistent. Future clinical studies are needed to re-evaluate the potential benefits of pre-emptive antifungal therapy for preventing VAP. Y-TY and JL: conception and design. D-CC: administrative support. C-HX: provision of study materials or patients. Y-TY and C-HX: data analysis and interpretation. All authors: collection and assembly of data, manuscript writing, and final approval of manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmed.2020.598037/full#supplementary-material 1. Epelbaum O, Chasan R. Candidemia in the intensive care unit. Clin Chest Med. (2017) 38:493–509. doi: 10.1016/j.ccm.2017.04.010 PubMed Abstract | CrossRef Full Text | Google Scholar 2. Pappas PG, Kauffman CA, Andes DR, Clancy CJ, Marr KA, Ostrosky-Zeichner L, et al. Clinical practice guideline for the management of Candidiasis: 2016 update by the Infectious Diseases Society of America. Clin Infect Dis. (2016) 62:e1–50. doi: 10.1093/cid/civ933 CrossRef Full Text | Google Scholar 3. Hamet M, Pavon A, Dalle F, Pechinot A, Prin S, Quenot JP, et al. Candida spp. airway colonization could promote antibiotic-resistant bacteria selection in patients with suspected ventilator-associated pneumonia. Intensive Care Med. (2012) 38:1272–9. doi: 10.1007/s00134-012-2584-2 PubMed Abstract | CrossRef Full Text | Google Scholar 4. Gabrilska RA, Rumbaugh KP. Biofilm models of polymicrobial infection. Future Microbiol. (2015) 10:1997–2015. doi: 10.2217/fmb.15.109 PubMed Abstract | CrossRef Full Text | Google Scholar 5. Kalil AC, Metersky ML, Klompas M, Muscedere J, Sweeney DA, Palmer LB, et al. Management of adults with hospital-acquired and ventilator-associated pneumonia: 2016 clinical practice guidelines by the Infectious Diseases Society of America and the American Thoracic Society. Clin Infect Dis. (2016) 63:e61–111. doi: 10.1093/cid/ciw353 CrossRef Full Text | Google Scholar 6. Barenfanger J, Arakere P, Cruz RD, Imran A, Drake C, Lawhorn J, et al. Improved outcomes associated with limiting identification of Candida spp. in respiratory secretions. J Clin Microbiol. (2003) 41:5645–9. doi: 10.1128/jcm.41.12.5645-5649.2003 PubMed Abstract | CrossRef Full Text | Google Scholar 7. Martin-Loeches I, Antonelli M, Cuenca-Estrella M, Dimopoulos G, Einav S, De Waele JJ, et al. ESICM/ESCMID task force on practical management of invasive candidiasis in critically ill patients. Intensive Care Med. (2019) 45:789–805. doi: 10.1007/s00134-019-05599-w PubMed Abstract | CrossRef Full Text | Google Scholar 8. Shao TY, Ang WXG, Jiang TT, Huang FS, Andersen H, Kinder JM, et al. Commensal Candida albicans positively calibrates systemic Th17 immunological responses. Cell Host Microbe. (2019) 25:404–17 e6. doi: 10.1016/j.chom.2019.02.004 PubMed Abstract | CrossRef Full Text | Google Scholar 9. Meersseman W, Lagrou K, Spriet I, Maertens J, Verbeken E, Peetermans WE, et al. Significance of the isolation of Candida species from airway samples in critically ill patients: a prospective, autopsy study. Intensive Care Med. (2009) 35:1526–31. doi: 10.1007/s00134-009-1482-8 PubMed Abstract | CrossRef Full Text | Google Scholar 10. Netea MG, Brown GD, Kullberg BJ, Gow NA. An integrated model of the recognition of Candida albicans by the innate immune system. Nat Rev Microbiol. (2008) 6:67–78. doi: 10.1038/nrmicro1815 PubMed Abstract | CrossRef Full Text | Google Scholar 11. Sedlmayer F, Hell D, Muller M, Auslander D, Fussenegger M. Designer cells programming quorum-sensing interference with microbes. Nat Commun. (2018) 9:1822. doi: 10.1038/s41467-018-04223-7 PubMed Abstract | CrossRef Full Text | Google Scholar 12. Perez-Rodriguez G, Dias S, Perez-Perez M, Fdez-Riverola F, Azevedo NF, Lourenco A. Agent-based model of diffusion of N-acyl homoserine lactones in a multicellular environment of Pseudomonas aeruginosa and Candida albicans. Biofouling. (2018) 34:335–45. doi: 10.1080/08927014.2018.1440392 PubMed Abstract | CrossRef Full Text | Google Scholar 13. Meto A, Colombari B, Sala A, Pericolini E, Meto A, Peppoloni S, et al. Antimicrobial and antibiofilm efficacy of a copper/calcium hydroxide-based endodontic paste against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. Dent Mater J. (2019) 38:591–603. doi: 10.4012/dmj.2018-252 PubMed Abstract | CrossRef Full Text | Google Scholar 14. Gulati M, Nobile CJ. Candida albicans biofilms: development, regulation, and molecular mechanisms. Microbes Infect. (2016) 18:310–21. doi: 10.1016/j.micinf.2016.01.002 PubMed Abstract | CrossRef Full Text | Google Scholar 15. Lohse MB, Gulati M, Johnson AD, Nobile CJ. Development and regulation of single- and multi-species Candida albicans biofilms. Nat Rev Microbiol. (2018) 16:19–31. doi: 10.1038/nrmicro.2017.107 PubMed Abstract | CrossRef Full Text | Google Scholar 16. Ardizzoni A, Pericolini E, Paulone S, Orsi CF, Castagnoli A, Oliva I, et al. In vitro effects of commercial mouthwashes on several virulence traits of Candida albicans, viridans streptococci and Enterococcus faecalis colonizing the oral cavity. PLoS ONE. (2018) 13:e0207262. doi: 10.1371/journal.pone.0207262 PubMed Abstract | CrossRef Full Text | Google Scholar 17. Tan X, Chen R, Zhu S, Wang H, Yan D, Zhang X, et al. Candida albicans airway colonization facilitates subsequent Acinetobacter baumannii pneumonia in a rat model. Antimicrob Agents Chemother. (2016) 60:3348–54. doi: 10.1128/AAC.02180-15 PubMed Abstract | CrossRef Full Text | Google Scholar 18. Green IM, Margoni I, Nair SP, Petridis H. Adhesion of methicillin-resistant Staphylococcus aureus and Candida albicans to parylene-C-coated polymethyl methacrylate. Int J Prosthodont. (2019) 32:193–5. doi: 10.11607/ijp.5918 PubMed Abstract | CrossRef Full Text | Google Scholar 19. Waters NR, Samuels DJ, Behera RK, Livny J, Rhee KY, Sadykov MR, et al. A spectrum of CodY activities drives metabolic reorganization and virulence gene expression in Staphylococcus aureus. Mol Microbiol. (2016) 101:495–514. doi: 10.1111/mmi.13404 PubMed Abstract | CrossRef Full Text | Google Scholar 20. Padder SA, Prasad R, Shah AH. Quorum sensing: a less known mode of communication among fungi. Microbiol Res. (2018) 210:51–8. doi: 10.1016/j.micres.2018.03.007 PubMed Abstract | CrossRef Full Text | Google Scholar 21. Albert M, Williamson D, Muscedere J, Lauzier F, Rotstein C, Kanji S, et al. Candida in the respiratory tract secretions of critically ill patients and the impact of antifungal treatment: a randomized placebo controlled pilot trial (CANTREAT study). Intensive Care Med. (2014) 40:1313–22. doi: 10.1007/s00134-014-3352-2 PubMed Abstract | CrossRef Full Text | Google Scholar 22. Delisle MS, Williamson DR, Perreault MM, Albert M, Jiang X, Heyland DK. The clinical significance of Candida colonization of respiratory tract secretions in critically ill patients. J Crit Care. (2008) 23:11–7. doi: 10.1016/j.jcrc.2008.01.005 PubMed Abstract | CrossRef Full Text | Google Scholar 23. Huang Y, Jiao Y, Zhang J, Xu J, Cheng Q, Li Y, et al. Microbial etiology and prognostic factors of ventilator-associated pneumonia: a multicenter retrospective study in Shanghai. Clin Infect Dis. (2018) 67:S146–52. doi: 10.1093/cid/ciy686 PubMed Abstract | CrossRef Full Text | Google Scholar 24. Delisle MS, Williamson DR, Albert M, Perreault MM, Jiang X, Day AG, et al. Impact of Candida species on clinical outcomes in patients with suspected ventilator-associated pneumonia. Can Respir J. (2011) 18:131–6. doi: 10.1155/2011/827692 PubMed Abstract | CrossRef Full Text | Google Scholar 25. Mear JB, Kipnis E, Faure E, Dessein R, Schurtz G, Faure K, et al. Candida albicans and Pseudomonas aeruginosa interactions: more than an opportunistic criminal association? Med Mal Infect. (2013) 43:146–51. doi: 10.1016/j.medmal.2013.02.005 PubMed Abstract | CrossRef Full Text | Google Scholar 26. van der Geest PJ, Dieters EI, Rijnders B, Groeneveld JA. Safety and efficacy of amphotericin-B deoxycholate inhalation in critically ill patients with respiratory Candida spp. colonization: a retrospective analysis. BMC Infect Dis. (2014) 14:575. doi: 10.1186/s12879-014-0575-3 PubMed Abstract | CrossRef Full Text | Google Scholar 27. Dadar M, Tiwari R, Karthik K, Chakraborty S, Shahali Y, Dhama K. Candida albicans- biology, molecular characterization, pathogenicity, and advances in diagnosis and control - an update. Microb Pathog. (2018) 117:128–38. doi: 10.1016/j.micpath.2018.02.028 PubMed Abstract | CrossRef Full Text | Google Scholar 28. Timsit JF, Azoulay E, Schwebel C, Charles PE, Cornet M, Souweine B, et al. Empirical micafungin treatment and survival without invasive fungal infection in adults with ICU-acquired sepsis, candida colonization, and multiple organ failure: the EMPIRICUS randomized clinical trial. JAMA. (2016) 316:1555–64. doi: 10.1001/jama.2016.14655 PubMed Abstract | CrossRef Full Text | Google Scholar 29. Timsit JF, Schwebel C, Styfalova L, Cornet M, Poirier P, Forrestier C, et al. Impact of bronchial colonization with Candida spp. on the risk of bacterial ventilator-associated pneumonia in the ICU: the FUNGIBACT prospective cohort study. Intensive Care Med. (2019) 45:834–43. doi: 10.1007/s00134-019-05622-0 PubMed Abstract | CrossRef Full Text | Google Scholar 30. Yu Y, Li J, Wang S, Gao Y, Shen H, Lu L. Effect of Candida albicans bronchial colonization on hospital-acquired bacterial pneumonia in patients with systemic lupus erythematosus. Ann Transl Med. (2019) 7:673. doi: 10.21037/atm.2019.10.44 PubMed Abstract | CrossRef Full Text | Google Scholar 31. Huang D, Qi M, Hu Y, Yu M, Liang Z. The impact of Candida spp airway colonization on clinical outcomes in patients with ventilator-associated pneumonia: a systematic review and meta-analysis. Am J Infect Control. (2020) 48:695–701. doi: 10.1016/j.ajic.2019.11.002 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: Candida, colonization, ventilator associate pneumonia, critical ill patients, bioflim Citation: Liu J, Yu Y-T, Xu C-H and Chen D-C (2021) Candida Colonization in the Respiratory Tract: What Is the Significance? Front. Med. 7:598037. doi: 10.3389/fmed.2020.598037 Received: 23 August 2020; Accepted: 18 December 2020; Published: 04 February 2021. Edited by: Reviewed by: Copyright © 2021 Liu, Yu, Xu and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: De-Chang Chen, [email protected] These authors have contributed equally to this work
Corrigendum
Haiyang Xia, Xiaofang Li, Zhangqun Li, Xinqiao Zhan, Xuming Mao, Yongquan Li
Published: 3 February 2021
Frontiers in Microbiology, Volume 11; https://doi.org/10.3389/fmicb.2020.614274

Abstract:
A Corrigendum onThe Application of Regulatory Cascades in Streptomyces: Yield Enhancement and Metabolite Miningby Xia, H., Li, X., Li, Z., Zhan, X., Mao, X., and Li, Y. (2020). Front. Microbiol. 11:406. doi: 10.3389/fmicb.2020.00406 In the original article, there was some errors. A portion of this text was reproduced from another article, this has now been rephrased and appropriately attributed. A correction has been made to The Regulatory Cascades of Antibiotic Production in Streptomyces, paragraph 5: The fourth level is the feedback regulation which is brought by antibiotic and/or intermediates to coordinate antibiotic production and transport. Evidence has shown that antibiotic functions as signals to regulate the production of antibiotic besides as feedback substances for the enzymatic reactions. Antibiotic, as ligand for proper regulator, affects the final production in Streptomyces. The expression of antibiotic biosynthetic genes was modulated by the RedZ and undecylprodigiosin complex (Wang et al., 2009). The activity of AtrA, which regulates primary and secondary metabolism, is reduced by lidamycin of Streptomyces globisporus and actinorhodin (ACT) of S. coelicolor (Li et al., 2015). The biosynthesis of jadomycin is dynamically modulated by the interaction among jadomycin B, chloramphenicol, JadR1 and JadR2 in Streptomyces venezuelae (Wang et al., 2009; Xu et al., 2010). Daunorubicin (DNR) biosynthesis is regulated by three DNA binding regulatory proteins (DnrI, DnrN, and DnrO). The DNA binding activity of DnrO can be modulated by Rhodomycin D, a glycosylated precursor of DXR (Jiang and Hutchinson, 2006). Simocyclinone and its precursors inhibit the binding activity of SimReg1 to several promoter regions of simocyclinone biosynthesis genes and SimReg1 encoding gene (Horbal et al., 2012). As a GBL receptor-like protein, PapR5, which is the major regulator of pristinamycin biosynthesis, may sense pristinamycin or intermediate(s) of the pathway (Mast et al., 2015). SsaA can activate sansanmycin biosynthesis by binding to five different regions within the sansanmycin BGC. The sansanmycins A and H inhibit DNA-binding activity of SsaA in a concentration-dependent manner (Li et al., 2013). The rifamycin B, the end product of rifamycin biosynthesis, can relieve the repression of RifQ on the transcription of the rifamycin efflux pump (RifP) (Lei et al., 2018). Transporters may affect product maturation. Deletion of nysG and nysH, two ABC transporters encoding genes, resulted in ca. 35% reduction of nystatin production and accumulation of its deoxy precursor in Streptomyces noursei. NysGH complex is prone to export nystatin. Its activity would enhance the last biosynthetic step by relief of the feedback through final product removal (Sletta et al., 2005). ‘LanT,’ the dedicated ABC transporter for both class I and II lantibiotics, plays an important role in production of the final product (Gebhard, 2012). A correction has been made to Enhancing Antibiotic Production by Overexpression of Positive Regulator Genes, paragraph 1: The regulators also can be defined as positive and negative regulators according their effect on the antibiotic production. The positive regulators (activators) can promote the biosynthesis of antibiotics. But the negative ones (repressors) can repress the biosynthesis of antibiotics (Martin and Liras, 2010). Since the positive regulators activate the transcription of antibiotic BGCs, they can be manipulated to enhance the production of antibiotic in Streptomyces. The titer improvement can efficiently and simply be achieved by over-expression of genes encoding activators with proper promoters. As listed in Table 1, overexpression of genes encoding LAL family regulators, such as MilR, NemR, and AveR, has been used to increase production of milbemycin in S. bingchenggensis BC04, nemadectin in S. cyaneogriseus subsp. non-cyanogenus NMWT1 and avermectin in S. avermitilis, respectively (Guo et al., 2010; Zhang et al., 2016; Li et al., 2019). Overexpression of sanG, a CSR activator encoding gene, led to improvement of nikkomycin production (Liu et al., 2005). Tandem copies of otcR (a CSR activator gene), whose expression is driven by the SF14 promoter, can greatly enhance the production of oxytetracycline (OTC) (Yin et al., 2015). There are many examples in similar strategies to improve antibiotic production in Streptomyces. Overexpression of bulZ, fkbR1, wysR, and lnmO led to overproduction of tacrolimus (FK506), ascomycin, wuyiencin, and leinamycin, respectively (Liu et al., 2014; Huang et al., 2016; Song et al., 2017; Ma et al., 2018). A correction has been made to Enhancing Antibiotic Production by Manipulation of Feedback and Transport, Paragraph 1: Genes encoding exporters, which are responsible for the secretion of antibiotic, often situate in their BGCs. Various BGC-linked transporters, belonging to ATP-binding cassette (ABC) superfamily and major facilitator superfamily (MFS) are responsible for secreting antibiotics. Pumping out of toxic end-products can achieve more durable and sustainable productivity. In the section Enhancing Antibiotic Production by Manipulation of Feedback and Transport, Paragraphs 2, 3, and 4 should be replaced with the following text; It has been proved that the expression of BGCs was greatly affected by the secretion of end-products, even without toxicity. ActA (ActII-ORF2) and ActB (ActIIORF3), activate the transcription of BGCs in a feed-forward by transportation of the end-products (Tahlan et al., 2007; Xu et al., 2012). Only one fifth of ACT was produced by the actAB mutant. There are two waves for ACT production. The expression of key act genes is initially induced by an ACT biosynthetic intermediate. The ACT production is fully induced only when the inner ACT is pumped out. Overexpression of AvtAB, an ABC transporter, enhance the production of avermectin B1a with two-folds. But the production level of oligomycin A, another product from S. avermitilis, was found unaltered. The production promotion effects of avtAB could be specific to avermectin in S. avermitilis (Qiu et al., 2011). Co-overexpression of three OTC resistance genes, including otrA (encoding a ribosomal protection protein), otrB and otrC (encoding two efflux proteins), led to 179% increase of OTC production in Streptomyces rimosus M4018 (Yin et al., 2017). The biosynthesis of BGCs for the actinobacterial ribosomally synthesized and posttranslationally modified peptides (RiPPs), like planosporicin and microbisporicin, is probably regulated in a feed-forward way. Their production and self-immunity is seemed to be modulated by the multiple ABC transporter genes in these BGCs (Foulston and Bibb, 2010; Sherwood et al., 2013). GouM, the MFS transporter, is responsible for the secretion of gougerotin outside of Streptomyces graminearus (Wei et al., 2014). The overexpression of BotT, a putative efflux pump encoded in the bottromycin BGC, increased bottromycin production about 20 times in a heterologous host (Huo et al., 2012). The authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way. The original article has been updated. Foulston, L. C., and Bibb, M. J. (2010). Microbisporicin gene cluster reveals unusual features of lantibiotic biosynthesis in actinomycetes. Proc. Natl. Acad. Sci. U.S.A. 107, 13461–13466. doi: 10.1073/pnas.1008285107 PubMed Abstract | CrossRef Full Text | Google Scholar Gebhard, S. (2012). ABC transporters of antimicrobial peptides in Firmicutes bacteria – Phylogeny, function and regulation. Mol. Microbiol. 86, 1295–1317. doi: 10.1111/mmi.12078 PubMed Abstract | CrossRef Full Text | Google Scholar Guo, J., Zhao, J., Li, L., Chen, Z., Wen, Y., and Li, J. (2010). The pathway-specific regulator AveR from Streptomyces avermitilis positively regulates avermectin production while it negatively affects oligomycin biosynthesis. Mol. Genet. Genomics 283, 123–133. doi: 10.1007/s00438-009-0502-2 PubMed Abstract | CrossRef Full Text | Google Scholar Horbal, L., Rebets, Y., Rabyk, M., Makitrynskyy, R., Luzhetskyy, A., Fedorenko, V., et al. (2012). SimReg1 is a master switch for biosynthesis and export of simocyclinone D8 and its precursors. AMB Express. 2:1. doi: 10.1186/2191-0855-2-1 PubMed Abstract | CrossRef Full Text | Google Scholar Huang, Y., Yang, D., Pan, G., Tang, G. L., and Shen, B. (2016). Characterization of LnmO as a pathway-specific Crp/Fnr-type positive regulator for leinamycin biosynthesis in Streptomyces atroolivaceus and its application for titer improvement. Appl. Microbiol. Biotechnol. 100, 10555–10562. doi: 10.1007/s00253-016-7864-2 PubMed Abstract | CrossRef Full Text | Google Scholar Huo, L., Rachid, S., Stadler, M., Wenzel, S. C., and Muller, R. (2012). Synthetic biotechnology to study and engineer ribosomal bottromycin biosynthesis. Chem. Biol. 19, 1278–1287. doi: 10.1016/j.chembiol.2012.08.013 PubMed Abstract | CrossRef Full Text | Google Scholar Jiang, H., and Hutchinson, C. R. (2006). Feedback regulation of doxorubicin biosynthesis in Streptomyces peucetius. Res. Microbiol. 157, 666–674. doi: 10.1016/j.resmic.2006.02.004 PubMed Abstract | CrossRef Full Text | Google Scholar Lei, C., Wang, J., Liu, Y., Liu, X., Zhao, G., and Wang, J. (2018). A feedback regulatory model for RifQ-mediated repression of rifamycin export in Amycolatopsis mediterranei. Microb. Cell Fact. 17:14. doi: 10.1186/s12934-018-0863-5 PubMed Abstract | CrossRef Full Text | Google Scholar Li, C., He, H., Wang, J., Liu, H., Wang, H., Zhu, Y., et al. (2019). Characterization of a LAL-type regulator NemR in nemadectin biosynthesis and its application for increasing nemadectin production in Streptomyces cyaneogriseus. Sci. China Life Sci. 62, 394–405. doi: 10.1007/s11427-018-9442-9 PubMed Abstract | CrossRef Full Text | Google Scholar Li, Q., Wang, L., Xie, Y., Wang, S., Chen, R., and Hong, B. (2013). SsaA, a member of a novel class of transcriptional regulators, controls sansanmycin production in Streptomyces sp. strain SS through a feedback mechanism. J. Bacteriol. 195, 2232–2243. doi: 10.1128/jb.00054-13 PubMed Abstract | CrossRef Full Text | Google Scholar Li, X., Yu, T., He, Q., McDowall, K. J., Jiang, B., Jiang, Z., et al. (2015). Binding of a biosynthetic intermediate to AtrA modulates the production of lidamycin by Streptomyces globisporus. Mol. Microbiol. 96, 1257–1271. doi: 10.1111/mmi.13004 PubMed Abstract | CrossRef Full Text | Google Scholar Liu, G., Tian, Y., Yang, H., and Tan, H. (2005). A pathway-specific transcriptional regulatory gene for nikkomycin biosynthesis in Streptomyces ansochromogenes that also influences colony development. Mol. Microbiol. 55, 1855–1866. doi: 10.1111/j.1365-2958.2005.04512.x PubMed Abstract | CrossRef Full Text | Google Scholar Liu, Y., Ryu, H., Ge, B., Pan, G., Sun, L., Park, K., et al. (2014). Improvement of Wuyiencin biosynthesis in Streptomyces wuyiensis CK-15 by identification of a key regulator, WysR. J. Microbiol. Biotechnol. 24, 1644–1653. doi: 10.4014/jmb.1405.05017 PubMed Abstract | CrossRef Full Text | Google Scholar Ma, D., Wang, C., Chen, H., and Wen, J. (2018). Manipulating the expression of SARP family regulator BulZ and its target gene product to increase tacrolimus production. Appl. Microbiol. Biotechnol. 102, 4887–4900. doi: 10.1007/s00253-018-8979-4 PubMed Abstract | CrossRef Full Text | Google Scholar Martin, J.-F., and Liras, P. (2010). Engineering of regulatory cascades and networks controlling antibiotic biosynthesis in Streptomyces. Curr. Opin. Microbiol. 13, 263–273. doi: 10.1016/j.mib.2010.02.008 PubMed Abstract | CrossRef Full Text | Google Scholar Mast, Y., Guezguez, J., Handel, F., and Schinko, E. (2015). A complex signaling cascade governs pristinamycin biosynthesis in Streptomyces pristinaespiralis. Appl. Environ. Microbiol. 81, 6621–6636. doi: 10.1128/aem.00728-15 PubMed Abstract | CrossRef Full Text | Google Scholar Qiu, J., Zhuo, Y., Zhu, D., Zhou, X., Zhang, L., Bai, L., et al. (2011). Overexpression of the ABC transporter AvtAB increases avermectin production in Streptomyces avermitilis. Appl. Microbiol. Biotechnol. 92, 337–345. doi: 10.1007/s00253-011-3439-4 PubMed Abstract | CrossRef Full Text | Google Scholar Sherwood, E. J., Hesketh, A. R., and Bibb, M. J. (2013). Cloning and analysis of the planosporicin lantibiotic biosynthetic gene cluster of Planomonospora alba. J. Bacteriol. 195, 2309–2321. doi: 10.1128/jb.02291-12 PubMed Abstract | CrossRef Full Text | Google Scholar Sletta, H., Borgos, S. E., Bruheim, P., Sekurova, O. N., Grasdalen, H., Aune, R., et al. (2005). Nystatin biosynthesis and transport: nysH and nysG genes encoding a putative ABC transporter system in Streptomyces noursei ATCC 11455 are required for efficient conversion of 10-deoxynystatin to nystatin. Antimicrob. Agents Chemother. 49, 4576–4583. doi: 10.1128/AAC.49.11.4576-4583.2005 PubMed Abstract | CrossRef Full Text | Google Scholar Song, K., Wei, L., Liu, J., Wang, J., Qi, H., and Wen, J. (2017). Engineering of the LysR family transcriptional regulator FkbR1 and its target gene to improve ascomycin production. Appl. Microbiol. Biotechnol. 101, 4581–4592. doi: 10.1007/s00253-017-8242-4 PubMed Abstract | CrossRef Full Text | Google Scholar Tahlan, K., Ahn, S. K., Sing, A., Bodnaruk, T. D., Willems, A. R., Davidson, A. R., et al. (2007). Initiation of actinorhodin export in Streptomyces coelicolor. Mol. Microbiol. 63, 951–961. doi: 10.1111/j.1365-2958.2006.05559.x PubMed Abstract | CrossRef Full Text | Google Scholar Wang, L., Tian, X., Wang, J., Yang, H., Fan, K., Xu, G., et al. (2009). Autoregulation of antibiotic biosynthesis by binding of the end product to an atypical response regulator. Proc. Natl. Acad. Sci. U.S.A. 106, 8617–8622. doi: 10.1073/pnas.0900592106 PubMed Abstract | CrossRef Full Text | Google Scholar Wei, J., Tian, Y., Niu, G., and Tan, H. (2014). GouR, a TetR family transcriptional regulator, coordinates the biosynthesis and export of gougerotin in Streptomyces graminearus. Appl. Environ. Microbiol. 80, 714–722. doi: 10.1128/aem.03003-13 PubMed Abstract | CrossRef Full Text | Google Scholar Xu, D., Seghezzi, N., Esnault, C., and Virolle, M. J. (2010). Repression of antibiotic production and sporulation in Streptomyces coelicolor by overexpression of a TetR family transcriptional regulator. Appl. Environ. Microbiol. 76, 7741–7753. doi: 10.1128/aem.00819-10 PubMed Abstract | CrossRef Full Text | Google Scholar Xu, Y., Willems, A., Au-Yeung, C., Tahlan, K., and Nodwell, J. R. (2012). A two-step mechanism for the activation of actinorhodin export and resistance in Streptomyces coelicolor. MBio. 3, e191–e112. PubMed Abstract | Google Scholar Yin, S., Wang, W., Wang, X., Zhu, Y., Jia, X., Li, S., et al. (2015). Identification of a cluster-situated activator of oxytetracycline biosynthesis and manipulation of its expression for improved oxytetracycline production in Streptomyces rimosus. Microb. Cell Fact. 14, 46. PubMed Abstract | Google Scholar Yin, S., Wang, X., Shi, M., Yuan, F., Wang, H., Jia, X., et al. (2017). Improvement of oxytetracycline production mediated via cooperation of resistance genes in Streptomyces rimosus. Sci. China Life Sci. 60, 992–999. doi: 10.1007/s11427-017-9121-4 PubMed Abstract | CrossRef Full Text | Google Scholar Zhang, Y., He, H., Liu, H., Wang, H., Wang, X., and Xiang, W. (2016). Characterization of a pathway-specific activator of milbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces bingchenggensis. Microb. Cell Fact. 15:152. doi: 10.1186/s12934-016-0552-1 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: antibiotic production, regulatory cascades, rewiring regulatory network, unlocking cryptic metabolites, Streptomyces Citation: Xia H, Li X, Li Z, Zhan X, Mao X and Li Y (2021) Corrigendum: The Application of Regulatory Cascades in Streptomyces: Yield Enhancement and Metabolite Mining. Front. Microbiol. 11:614274. doi: 10.3389/fmicb.2020.614274 Received: 05 October 2020; Accepted: 15 December 2020; Published: 03 February 2021. Edited by: Reviewed by: Copyright © 2021 Xia, Li, Li, Zhan, Mao and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Xuming Mao, [email protected]; Yongquan Li, [email protected]
, Farhana Sarker, Tom Chau, Khondaker A. Mamun
Abstract:
Augmentative and Alternative Communication (AAC) emerged as a combination of methods or strategies that constitute any device, such as Speech Generating Device (SGD), Program (mobile applications), Procedure (PECS, Picture Exchange Communication System), which enhances individual’s communication ability. Autism Spectrum Disorder (ASD) is a spectrum of comprehensive neurodevelopment disorder that leads to speech impairments, repetitive behavior, and social communication difficulties; therefore, it is imperative to underscore that at the core of all impediments are communication impairment. This article represents a systematic review of research initiatives that investigate multi-modal AAC strategies and functionality, features of mobile applications to reinforce communication and communal skills in verbally challenged ASD children because other researches are focused only on low or high-tech AAC or interventions to provide insights on ASD children respond to a particular approach. Following the PRISMA method, a total of 60 (January 2015 to October 2020) research articles were reviewed, indexed by Springer, Science Direct, Scopus, ACM, IEEE databases, and published in the AAC journal. The selected research articles are categorized into different themes where most of them focused on interactive mobile applications to improve emotional, social, learning, and overall communication skills in verbally challenged ASD children. This systematic review provides an outline of the paradigm shift in AAC modalities from PECS to Artificial Intelligence (AI), Machine Learning (ML), and Augmented Reality (AR) based applications. It opens up underline future opportunities to integrate intelligent analytics features in mobile applications to strengthen communication skills in verbally undermined ASD children.
Published: 20 January 2021
Frontiers in Public Health, Volume 8; https://doi.org/10.3389/fpubh.2020.629120

Abstract:
A Commentary onMicrobial Resistance Movements: An Overview of Global Public Health Threats Posed by Antimicrobial Resistance, and How Best to Counterby Dhingra, S., Rahman, N. R. A., Peile, E., Rahman, M., Sartelli, M., Hassali, M. A., et al. (2020). Front. Public Health 8:535668. doi: 10.3389/fpubh.2020.535668 “Superbugs” are the antimicrobial-resistant microorganisms. Bacteria, viruses, fungi, and parasites acquire the ability to evade the antimicrobial drug effects, not only to survive but also, in some cases, become more virulent. As such, the existing antimicrobial drugs are no longer effective and useful in treating the infections (used to be treatable). Superbug-induced infections are the major worldwide health concern with higher human mortality and an increased financial burden on society. The underlying mechanism of the evolvement of drug-sensitive to drug-resistant microorganisms is an extremely complex phenomenon. It is partly related to microorganism's unique ability to modify their genetic structures and biochemical functionality to survive and keep growing even in the presence of antimicrobial drugs. Since there are multiple factors involved in developing antimicrobial drug resistance, it cannot be reversed by adopting a single prevention strategy. Of importance, certain bacterium may not require antimicrobial drug exposure to develop resistance, as surrounding environmental exposure can facilitate the resistance. Consistent misuse and overuse of antimicrobial drugs by healthcare professionals and consumers with its extensive use in food and meat production have put human health at risk. Lack of resources for research and low interest in developing the newer generation of antimicrobial drugs are also contributing to the evolution of superbugs. Without global involvement, partnership and collaboration, superbug-induced morbidity and mortality will be unmanageable in the future. To address this impending global health crisis, in May 2015, the World Health Organization (WHO) assembly adopted a global action plan to combat the antimicrobial resistance, that include (1) to increase the awareness of antimicrobial resistance, (2) to advance research and surveillance, (3) to cut down the rate of infections through preventive measures, (4) to ensure the optimal use of antimicrobial drugs, and (5) to develop sustainable investment, taking into account the needs of the countries, to develop novel interventions. Unfortunately, implementing such WHO measures have both financial and logistic hurdles, and the rates of superbug-induced infections are alarmingly increasing. A recently published article in “Frontiers in Public Health” has highlighted the importance of microbial resistance movements to reduce the burden of superbug-induced infections (1). The authors have elaborated on the genesis of antimicrobial drugs and listed the challenges of producing the newer generation of drugs to combat existing drug-resistant pathogens (1). Although the authors briefly touched on the irrational prescription of antimicrobial drugs in the both developed and developing countries, the review article did not discuss in depth the regulatory enforcement procedures to minimize the antimicrobial resistance (1). The opportunities and challenges of global standardization of the antimicrobial prescription processes were not explained in detail in the publication (1). Another critical area that was not emphasized enough in the article was the use of the antimicrobial drugs on animals and its potential consequences in developing antimicrobial resistance. This commentary will briefly elaborate on the cross-species transmission of resistant pathogens from animal to human. Appreciating that one single review article cannot cover all the aspects of antimicrobial resistance, the authors have fittingly highlighted the scientific and economic challenges that are hindering the novel antimicrobial drug development (1). The publication has justifiably concluded that antimicrobial resistance is a multifaced issue driven by numerous interrelated factors, and therefore, the use of any single intervention would have limited success (1). Recent publications have also emphasized why implementing an antimicrobial stewardship program is necessary to prepare the future medical professionals to enhance their awareness and knowledge of antimicrobial resistance to reduce the disease burden related to superbug-mediated infections (Figure 1) (2, 3). Figure 1. Schematic diagram showing the main steps needed to be implemented to minimize the antimicrobial drug resistance (2). Antimicrobial stewardship program is one of the most effective approaches to educate healthcare professionals to select suitable antimicrobial medications for required patients for the right period to lower the emergence of antimicrobial resistance (4). Of concern, antimicrobial drugs are commonly used in clinical practice, and around 50% of antibiotics prescribed in the hospitals are unnecessary (5). In a similar line of observation, the Centers for Disease Control and Prevention (CDC) reported that during 2010–2011, around 154 million times, antibiotics were prescribed in the ambulatory care settings in the U.S., of which ~47 million were estimated as unnecessary or inappropriate prescriptions (6). Such an irrational use of antimicrobial drugs partly contributed to the development of resistance against the microorganisms, once treatable before the emergence of resistance (7, 8). One unfortunate example would be the treatment of gonorrhea. Azithromycin and ceftriaxone were very effective in treating gonorrhea. However, gonorrhea is no longer responsive to azithromycin and ceftriaxone treatment due to their overuse/ misuse and the subsequent development of antimicrobial resistance. The CDC categorized gonorrhea as an “urgent threat” (9, 10). Therefore, it is of utmost importance to implement antimicrobial stewardship programs to educate future healthcare professionals on the responsible use of antimicrobial drugs to improve their effectiveness and sustainability (11). As mentioned, in the U. S., the annual (2010–2011) antibiotic prescription was 506 per 1,000 population, but only 353 antibiotic prescriptions were estimated to be appropriate (6). Implementing an antimicrobial stewardship program makes it possible to reduce around 30% fewer antibiotic prescriptions yearly, which will have far-reaching impacts and benefits on human health. Although the unnecessary antimicrobial prescriptions in humans with subsequent drug exposure are among the major causes of antimicrobial resistance for specific strains, (needless) prescriptions alone are not the cause of antimicrobial resistance for all bacterial strains (12). Another important area that requires intense focus to reduce antimicrobial resistance is the use of antibiotics to promote animal growth for meat consumption. In the U.S., around 70–80% of clinically important and useful antibiotics are sold and utilized for the maintenance and growth of the livestock (13). Such massive use of antibiotics on meat-producing animals is likely to promote antimicrobial resistance, although animal to the human association is not universally accepted and contested by certain groups. However, studies have shown that when foodborne pathogens (Campylobacter) were challenged with antimicrobial drugs, resistant pathogens could be isolated from the exposed animals (14). Of more concern, antimicrobial resistance remained higher for years, even after discontinuation of antibiotics (14). How quickly microorganisms can gain resistance usually depends on the microbial strains and the drugs used (15). Even though the resistance of animal pathogens on farms can risk human health is an ongoing area of research; a U.S. study found that tetracycline-treated birds (broiler chicken) resulted in the emergence of tetracycline resistance pathogens, which were traced in the birds, and isolated from 11 members of the farms who were exposed to those housed birds (16). The study suggests the chain of events of how antimicrobial resistance can develop in the birds, and can eventually be transmitted to humans. The cross-species transmission of resistant pathogens from animal to human can be transmitted by direct contact between humans and animals by consuming contaminated food or sharing pathogen-polluted water (17). According to the CDC, more than 35,000 yearly deaths are estimated to be related to antibiotic-resistant infections in the U.S., costing more than US$50 billion (18). It is essential to realize that even if unnecessary antimicrobial prescriptions in humans are reduced, it may not necessarily eliminate the antimicrobial resistance altogether, as the use of antimicrobial drugs on animals can also initiate and propagate antimicrobial resistance. From the available evidence, it appears to be rational to advocate reducing or more preferably eliminating growth-promoting antimicrobial use in animals and birds to minimize the occurrence of antimicrobial resistance (19). Of relevance, such initiatives are partially implemented in Europe and should be executed globally. Decreasing antibiotic use reduces the prevalence of antimicrobial resistance in animals by around 15% and multidrug-resistant bacteria by ~24–32% (19). The subsequent effect on humans is difficult to determine, but it is estimated that around a 24% reduction in the prevalence of antimicrobial resistance in humans with reduced use of antimicrobial drugs in animals (19). Naturally occurring antimicrobial resistance is a prolonged and slow process. On the other hand, the overuse or misuse of antimicrobial drugs can rapidly induce antimicrobial resistance, and these superbugs are clinically challenging to eradicate. Through the antimicrobial stewardship program, necessary training should be provided to the healthcare professionals to reduce the unnecessary use of antimicrobial drugs to delay the occurrence of antimicrobial-resistant organisms. Also, alternatives to the existing antimicrobial drugs or supplementary agents are needed to reduce the burden of antimicrobial drug resistance. For instance, a recent publication from McGill University (in Montreal, Canada) has claimed that a cranberry extract can make the bacteria more sensitive to antibiotic treatment (20). Developing clinically viable quorum sensing inhibitors to supplement existing antimicrobial agents would be another research avenue to pursue (21, 22). Finally, the empiric antibacterial therapy during the ongoing COVID-19 pandemic is likely to enhance antibiotic-resistant microorganisms (19–21). There is an urgent need to develop more potent antibiotics and/or innovative therapeutic strategies to deal with such emerging microbial resistance (23–25). The newly developed therapeutics need to be strictly regulated to avoid the past error of overuse-antimicrobial resistance. The coordinated and collaborative efforts among the national and international governmental and private agencies are required to achieve substantial progress in reducing or delaying the occurrence of antimicrobial resistance. MR: conceptualized and wrote the article. The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. I want to express my sincere gratitude to Dr. Nuraly Akimbekov (Al-Farabi Kazakh National University, Kazakhstan) for his help in drawing the illustration. I also wish to thank Dr. Arafat Tannum, Mr. Muhit Razzaque, and Ms. Peace Uwambaye for proofreading the manuscript and providing useful suggestions. 1. Dhingra S, Rahman NAA, Peile E, Rahman M, Sartelli M, Hassali MA, et al. Microbial resistance movements: an overview of global public health threats posed by antimicrobial resistance, and how best to counter. Front Public Health. (2020) 8:535668. doi: 10.3389/fpubh.2020535668 PubMed Abstract | CrossRef Full Text | Google Scholar 2. Razzaque MS. Implementation of antimicrobial stewardship to reduce antimicrobial drug resistance. Expert Rev Anti Infect Ther. (2020). doi: 10.1080/14787210.20211840977 PubMed Abstract | CrossRef Full Text | Google Scholar 3. Majumder MAA, Singh K, Hilaire MG, Rahman S, Sa B, Haque M. Tackling antimicrobial resistance by promoting antimicrobial stewardship in medical and allied health professional curricula. Expert Rev Anti Infect Ther. (2020) 18:1245–58. doi: 10.1080/14787210.20201796638 PubMed Abstract | CrossRef Full Text | Google Scholar 4. Moody J, Cosgrove SE, Olmsted R, Septimus E, Aureden K, Oriola S, et al. Antimicrobial stewardship: a collaborative partnership between infection preventionists and healthcare epidemiologists. Infect Control Hosp Epidemiol. (2012) 33:328–30. doi: 10.1086/665037 PubMed Abstract | CrossRef Full Text | Google Scholar 5. Pulcini C, Cua E, Lieutier F, Landraud L, Dellamonica P, Roger PM. Antibiotic misuse: a prospective clinical audit in a French university hospital. Eur J Clin Microbiol Infect Dis. (2007) 26:277–80. doi: 10.1007/s10096-007-0277-5 PubMed Abstract | CrossRef Full Text | Google Scholar 6. Fleming-Dutra KE, Hersh AL, Shapiro DJ, Bartoces M, Enns EA, File TM Jr, et al. Prevalence of inappropriate antibiotic prescriptions among us ambulatory care visits, 2010–2011. JAMA. (2016) 315:1864–73. doi: 10.1001/jama.2016.4151 PubMed Abstract | CrossRef Full Text | Google Scholar 7. Maduna LD, Kock MM, van der Veer B, Radebe O, McIntyre J, van Alphen LB, et al. Antimicrobial resistance of Neisseria gonorrhoeae isolates from high risk men in Johannesburg, South Africa. Antimicrob Agents Chemother. (2020) 64:e00906–20. doi: 10.1128/AAC00906-20 PubMed Abstract | CrossRef Full Text | Google Scholar 8. Bodie M, Gale-Rowe M, Alexandre S, Auguste U, Tomas K, Martin I. Addressing the rising rates of gonorrhea and drug-resistant gonorrhea: there is no time like the present. Can Commun Dis Rep. (2019) 45:54–62. doi: 10.14745/ccdr.v45i23a02 PubMed Abstract | CrossRef Full Text | Google Scholar 9. CDC. Drug-resistant N. gonorrhoeae pathogen page. U.S. Department of Health & Human Services. (2019). Available online at: https://www.cdc.gov/drugresistance/biggest-threats.html (accessed November 10, 2020). 10. Lahra MM, Shoushtari M, George CRR, Armstrong BH, Hogan TR. Australian gonococcal surveillance programme annual report, 2019. Commun Dis Intell (2018). (2019) 43:1–12. doi: 10.33321/cdi.2019.43.13 PubMed Abstract | CrossRef Full Text | Google Scholar 11. Mendelson M, Balasegaram M, Jinks T, Pulcini C, Sharland M. Antibiotic resistance has a language problem. Nature. (2017) 545:23–5. doi: 10.1038/545023a PubMed Abstract | CrossRef Full Text | Google Scholar 12. Hoelzer K, Wong N, Thomas J, Talkington K, Jungman E, Coukell A. Antimicrobial drug use in food-producing animals and associated human health risks: what, and how strong, is the evidence? BMC Vet Res. (2017) 13:211. doi: 10.1186/s12917-017-1131-3 PubMed Abstract | CrossRef Full Text | Google Scholar 13. FDA. Antimicrobials Sold or Distributed for Use in Food-Producing Animals. Food and Drug Administration. (2014). Available online at: https://www.fda.gov/media/79581/download (accessed November 10, 2020). 14. Price LB, Lackey LG, Vailes R, Silbergeld E. The persistence of fluoroquinolone-resistant Campylobacter in poultry production. Environ Health Perspect. (2007) 115:1035–9. doi: 10.1289/ehp.10050 PubMed Abstract | CrossRef Full Text | Google Scholar 15. Pechère JC, Marchou B, Michéa-Hamzehpour M, Auckenthaler R. Emergence of resistance after therapy with antibiotics used alone or combined in a murine model. J Antimicrob Chemother. (1986) 17:(Suppl A):11–8. PubMed Abstract | Google Scholar 16. Levy SB, FitzGerald GB, Macone AB. Changes in intestinal flora of farm personnel after introduction of a tetracycline-supplemented feed on a farm. N Engl J Med. (1976) 295:583–8. PubMed Abstract | Google Scholar 17. Landers TF, Cohen B, Wittum TE, Larson EL. A review of antibiotic use in food animals: perspective, policy, and potential. Public Health Rep. (2012) 127:4–22. doi: 10.1177/003335491212700103 PubMed Abstract | CrossRef Full Text | Google Scholar 18. CDC. U.S. Centers for Disease Control and Prevention. Antibiotic Resistance Threats in the United States, 2013. Atlanta: U.S. Department of Health and Human Services 2013 (2013). Available online at: https://www.cdc.gov/drugresistance/pdf/ar-threats-2013-2508.pdf (accessed November 10, 2020). Google Scholar 19. Tang KL, Caffrey NP, Nóbrega DB, Cork SC, Ronksley PE, Barkema HW, et al. Restricting the use of antibiotics in food-producing animals and its associations with antibiotic resistance in food-producing animals and human beings: a systematic review and meta-analysis. Lancet Planet Health. (2017) 1:e316–27. doi: 10.1016/S2542-5196(17)30141-9 PubMed Abstract | CrossRef Full Text | Google Scholar 20. Maisuria VB, Okshevsky M, Deziel E, Tufenkji N. Proanthocyanidin interferes with intrinsic antibiotic resistance mechanisms of gram-negative bacteria. Adv Sci (Weinh). (2019) 6:1802333. doi: 10.1002/advs.201802333 PubMed Abstract | CrossRef Full Text | Google Scholar 21. Haque M, Islam S, Sheikh MA, Dhingra S, Uwambaye P, Labricciosa FM, et al. Quorum sensing: a new prospect for the management of antimicrobial-resistant infectious diseases. Expert Rev Anti Infect Ther. (2020). doi: 10.1080/14787210.20211843427 PubMed Abstract | CrossRef Full Text | Google Scholar 22. Razzaque MS. Exacerbation of antimicrobial resistance: another casualty of the COVID-19 pandemic? Expert Rev Anti Infect Ther. (2020). doi: 10.1080/14787210.2021.1865802 PubMed Abstract | CrossRef Full Text | Google Scholar 23. Buetti N, Mazzuchelli T, Lo Priore E, Balmelli C, Llamas M, Pallanza M, et al. Early administered antibiotics do not impact mortality in critically ill patients with COVID-19. J Infect. (2020) 81:e148–9. doi: 10.1016/j.jinf.2020.06.004 PubMed Abstract | CrossRef Full Text | Google Scholar 24. Chang CY, Chan KG. Underestimation of co-infections in COVID-19 due to non-discriminatory use of antibiotics. J Infect. (2020) 81:e29–30. doi: 10.1016/j.jinf.2020.06.077 PubMed Abstract | CrossRef Full Text | Google Scholar 25. Miranda C, Silva V, Capita R, Alonso-Calleja C, Igrejas G, Poeta P. Implications of antibiotics use during the COVID-19 pandemic: present and future. J Antimicrob Chemother. (2020) 75:3413–6. doi: 10.1093/jac/dkaa350 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: microbial resistance, antibiotics, Public Health, antimicrobial stewardship, global healh Citation: Razzaque MS (2021) Commentary: Microbial Resistance Movements: An Overview of Global Public Health Threats Posed by Antimicrobial Resistance, and How Best to Counter. Front. Public Health 8:629120. doi: 10.3389/fpubh.2020.629120 Received: 13 November 2020; Accepted: 11 December 2020; Published: 20 January 2021. Edited by: Reviewed by: Copyright © 2021 Razzaque. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Mohammed S. Razzaque, [email protected]; [email protected]
Pedro Réquio
Cadernos de Literatura Comparada pp 275-290; https://doi.org/10.21747/2183-2242/cad44a16

Abstract:
This article aims to evaluate the creation and literary dissemination of students from the Academic Association of Coimbra (AAC) during the period between 1958 and 1962. The chronological limits are justified by Humberto Delgado’s candidacy to the 1958 presidential elections, which brings with it the politicization of different sectors of society, and the first academic crisis, in 1962. During the period mentioned in the AAC magazine, Via Latina, many short stories and literary essays by university students were published. This production denotes a clear influence of the artistic currents that dominated the Portuguese cultural panorama at the time. Inspired by neo-realism and existentialism, as well as by democratizing political opportunities, these authors carried out a transformation of the university and Coimbra’s cultural panorama. For a richer analysis, Via Latina is compared with other academic journals of the time. This article has used interviews with members of the magazine.
Temistocles Diaz, Barry H. Trachtenberg, Samuel J. K. Abraham, Rao Kosagisharaf,
Frontiers in Cardiovascular Medicine, Volume 7; https://doi.org/10.3389/fcvm.2020.562708

Abstract:
The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has come to be one of the gravest pandemics of the last two centuries. WHO epidemiological records on COVID-19 outbreak confirmed more than 35 million cases and 1 million deaths worldwide since the disease have originated in Wuhan, China (1, 2). Although clinical treatment of COVID-19 patients focuses on the pulmonary complications and acute respiratory distress syndrome (ARDS), medical reports have also pointed toward the severe deterioration of the patient's state of health due to cardiovascular complications. Furthermore, cardiovascular comorbidities have been determined as key factors of mortality for SARS-CoV-2 infected patients, which present high blood levels of cardiac-specific proteins troponin I and/or T, indicative signs of hypoxia, tachyarrhythmia, myocarditis, and myocardial injury (3–8). Other cardiovascular injuries associated with COVID-19 are venous and arterial thrombosis, and venous thromboembolism (VTE). Case studies performed on COVID-19 patients, and autopsies conducted on those who died due to cardiovascular complications such as stroke and acute coronary syndromes, point to thrombotic disease as a critical factor of mortality in severe cases of COVID-19 (9–12). Severe cases of SARS-COV-2 infected patients experience lymphocytopenia and a high activation of metabolic proinflammatory cytokines mechanisms which leads to an elevated blood concentration of interleukin (IL) 2 (IL-2), IL-6, IL-7, interferon gamma (IFN-γ), macrophage inflammatory protein- 1 alpha (MIP1A), and tumor necrosis factor alpha (TNF-α) pro-inflammatory cytokines (4, 13). This high level of cytokines, known as cytokine storm syndrome (CSS), tends to be a critical factor of morbidity and mortality for COVID-19 patients. CSS contributes to the upregulation of metabolic coagulation pathways resulting in damage to the endothelium, and therefore to the cardiovascular system (14–16). Furthermore, oxygen deprivation seems to mediate the hypercoagulability in COVID-19 (9). Patients with severe COVID-19 pneumonia progress to ARDS, accompanied by disseminated intravascular coagulation (DIC) (17), which may upregulate the coagulation pathways by activation of procoagulant factors, such as tissue factor, leading to both arterial and venous thrombotic disease. These biochemical mechanisms are main factors associated with disturbance of blood coagulation in COVID-19 patients. On the other hand, there is a potential risk of VTE associated with administration of some medications to severe cases of COVID-19 (18). Clinical reports have indicated thrombotic disease in around 25–30% SARS-CoV-2 infected patients, mainly in seriously ill patients (9, 10, 12, 19–22). Therefore, early anticoagulant treatment certainly leads to a better prognosis. In this sense, the antithrombotic properties of aspirin make it a plausible drug for thrombotic disease prevention, the efficacy of which requires to be validated in COVID-19 patients. Acetylsalicylic acid, aspirin, is an antiplatelet drug that inhibits platelet aggregation. The main biochemical mechanism by which aspirin inhibits thrombotic damage is through irreversible inactivation of cyclooxygenase 1 (COX-1) enzyme. In this respect, aspirin acetyl group attaches to the active side of COX-1 at S529, inhibiting the biosynthesis of prostaglandin H2 (PGH2), which is the substrate of thromboxane-A synthase that catalyzes the generation of the prothrombotic eicosanoid thromboxane A2 (TXA2) (Figure 1) suppressing platelet aggregation leading to the prevention of VTE without significant alterations in the endothelial function. On the other hand, aspirin acetylates fibrinogen and other proteins involved in blood coagulation, also preventing thrombus formation. These biochemical mechanisms cause a decrease of dense granule release from platelets (23, 24). Activated platelets and endothelial cells biosynthesize P-selectin, a cell-adhesion glycoprotein that promotes leukocyte and platelet adhesion, and the attachment of leukocytes to the vascular endothelium. Aspirin inhibits P-selectin, which results in the reduction of deep vein thrombosis (DVT) (25). Other antithrombotic mechanism of aspirin involves upregulation of nitric oxide (NO) metabolic production by endothelial cells, through inhibition prostacyclin synthesis, which leads to platelet inactivation (26). Additionally, aspirin prevents the formation of the serine protease enzyme thrombin, which catalyzes the transformation of fibrinogen to fibrin and, hence, leads to the formation of blood clot. Furthermore, thrombin is a potent mediator of platelet activation and aggregation. Thrombin promotes its biosynthesis by feedback activation of prothrombinase complex coenzymes (factors V and VIII). Aspirin inhibits tissue factor (TF): Factor VIIa (FVIIa) complex that catalyzes thrombin formation and, therefore, thrombin-mediated coagulation pathways (24). Figure 1. Putative mechanism of thrombotic disease associated with cytokine storms in COVID-19 patients, and biochemical mechanism of aspirin to prevent arterial/venous thrombosis and thromboembolism. Aspirin is certainly one of the most used drugs in medicine. In addition to its antithrombotic activity, aspirin is very well-known for its antipyretic, antiviral, and analgesic properties (27). Aspirin bioactivity inhibits virus replication such as influenza virus, hepatitis C virus, and flavivirus. Among the reported metabolic mechanisms that produces inhibition is the activation of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase ½ and p38 mitogen-activated protein kinase. Furthermore, an additional mechanism is the inhibition of the proinflammatory transcription factor NF-κB which is relevant for viral genes expression (28). Clinical studies have reported reduction in the rate of stroke, peripheral artery disease, thromboembolism, and myocardial infarction (MI). At present, aspirin is recommended for primary and secondary prevention of stroke, anterior MI with left ventricular thrombus, peripheral artery disease, and acute coronary syndrome (29, 30). A multicenter, double-blind, placebo-controlled study WARFASA (the Aspirin for the Prevention of Recurrent Venous Thromboembolism Warfarin and Aspirin) for the assessment of the efficacy of aspirin for prevention and treatment of VTE, which included 403 patients, showed a lower VTE in patients who received aspirin than in patients not receiving antithrombotic treatment (28 [6.6%] of 205 patients vs. 43 [11.2%] of 197 patients) (31). Furthermore, in a more recent clinical trial of 1,224 patients on the prevention of recurrent unprovoked venous thromboembolism by aspirin, performed by the International Collaboration of Aspirin Trials for Recurrent Venous Thromboembolism (INSPIRE), it was found that aspirin reduces the risk of recurrence of DVT by 34%, without significantly increasing the risk of bleeding (32). Clinical studies from China showed that ARDS developed in a short period of time in COVID-19 patients, which leads to a high number of deaths in severe cases of the disease (74%) (33, 34). ARDS causes uncontrolled coagulation disfunction in severely ill patients (35). Clinical trials reported a decrease in number of cases of ARDS in patients treated with aspirin, which is can be explained by the antithrombotic properties of the drug (36). On the other hand, there are an important number of clinical investigations, registered at ClinicalTrials.gov, which are currently studying the bioactivities of aspirin in COVID-19 patients (NCT04365309; NCT04363840; NCT04333407; NCT04343001; NCT04324463; NCT04368377; NCT04410328; NCT04425863; NCT04466670; NCT04498273) (37). In the light of the evidence discussed in this article, it is clear that patients infected with SARS-CoV-2 virus experience an increment of proinflammatory cytokine molecules, which is a main factor that leads to abnormal platelet aggregation, which causes thrombosis and thromboembolism in COVID-19 patients. Thrombotic disease leads to alterations of many organs, mainly the lung, and the cardiovascular system. Treatment of patients in early stages of COVID-19 with low-dose aspirin (75–100 mg) represents an important pharmacological strategy for prevention of platelet aggregation, which leads to a predictable potential disease progression of arterial/venous thrombosis and thromboembolism, based on the metabolic mechanisms of action of this drug. The incorporation of aspirin therapeutical evaluation into clinical trials that studies the effectiveness of the drug on early stages patients with COVID-19, as well as its concomitant use with antiviral drugs, will enhance our knowledge regarding the physiological factors which promotes health and the development of more accurate therapeutical protocols. RK and AD-A started the debate topic. TD, BT, SA, RK, and AD-A participated in discussions and made substantial contributions to the conception of the work, and writing of the manuscript. All authors read and agreed to its submission. All authors contributed to the article and approved the submitted version. This work was supported by National System of Research Awards (SNI) of Panama; Institute of Scientific Research and High Technology Services. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. RK and AD-A gratefully acknowledge the support received through the National System of Research Awards (SNI) of Panama. We acknowledge Rita Marissa Giovani for technical support. 1. World Health Organization. Coronavirus Disease 2019 (COVID-19). Available online at: www.who.int/emergencies/diseases/novel-coronavirus-2019 (accessed October 6, 2020). Google Scholar 2. Olum R, Chekwech G, Wekha G, Nassozi DR, Bongomin F. Coronavirus disease-2019: knowledge, attitude, and practices of health care workers at makerere university teaching hospitals, Uganda. Front Public Health. (2020) 8:181. doi: 10.3389/fpubh.2020.00181 PubMed Abstract | CrossRef Full Text | Google Scholar 3. Banerjee A, Pasea L, Harris S, Gonzalez-Izquierdo A, Torralbo A, Shallcross L, et al. Estimating excess 1-year mortality associated with the COVID-19 pandemic according to underlying conditions and age: a population-based cohort study. Lancet. (2020). doi: 10.1016/S0140-6736(20)30854-0 PubMed Abstract | CrossRef Full Text | Google Scholar 4. Wang L, Zhang Y, Zhang S. Cardiovascular impairment in COVID-19: learning from current options for cardiovascular anti-inflammatory therapy. Front Cardiovasc Med. (2020) 7:78. doi: 10.3389/fcvm.2020.00078 PubMed Abstract | CrossRef Full Text | Google Scholar 5. Li B, Yang J, Zhao F, Zhi L, Wang X, Liu L, et al. Prevalence and impact of cardiovascular metabolic diseases on COVID-19 in China. Clin Res Cardiol. (2020) 109:531–8. doi: 10.1007/s00392-020-01626-9 PubMed Abstract | CrossRef Full Text | Google Scholar 6. Kang Y, Chen T, Mui D, Ferrari V, Jagasia D, Scherrer-Crosbie M, et al. Cardiovascular manifestations and treatment considerations in COVID-19. Heart. (2020) 106:1132–41. doi: 10.1136/heartjnl-2020-317056 PubMed Abstract | CrossRef Full Text | Google Scholar 7. Guzik TJ, Mohiddin SA, Dimarco A, Patel V, Savvatis K, Marelli-Berg FM, et al. COVID-19 and the cardiovascular system: implications for risk assessment, diagnosis, and treatment options. Cardiovasc Res. (2020) 116:1666–87. doi: 10.1093/cvr/cvaa106 PubMed Abstract | CrossRef Full Text | Google Scholar 8. Guo T, Fan Y, Chen M, Wu X, Zhang L, He T, et al. Cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (COVID-19). JAMA Cardiol. (2020) 5:811–18. doi: 10.1001/jamacardio.2020.1017 PubMed Abstract | CrossRef Full Text | Google Scholar 9. Klok FA, Kruip MJHA, van der Meer NJM, Arbous MS, Gommers DAMPJ, Kant KM, et al. Incidence of thrombotic complications in critically ill ICU patients with COVID-19. Thromb Res. (2020) 191:145–7. doi: 10.1016/j.thromres.2020.04.013 CrossRef Full Text | Google Scholar 10. Ranucci M, Ballotta A, Di Dedda U, Bayshnikova E, Dei Poli M, Resta M, et al. The procoagulant pattern of patients with COVID-19 acute respiratory distress syndrome. J Thromb Haemost. (2020) 18:1747–51. doi: 10.1111/jth.14854 PubMed Abstract | CrossRef Full Text | Google Scholar 11. Bikdeli B, Madhavan MV, Jimenez D, Chuich T, Dreyfus I, Driggin E, et al. COVID-19 and Thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up. J Am Coll Cardiol. (2020) 75:2950–73. doi: 10.1016/j.jacc.2020.04.031 CrossRef Full Text | Google Scholar 12. Wichmann D, Sperhake JP, Lütgehetmann M, Steurer S, Edler C, et al. Autopsy findings and venous thromboembolism in patients with COVID-19: a prospective cohort study. Ann Intern Med. (2020). doi: 10.7326/M20-2003 PubMed Abstract | CrossRef Full Text | Google Scholar 13. Coperchini F, Chiovato L, Croce L, Magri F, Rotondi M. The cytokine storm in COVID-19: an overview of the involvement of the chemokine/chemokine-receptor system. Cytokine Growth Factor Rev. (2020) 53:25–32. doi: 10.1016/j.cytogfr.2020.05.003 PubMed Abstract | CrossRef Full Text | Google Scholar 14. Levi M, van der Poll T, Büller H R. Bidirectional relation between inflammation and coagulation. Circulation. (2004) 109:2698–704. doi: 10.1161/01.CIR.0000131660.51520.9A PubMed Abstract | CrossRef Full Text | Google Scholar 15. Ozeren A, Aydin M, Tokac M, Demircan N, Unalacak M, Gurel A, et al. Levels of serum IL-1beta, IL-2, IL-8 and tumor necrosis factor-alpha in patients with unstable angina pectoris. Mediators Inflamm. (2003) 12:361–5. doi: 10.1080/09629350310001633360 PubMed Abstract | CrossRef Full Text | Google Scholar 16. Levi M, Poll T. Coagulation in patients with severe sepsis. Semin Thromb Hemost. (2015) 41:9–15. doi: 10.1055/s-0034-1398376 CrossRef Full Text | Google Scholar 17. Whyte C, Morrow G, Mitchell J, Chowdary P, Mutch N. Fibrinolytic abnormalities in acute respiratory distress syndrome (ARDS) and versatility of thrombolytic drugs to treat COVID-19. J Thromb Haemost. (2020) 18:1548–55. doi: 10.1111/jth.14872 PubMed Abstract | CrossRef Full Text | Google Scholar 18. Tang N, Bai H, Chen X, Gong J, Li D, Sun Z. Anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy. J Thromb Haemost. (2020) 18:1094–9. doi: 10.1111/jth.14817 CrossRef Full Text | Google Scholar 19. Levi M, Thachil J, Iba T, Levy J. Coagulation abnormalities and thrombosis in patients with COVID-19. Lancet Haematol. (2020) 7:e438–40. doi: 10.1016/S2352-3026(20)30145-9 PubMed Abstract | CrossRef Full Text | Google Scholar 20. Cui S, Chen S, Li X, Liu S, Wang F. Prevalence of venous thromboembolism in patients with severe novel coronavirus pneumonia. J Thromb Haemost. (2020) 18:1421–4. doi: 10.1111/jth.14830 PubMed Abstract | CrossRef Full Text | Google Scholar 21. Bangalore S, Sharma A, Slotwiner A, Yatskar L, Harari R, Shah B. ST-segment elevation in patients with covid-19 - a case series. N Engl J Med. (2020) 382:2478–80. doi: 10.1056/NEJMc2009020 PubMed Abstract | CrossRef Full Text | Google Scholar 22. Lodigiani C, Iapichino G, Carenzo L, Cecconi M, Ferrazzi P, Sebastian T, et al. Venous and arterial thromboembolic complications in COVID-19 patients admitted to an academic hospital in Milan, Italy. Thromb Res. (2020) 191:9–14. doi: 10.1016/j.thromres.2020.04.024 PubMed Abstract | CrossRef Full Text | Google Scholar 23. Fuster V, Sweeny JM. Aspirin: a historical and contemporary therapeutic overview. Circulation. (2011) 123:768–78. doi: 10.1161/CIRCULATIONAHA.110.963843 PubMed Abstract | CrossRef Full Text | Google Scholar 24. Undas A, Brummel-Ziedins K, Mann K. Why does aspirin decrease the risk of venous thromboembolism? On old and novel antithrombotic effects of acetyl salicylic acid. J Thromb Haemost. (2014) 12:1776–87. doi: 10.1111/jth.12728 PubMed Abstract | CrossRef Full Text | Google Scholar 25. Myers D, Wrobleski S, Londy F, Fex B, Hawley A, Schaub R, et al. New and effective treatment of experimentally induced venous thrombosis with anti-inflammatory rPSGL-Ig. Thromb Haemost. (2002) 87:374–82. doi: 10.1055/s-0037-1613014 PubMed Abstract | CrossRef Full Text | Google Scholar 26. Ruggeri Z. Platelets in atherothrombosis. Nat Med. (2002) 8:1227–34. doi: 10.1038/nm1102-1227 PubMed Abstract | CrossRef Full Text | Google Scholar 27. Cadavid A. Aspirin: the mechanism of action revisited in the context of pregnancy complications. Front Immunol. (2017) 8:261. doi: 10.3389/fimmu.2017.00261 PubMed Abstract | CrossRef Full Text | Google Scholar 28. Zimmermann P, Curtis N. Antimicrobial effects of antipyretics. Antimicrob Agents Chemother. (2017) 61:e02268. doi: 10.1128/AAC.02268-16 PubMed Abstract | CrossRef Full Text | Google Scholar 29. Eikelboom J, Hirsh J, Spencer F, Baglin T, Weitz J. Antiplatelet drugs: antithrombotic therapy and prevention of thrombosis, 9th ed: American college of chest physicians evidence-based clinical practice guidelines. Chest. (2012) 141:e89S−119S. doi: 10.1378/chest.11-2293 CrossRef Full Text | Google Scholar 30. Brighton T, Eikelboom J, Mann K, Mister R, Gallus A, Ockelford P, et al. Low-dose aspirin for preventing recurrent venous thromboembolism. N Engl J Med. (2012) 367:1979–87. doi: 10.1056/NEJMoa1210384 PubMed Abstract | CrossRef Full Text | Google Scholar 31. Becattini C, Agnelli G, Schenone A, Eichinger S, Bucherini E, Silingardi M, et al. Aspirin for preventing the recurrence of venous thromboembolism. N Engl J Med. (2012) 366:1959–67. doi: 10.1056/NEJMoa1114238 PubMed Abstract | CrossRef Full Text | Google Scholar 32. Simes J, Becattini C, Agnelli G, Eikelboom J, Kirby A, Mister R, et al. Aspirin for the prevention of recurrent venous thromboembolism: the INSPIRE collaboration. Circulation. (2014) 130:1062–71. doi: 10.1161/CIRCULATIONAHA.114.008828 PubMed Abstract | CrossRef Full Text | Google Scholar 33. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. (2020) 395:497–506. doi: 10.1016/S0140-6736(20)30183-5 PubMed Abstract | CrossRef Full Text | Google Scholar 34. Du Y, Tu L, Zhu P, Mu M, Wang R, Yang P, et al. Clinical features of 85 fatal cases of COVID19 from Wuhan: a retrospective observational study. Am J Respir Crit Care Med. (2020) 201:1372–9. doi: 10.1164/rccm.202003-0543OC PubMed Abstract | CrossRef Full Text | Google Scholar 35. Yuki K, Fujiogi M, Koutsogiannaki S. COVID19 pathophysiology: a review. Clin Immunol. (2020) 215:108427. doi: 10.1016/j.clim.2020.108427 PubMed Abstract | CrossRef Full Text | Google Scholar 36. Panka B, de Grooth H-J, Spoelstra-de Man A, Looney M, Pieter-Roel Tuinman P-R. Prevention or treatment of ARDS with aspirin: a review of preclinical models and meta-analysis of clinical studies. Shock. (2017) 47:13–21. doi: 10.1097/SHK.0000000000000745 PubMed Abstract | CrossRef Full Text | Google Scholar 37. ClinicalTrials.gov. Available online at: www.clinicaltrials.gov (accessed October 6, 2020). Google Scholar Keywords: molecular mechanisms, aspirin, SARS-CoV-2, COVID-19, prevention of cardiovascular disease Citation: Diaz T, Trachtenberg BH, Abraham SJK, KosagiSharaf R and Durant-Archibold AA (2020) Aspirin Bioactivity for Prevention of Cardiovascular Injury in COVID-19. Front. Cardiovasc. Med. 7:562708. doi: 10.3389/fcvm.2020.562708 Received: 26 May 2020; Accepted: 10 November 2020; Published: 30 November 2020. Edited by: Reviewed by: Copyright © 2020 Diaz, Trachtenberg, Abraham, KosagiSharaf and Durant-Archibold. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Rao KosagiSharaf, [email protected]; Armando A. Durant-Archibold, [email protected]
Varun J Sharma, Minesh Prakash, Zaw Lin, Casey Lo
Interactive CardioVascular and Thoracic Surgery, Volume 32, pp 106-110; https://doi.org/10.1093/icvts/ivaa215

Abstract:
Summary A best evidence topic in cardiac surgery was written according to a structured protocol. The question addressed was ‘in patients with ascending aortic or aortic arch disease what are the outcomes with endovascular repair in terms of survival, complications and reintervention?’ Altogether 585 papers were found using the reported search, of which 9 represented the best evidence to answer the clinical question. The authors, journal, date and country of publication, patient group studied, study type, relevant outcomes and results of these papers are tabulated. We found that the endovascular operative techniques with the greatest evidence were ascending aortic chimney grafts (AACs), branched thoracic endovascular aortic repair (bTEVAR) aortic grafts and fenestrated TEVAR (fTEVAR) aortic grafts. The best evidence available were small case-series or retrospective cohort studies (n < 100), with 1 systematic review, at a short follow-up period (range 0–5 years). Intraoperatively, these techniques have a high technical success rate (84–100%). We found rates of endoleak comparable between AAC (7.4–16%) and bTEVAR/fenestrated TEVAR (11.1–21.4%). Stroke rates are higher in bTEVAR (3.1–42% vs 1–26% in AACs), attributed to more proximal pathology and technically challenging procedures. Following the immediate postoperative period, the 30-day mortality is 0–10.8% and patency is 97–100%. Stroke and reintervention rates remain higher in the bTEVAR group (3.1–42.0% and 0.5–33.3%) compared to the AAC group (1.0–11.1% and 6.7–16.7%). The 3- and 5-year survival ranges from 59% to 90%, but is driven by non-aortic pathology in a high-risk population; 3-year freedom from aortic death is 93–97%. Patency is 97–100% at up to 3 years, conformation and supra-aortic occlusions thereafter remain unknown. We conclude that AACs, bTEVARs and fenestrated TEVARs are safe endovascular options in high-risk elective patients, with results comparable to open or hybrid repair. They remain unverified in acute settings or in patients fit for open intervention.
Markus Blaess, Lars Kaiser, Oliver Sommerfeld, Simone Rentschler, René Csuk,
Published: 20 November 2020
Frontiers in Pharmacology, Volume 11; https://doi.org/10.3389/fphar.2020.584881

Abstract:
The pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the disease-causing pathogen of Coronavirus disease 2019 (COVID-19). (Pre)clinical research to identify rapidly available small molecules for the treatment of SARS-CoV-2 infections/COVID-19 has focused to date on the approved lysosomotropic antimalarials chloroquine and hydroxychloroquine, the investigational remdesivir (GS-5734, compassionate use), and the anti-inflammatory corticosteroid dexamethasone (COVID-19 Treatment Guidelines Panel, 2020). Lopinavir/ritonavir and other HIV protease inhibitors, however, were discontinued as treatment options in COVID-19 demonstrating no clinical benefit in clinical trials. Despite encouraging results in treating hospitalized patients with COVID-19 requiring supplemental oxygen, mechanical ventilation, or extracorporeal membrane oxygenation (ECMO) with remdesivir and dexamethasone, there is still a lack of active compounds exhibiting pan-coronavirus antiviral activity, tackling or preventing host cell infection, forming syncytia, endotheliitis, or the cytokine release syndrome (CRS)/cytokine storm syndrome in COVID-19. Target-oriented and in particular site of action-oriented drug repurposing of small molecules has the potential to close the gap in prophylaxis and treatment of mild and moderate COVID-19 and to reduce mortality in severe cases. Oxidative stress (e.g., enhanced ROS levels) has been demonstrated in animal models of SARS (Delgado-Roche and Mesta, 2020) and serves as a possible explanation why SARS-CoV-2 patients with Glucose-6-phosphate dehydrogenase (G6PD) deficiency develop intravascular hemolysis and methemoglobinemia (Palmer et al., 2020). Both, chloroquine and hydroxychloroquine, are supposed to trigger severe drug-induced hemolytic anemia in G6PD-deficient COVID-19 patients (Beauverd et al., 2020; Kuipers et al., 2020). Severe COVID-19 is associated with an atypical diffuse alveolar damage, ending in the acute respiratory distress syndrome (ARDS) (Huang et al., 2020), most likely accompanied by occurrence of syncytia as a result of a direct infection of cells by an infected neighboring cell without releasing a complete virus (Ou et al., 2020). Ceramides, in particular C18-ceramide, are present in (sepsis-induced) cardiac dysfunction (Chung et al., 2017), and are effective in triggering exocytosis in rat PC12 cells (Tang et al., 2007); further they may contribute to SARS-CoV-2-related cell–cell fusion by exocytosis of viral S protein fractions and development of multinucleate syncytia. Non-structural protein nsp2 of SARS-CoV-2 was associated with host cell cell cycle progression, and apoptosis in host cells, suggesting an impact on disrupting the host cell environment (Yoshimoto, 2020) and apoptosis of endothelial cells (Varga et al., 2020). According to current knowledge, cleavage-mediated fusion of viral S protein with host cells can occur either immediately at the cell surface by TMPRSS2 or within the lysosome catalyzed by lysosomal cathepsin L (Belouzard et al., 2012). The lysosomal cathepsin L induced fusion of SARS particles bound to ACE2 with host cells (Millet and Whittaker, 2015) is sensitive to lysosomal pH. Hence both, TMPRSS2 and cathepsin L, display promising targets of prophylaxis and treatment of SARS-CoV-2 infection/COVID-19. In severe COVID-19, SARS-CoV-2 is likely to cause both, pulmonary and systemic inflammation, thus leading to multi-organ dysfunction in high risk populations. Significantly higher concentrations of IL-8, TNFα, and IL-6 in deceased patients (Chen et al., 2020) are suggesting a rapid and severe deterioration during SARS-CoV-2 infection associated with CRS/cytokine storm syndrome (Mehta et al., 2020). Lysosomotropism is a biological characteristic of small molecules and always present in addition to intrinsic pharmacological effects. Various well-known approved drugs such as amitriptyline, chlorpromazine, sertraline, and imipramine share lysosomotropic characteristics (Figure 1A) (Kornhuber et al., 2008; Blaess et al., 2018). Regardless of their pharmacological effects, they are accumulating in lysosomes raising the lysosomal pH from 4.5–5 to 6–6.5, beyond the optimum of most of the lysosomal enzymes, including cathepsin L. Since no effects of lysosomotropic aminoglycoside antibiotics on free cathepsin L (Zhou et al., 2016) or other lysosomotropic drugs on lysosomal enzymes such as acid sphingomyelinase exist (Blaess et al., 2018), a selective inhibition is unlikely. FIGURE 1. (A) Variety of approved lysosomotropic compounds for various indications (Kornhuber et al., 2008; Blaess et al., 2018). Achievement of the desired lysosomotropic effect depends on the active compound, the dosage, and accumulation in lysosomes. Unless indicated, maximum daily doses are split into three applications. *Lysosomotropism very likely, but not yet confirmed, lysosomal drug concentration (effect) within the therapeutic margin expected; dosage: #single dose per day; xin vitro anti-SARS‐CoV tested, xxin vitro anti-SARS-CoV and anti-SARS-CoV-2 tested (Vincent et al., 2005; Kornhuber et al., 2008; Dyall et al., 2014; Zhou et al., 2016; Blaess et al., 2018; Liu et al., 2020; Weston et al., 2020). (B) Cellular targets, cellular effects, and effects related effects of lysosomotropic active compounds in SARS-CoV-2 infection/COVID-19 (Vincent et al., 2005; Masters, 2006; Mingo et al., 2015; Zhou et al., 2016; Blaess et al., 2018; Varga et al., 2020; Zhou et al., 2020). Lysosomotropic compounds target in mammalian cells three major targets related to SARS-CoV-2 infection/COVID-19: cathepsin L (1), gene expression of inflammation-relevant genes (2), C16-ceramide and C18-ceramide synthesis, and apoptosis of host cells (3). Addressing targets 1–3 results in various disease process interfering effects supposed to improve SARS-CoV-2 infection/COVID-19 outcome; (°) in viral infection and bacterial superinfection, (°°) only in bacterial superinfection. Lysosomotropic compounds are not limited to mediate inactivation of cathepsin L Figure 1B. Moreover, lysosomotropic compounds are assumed to suppress the CRS/cytokine storm syndrome and to attenuate the transition from mild to severe SARS-CoV-2 infection/COVID-19 (Zhou et al., 2020). Data of the lysosomotropic model compound NB 06 in LPS-induced inflammation in monocytic cells (Blaess et al., 2018) supports the hypothesis. NB 06 affects gene expression of the prominent inflammatory messengers IL1B, IL23A, CCL4, CCL20, and IL6; likewise, it has beneficial effects in (systemic) infections involving bacterial endotoxins by targeting the TLR4 receptor pathway in sepsis. Similarly, desipramine reduces endothelial stress response in systemic inflammation (Chung et al., 2017). Apoptosis of (infected) mammalian cells is characterized by an increase in C16-ceramide (Thomas et al., 1999) and can be blocked via lysosomotropic compounds such as NB 06, chlorpromazine, and imipramine (Blaess et al., 2018). Furthermore, C18-ceramide triggered exocytosis and forming of syncytia is blocked by chlorpromazine as well (Garner et al., 2010). According to current knowledge, in therapy inhibition of lysosomal pH dependent processes (e.g., cathepsin L dependent viral entry into host cells) can be obtained only through off-label use of lysosomotropic drugs. Systemic application in lysosomotropic drug concentrations and obtaining an efficacious blood level is sometimes accompanied by severe adverse effects and/or (in this case) undesirable (intrinsic) pharmacological effects. Chloroquine was among the first lysosomotropic active compounds exerting antiviral effects on SARS-CoV-2 (Liu et al., 2020) and during SARS-CoV pre- and post-infection conditions (Vincent et al., 2005). Owing to an unfavorable drug profile (G6PD patients, insufficient lysosomotropism, elimination half-life of 45 ± 15 days), a recommendation against (hydroxy)chloroquine, but not against lysosomotropic active compounds in principle was issued (COVID-19 Treatment Guidelines Panel, 2020). Chlorpromazine displayed anti-SARS-CoV-2 effects in vitro (Weston et al., 2020) and protective effects on COVID-19 in patients in a psychiatry hospital (NCT04366739). Consequently, chlorpromazine is rated as a promising candidate in COVID-19/CRS treatment. In case of treatment of people without mental illness, however, a premature termination of treatment due to severe side effects by systemic application of chlorpromazine is extremely likely. This raises the question of how to handle this issue to provide well tolerated lysosomotropic drugs in SARS-CoV-2 infection/COVID-19. Numerous available approved drugs with lysosomotropic characteristics permit tailor-made therapy. The individual pre-existing conditions are a criterion for the selection and combination of lysosomotropic drugs. For choosing suitable lysosomotropic drugs some issues have to be considered: Various lysosmotropic drugs in Figure 1A demonstrated anti-SARS-CoV(-2) efficacy (Dyall et al., 2014; Zhou et al., 2016; Liu et al., 2020; Weston et al., 2020), offer a more favorable drug profile than the initially investigated chloroquine and hydroxychloroquine. Imipramine and chlorpromazine are accumulating in isolated perfused lung tissue and imipramine in alveolar macrophages (Wilson et al., 1982; Macintyre and Cutler, 1988) suggesting that lysosomotropic drug concentrations in pulmonary alveoli and protective effects on SARS-CoV-2 infection of particular drugs are likely. Of the lysosomotropic in vitro anti-SARS-CoV-2 antibiotics teicoplanin, oritavancin, dalbavancin, and telavancin (Zhou et al., 2016), solely teicoplanin and telavancin are in accumulating pulmonary tissue and are expected to be a treatment option. Beside lysosomotropism certain intrinsic pharmacological effects are advantageously in SARS-CoV-2 infection/COVID-19. The incidence of CRS/cytokine storm syndrome associated with secondary gram-positive bacterial infections is likely to be minimized by using the pulmonary tissue accumulating antibacterials teicoplanin and telavancin or the antifungal itraconazole in systemic mycoses in appropriate systemic drug levels. Systemic application of chlorpromazine (NCT04366739) and fluoxetine (NCT04377308) as lysosomotropic drugs may provoke severe and unfavorable adverse effects in mental healthy patients. Since the respiratory tract is both, the gateway for SARS-CoV-2 infection/COVID-19 and an internal surface, the expedient is a local application in the airways and/or the respiratory tract. Local application of small molecules is possible, preferably as inhalant or via nebulizers to avoid (undesirable) systemic effects. The majority of lysosomotropic drugs should be suitable for inhalation. COVID-19 originates from a SARS-CoV-2 infection that could not be tackled successfully by the immune system. The antiviral remdesivir proved to be effective in infection prophylaxis (phase 0) (de Wit et al., 2020) and viral (SARS-CoV-2) infection (phase 1) within a limited period (5–6 days), shortly after the symptoms emerge and viral shedding occurs (Mitjà and Clotet, 2020). In severe COVID-19 neither a lower mortality nor a faster clearance of viruses was observed (Wang et al., 2020). As soon as the infection initiates a CRS/cytokine storm, it is likely that the transition toward COVID-19 (phase 2), a disseminated intravascular coagulation/thrombotic microangiopathy, or a bacterial secondary infection occurs. An effective multi-drug therapy, focusing on the progression of COVID-19 and emerging severe complications, can be implemented by lysosomotropic drugs, TMPRSS2 inhibitors and antivirals. Nafamostat is an approved protease inhibitor that inhibits TMPRSS2 (in vitro) (Hoffmann et al., 2020), prevents (sepsis-related) disseminated intravascular coagulation, and thrombotic microangiopathy (Okajima et al., 1995; Levi and Thachil, 2020), appears to be useful in SARS-CoV-2 infection and prophylaxis, and for patients subjected to extracorporeal circulation such as ECMO (Han et al., 2011). It is doubtful, however, whether the pulmonary concentration in therapeutically dosage (Ono Pharmaceuticals, 2020) is sufficient to generate a TMPRSS2 inhibition in vivo as demonstrated in vitro due to poor accumulation in pulmonary tissue (Midgley et al., 1994). Various clinical trials are currently under way using immunomodulatory IL-1 and IL-6 inhibitors or anti-IL-6R antibodies (anakinra, tocilizumab, siltuximab, and sarilumab) in patients with COVID-19 (COVID-19 Treatment Guidelines Panel, 2020); limited data, however, is yet available. In a retrospective study using tocilizumab and hydroxychloroquine, both demonstrated a limited benefit in survival (Ip et al., 2020). Tocilizumab shortens mechanical ventilation and hospital stay in severe COVID-19 (Eimer et al., 2020), while tocilizumab is often accompanied by bacterial pneumonia 2 days after application (23%) (Pettit et al., 2020). To improve outcome, antibody cocktails consisting of anti-IL-6, IL-1 receptor blocker, IL-1 type 1 receptor, and TNF-α are suggested (Harrison, 2020), irrespective of the risk of serious adverse effects (e.g., bacterial pneumonia) due to more pronounced interference with the immune defense. Such cocktails are intended to tackle the release of pro-inflammatory cytokines IL-1β and IL-6 mediating lung and tissue inflammation, fever, and fibrosis, as they are supposed to be responsible for the emergence of COVID-19. Although lysosomotropic drugs likewise interfere with the immune defense, such adverse effects are not reported. In contrast to antibodies, however, only the resynthesis of IL-6 and thus the available amount is reduced, but not completely obstructed, still allowing a moderate immune response. Multitargeting on core processes of the viral infection addressing the formation of multinucleate syncytia and alteration of tissue structure, ceramide metabolism, and the release of virions could be a key advantage of lysosomotropic drugs compared to current strategies. Daunting results of (hydroxy)chloroquine in clinical trials are closely related to their drug profile and minor lysosomotropism, but not to the mode of action (lysosomotropism) in general. Observations in patients treated with chlorpromazine and the extensive accumulation of imipramine in alveolar macrophages and of both, imipramine and chlorpromazine in isolated perfused lung tissue supports the benefits of lysosomotropic drugs that are accumulating in pulmonary tissue in SARS-CoV-2 infection/COVID-19. Promising candidates among lysosomotropic drugs in fact require more than adequate lysosomotropism; accumulation in pulmonary tissue is a prerequisite as well. It is, however, likely irrelevant whether the drug or its metabolite(s) is accumulating given the broad structural requirements for this activity. Since a large number of compounds has not yet been evaluated for lysosomotropism, many compounds beside those listed in Figure 1A are expected to meet the requirements described here and may (partially) be responsible for background immunity to SARS-CoV infection. MB conceived the work. MB, LK, OS, SR, RC, and H-PD wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding from the Institute of Precision Medicine, the Federal Ministry of Science, Research and Art of Baden-Wuerttemberg, Germany (researchership for LK) and the Institute for Applied Research (IAF, Furtwangen University, Schwenningen, Germany) is gratefully acknowledged. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank Edith Walther for her tremendous technical support in performing elaborate cell experiments and sample preparation in the initial stages of conceptual work, as well as Petra and Peter Bauer, the staff at Riesling Apotheke (Ellerstadt, Germany), and Apotheke im Markt (Heidelberg, Germany) for their tremendous support. Beauverd, Y., Adam, Y., Assouline, B., and Samii, K. (2020). COVID‐19 infection and treatment with hydroxychloroquine cause severe haemolysis crisis in a patient with glucose‐6‐phosphate dehydrogenase deficiency. Eur. J. Haematol. 105, 357. doi:10.1111/ejh.13432 PubMed Abstract | CrossRef Full Text | Google Scholar Belouzard, S., Millet, J. K., Licitra, B. N., and Whittaker, G. R. (2012). Mechanisms of coronavirus cell entry mediated by the viral spike protein. Viruses 4, 1011–1033. doi:10.3390/v4061011 PubMed Abstract | CrossRef Full Text | Google Scholar Blaess, M., Bibak, N., Claus, R. A., Kohl, M., Bonaterra, G. A., Kinscherf, R., et al. (2018). NB 06: from a simple lysosomotropic aSMase inhibitor to tools for elucidating the role of lysosomes in signaling apoptosis and LPS-induced inflammation. Eur. J. Med. Chem. 153, 73–104. doi:10.1016/j.ejmech.2017.09.021 PubMed Abstract | CrossRef Full Text | Google Scholar Chen, T., Wu, D., Chen, H., Yan, W., Yang, D., Chen, G., et al. (2020). Clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study. BMJ 368, m1091. doi:10.1136/bmj.m1091 PubMed Abstract | CrossRef Full Text | Google Scholar Chung, H.-Y., Kollmey, A., Schrepper, A., Kohl, M., Bläss, M., Stehr, S., et al. (2017). Adjustment of dysregulated ceramide metabolism in a murine model of sepsis-induced cardiac dysfunction. IJMS 18, 839. doi:10.3390/ijms18040839 PubMed Abstract | CrossRef Full Text | Google Scholar COVID-19 Treatment Guidelines Panel (2020). Coronavirus disease 2019 (COVID-19) treatment guidelines. COVID-19 treatment guidelines. Available at: https://www.covid19treatmentguidelines.nih.gov/ (Accessed July 17, 2020). PubMed Abstract | Google Scholar de Wit, E., Feldmann, F., Cronin, J., Jordan, R., Okumura, A., Thomas, T., et al. (2020). Prophylactic and therapeutic remdesivir (GS-5734) treatment in the rhesus macaque model of MERS-CoV infection. Proc. Natl. Acad. Sci. U.S.A. 117, 6771–6776. doi:10.1073/pnas.1922083117 PubMed Abstract | CrossRef Full Text | Google Scholar Delgado-Roche, L., and Mesta, F. (2020). Oxidative stress as key player in severe acute respiratory syndrome coronavirus (SARS-CoV) infection. Arch. Med. Res. 51, 384–387. doi:10.1016/j.arcmed.2020.04.019 PubMed Abstract| CrossRef Full Text | Google Scholar Dyall, J., Coleman, C. M., Hart, B. J., Venkataraman, T., Holbrook, M. R., Kindrachuk, J., et al. (2014). Repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection. Antimicrob. Agents Chemother. 58, 4885–4893. doi:10.1128/AAC.03036-14 PubMed Abstract | CrossRef Full Text | Google Scholar Eimer, J., Vesterbacka, J., Svensson, A. K., Stojanovic, B., Wagrell, C., Sönnerborg, A., et al. (2020). Tocilizumab shortens time on mechanical ventilation and length of hospital stay in patients with severe COVID‐19: a retrospective cohort study. J. Intern. Med., 13162. doi:10.1111/joim.13162 PubMed Abstract | CrossRef Full Text | Google Scholar Garner, O. B., Aguilar, H. C., Fulcher, J. A., Levroney, E. L., Harrison, R., Wright, L., et al. (2010). Endothelial galectin-1 binds to specific glycans on nipah virus fusion protein and inhibits maturation, mobility, and function to block syncytia formation. PLoS Pathog. 6, e1000993, doi:10.1371/journal.ppat.1000993 PubMed Abstract | CrossRef Full Text | Google Scholar Han, S. J., Kim, H. S., Kim, K. I., Whang, S. M., Hong, K. S., Lee, W. K., et al. (2011). Use of nafamostat mesilate as an anticoagulant during extracorporeal membrane oxygenation. J. Korean Med. Sci. 26, 945. doi:10.3346/jkms.2011.26.7.945 PubMed Abstract | CrossRef Full Text | Google Scholar Harrison, C. (2020). Focus shifts to antibody cocktails for COVID-19 cytokine storm. Nat. Biotechnol. 38, 905–908. doi:10.1038/s41587-020-0634-9 PubMed Abstract | CrossRef Full Text | Google Scholar Hoffmann, M., Schroeder, S., Kleine-Weber, H., Müller, M. A., Drosten, C., and Pöhlmann, S. (2020). Nafamostat mesylate blocks activation of SARS-CoV-2: new treatment option for COVID-19. Antimicrob. Agents Chemother. 64, e00754-20. doi:10.1128/AAC.00754-20 PubMed Abstract | CrossRef Full Text | Google Scholar Huang, C., Wang, Y., Li, X., Ren, L., Zhao, J., Hu, Y., et al. (2020). Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395, 497–506. doi:10.1016/S0140-6736(20)30183-5 PubMed Abstract | CrossRef Full Text | Google Scholar Ip, A., Berry, D. A., Hansen, E., Goy, A. H., Pecora, A. L., Sinclaire, B. A., et al. (2020). Hydroxychloroquine and tocilizumab therapy in COVID-19 patients—an observational study. PLoS One 15, e0237693. doi:10.1371/journal.pone.0237693 PubMed Abstract | CrossRef Full Text | Google Scholar Kornhuber, J., Tripal, P., Reichel, M., Terfloth, L., Bleich, S., Wiltfang, J., et al. (2008). Identification of new functional inhibitors of acid sphingomyelinase using a structure-property-activity relation model. J. Med. Chem. 51, 219–237. doi:10.1021/jm070524a PubMed Abstract | CrossRef Full Text | Google Scholar Kuipers, M. T., Zwieten, R., Heijmans, J., Rutten, C. E., Heer, K., Kater, A. P., et al. (2020). Glucose‐6‐phosphate dehydrogenase deficiency‐associated hemolysis and methemoglobinemia in a COVID ‐19 patient treated with chloroquine. Am. J. Hematol. 95. doi:10.1002/ajh.25862 PubMed Abstract | CrossRef Full Text | Google Scholar Levi, M., and Thachil, J. (2020). Coronavirus disease 2019 coagulopathy: disseminated intravascular coagulation and thrombotic microangiopathy-either, neither, or both. Semin. Thromb. Hemost. doi:10.1055/s-0040-1712156 PubMed Abstract | CrossRef Full Text | Google Scholar Liu, J., Cao, R., Xu, M., Wang, X., Zhang, H., Hu, H., et al. (2020). Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro. Cell Discov. 6, 16. doi:10.1038/s41421-020-0156-0 PubMed Abstract | CrossRef Full Text | Google Scholar Macintyre, A. C., and Cutler, D. J. (1988). The potential role of lysosomes in tissue distribution of weak bases. Biopharm. Drug Dispos. 9, 513–526. doi:10.1002/bod.2510090602 PubMed Abstract | CrossRef Full Text | Google Scholar Masters, P. S. (2006). The molecular biology of coronaviruses. Adv. Virus Res. 66, 193–292. doi:10.1016/S0065-3527(06)66005-3 PubMed Abstract | CrossRef Full Text | Google Scholar Mehta, P., McAuley, D. F., Brown, M., Sanchez, E., Tattersall, R. S., and Manson, J. J. (2020). COVID-19: consider cytokine storm syndromes and immunosuppression. Lancet 395, 1033–1034. doi:10.1016/S0140-6736(20)30628-0 PubMed Abstract | CrossRef Full Text | Google Scholar Midgley, I., Hood, A. J., Proctor, P., Chasseaud, L. F., Irons, S. R., Cheng, K. N., et al. (1994). Metabolic fate of 14C-camostat mesylate in man, rat and dog after intravenous administration. Xenobiotica 24, 79–92. doi:10.3109/00498259409043223 PubMed Abstract | CrossRef Full Text | Google Scholar Millet, J. K., and Whittaker, G. R. (2015). Host cell proteases: critical determinants of coronavirus tropism and pathogenesis. Virus Res. 202, 120–134. doi:10.1016/j.virusres.2014.11.021 PubMed Abstract | CrossRef Full Text | Google Scholar Mingo, R. M., Simmons, J. A., Shoemaker, C. J., Nelson, E. A., Schornberg, K. L., D'Souza, R. S., et al. (2015). Ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to NPC1+endolysosomes is a rate-defining step. J. Virol. 89, 2931–2943. doi:10.1128/JVI.03398-14 PubMed Abstract | CrossRef Full Text | Google Scholar Mitjà, O., and Clotet, B. (2020). Use of antiviral drugs to reduce COVID-19 transmission. Lancet Glob. Health 8, e639–e640. doi:10.1016/S2214-109X(20)30114-5 PubMed Abstract | CrossRef Full Text | Google Scholar Okajima, K., Uchiba, M., and Murakami, K. (1995). Nafamostat mesilate. Cardiovasc. Drug Rev. 13, 51–65. doi:10.1111/j.1527-3466.1995.tb00213.x PubMed Abstract | CrossRef Full Text | Google Scholar Ono Pharmaceuticals (2020). Foipan Camostat Mesilate Oral Tablets (Ono Pharmaceuticals). Available at: http://www.shijiebiaopin.net/upload/product/201272318373223.PDF (Accessed May 6, 2020). Google Scholar Ou, X., Liu, Y., Lei, X., Li, P., Mi, D., Ren, L., et al. (2020). Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat. Commun. 11, 1620. doi:10.1038/s41467-020-15562-9 PubMed Abstract | CrossRef Full Text | Google Scholar Palmer, K., Dick, J., French, W., Floro, L., and Ford, M. (2020). Methemoglobinemia in patient with G6PD deficiency and SARS-CoV-2 infection. Emerg. Infect. Dis. 26, 2279. doi:10.3201/eid2609.202353 PubMed Abstract | CrossRef Full Text | Google Scholar Pettit, N. N., Nguyen, C. T., Mutlu, G. M., Wu, D., Kimmig, L., Pitrak, D., et al. (2020). Late onset infectious complications and safety of tocilizumab in the management of COVID‐19. J. Med. Virol., 26429. doi:10.1002/jmv.26429 PubMed Abstract | CrossRef Full Text | Google Scholar Tang, N., Ong, W.-Y., Zhang, E.-M., Chen, P., and Yeo, J.-F. (2007). Differential effects of ceramide species on exocytosis in rat PC12 cells. Exp. Brain Res. 183, 241–247. doi:10.1007/s00221-007-1036-7 PubMed Abstract | CrossRef Full Text | Google Scholar Thomas, R. L., Matsko, C. M., Lotze, M. T., and Amoscato, A. A. (1999). Mass spectrometric identification of increased C16 ceramide levels during apoptosis. J. Biol. Chem. 274, 30580–30588. doi:10.1074/jbc.274.43.30580 PubMed Abstract | CrossRef Full Text | Google Scholar Varga, Z., Flammer, A. J., Steiger, P., Haberecker, M., Andermatt, R., Zinkernagel, A. S., et al. (2020). Endothelial cell infection and endotheliitis in COVID-19. Lancet 395, 1417–1418. doi:10.1016/S0140-6736(20)30937-5 PubMed Abstract | CrossRef Full Text | Google Scholar Vincent, M. J., Bergeron, E., Benjannet, S., Erickson, B. R., Rollin, P. E., Ksiazek, T. G., et al. (2005). Chloroquine is a potent inhibitor of SARS coronavirus infection and spread. Virol. J. 2, 69. doi:10.1186/1743-422X-2-69 PubMed Abstract | CrossRef Full Text | Google Scholar Wang, Y., Zhang, D., Du, G., Du, R., Zhao, J., Jin, Y., et al. (2020). Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trial. Lancet 395, 1569–1578. doi:10.1016/S0140-6736(20)31022-9 PubMed Abstract | CrossRef Full Text | Google Scholar Weston, S., Coleman, C. M., Haupt, R., Logue, J., Matthews, K., and Frieman, M. B. (2020). Broad anti-coronaviral activity of FDA approved drugs against SARS-CoV-2 in vitro and SARS-CoV in vivo. J. Virol. 94 (21), e01218–e01220. doi:10.1101/2020.03.25.008482 PubMed Abstract | CrossRef Full Text | Google Scholar Wilson, A. G., Sar, M., and Stumpf, W. E. (1982). Autoradiographic study of imipramine localization in the isolated perfused rabbit lung. Drug Metab. Dispos. 10, 281–283. PubMed Abstract | Google Scholar Yoshimoto, F. K. (2020). The proteins of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2 or n-COV19), the cause of COVID-19. Protein J. 39 (3), 198–216. doi:10.1007/s10930-020-09901-4 CrossRef Full Text | Google Scholar Zhou, D., Dai, S.-M., and Tong, Q. (2020). COVID-19: a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression. J. Antimicrob. Chemother. 75, 1667–1670. doi:10.1093/jac/dkaa114 PubMed Abstract | CrossRef Full Text | Google Scholar Zhou, N., Pan, T., Zhang, J., Li, Q., Zhang, X., Bai, C., et al. (2016). Glycopeptide antibiotics potently inhibit cathepsin L in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus (SARS-CoV). J. Biol. Chem. 291, 9218–9232. doi:10.1074/jbc.M116.716100 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: SARS-CoV-2, COVID-19, lysosomotropic compounds, approved active compounds, cytokine storm syndrome, lysosomotropism, repurposing approved drugs, lysosome Citation: Blaess M, Kaiser L, Sommerfeld O, Rentschler S, Csuk R and Deigner H-P (2020) Rational Drug Repurposing: Focus on Lysosomotropism, Targets in Disease Process, Drug Profile, and Pulmonary Tissue Accumulation in SARS-CoV-2 Infection/COVID-19. Front. Pharmacol. 11:584881. doi: 10.3389/fphar.2020.584881 Received: 18 July 2020; Accepted: 06 October 2020; Published: 20 November 2020. Edited by: Reviewed by: Copyright © 2020 Blaess, Kaiser, Sommerfeld, Rentschler, Csuk and Deigner. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Hans-Peter Deigner, [email protected]
, Catherine P Bondonno, , Lauren C Blekkenhorst, Reindolf Anokye, Emma Connolly, Nicola P Bondonno, John T Schousboe, Richard J Woodman, Kun Zhu, et al.
Published: 11 November 2020
by BMJ
Abstract:
IntroductionMost cardiovascular disease (CVD)-related events could be prevented or substantially delayed with improved diet and lifestyle. Providing information on structural vascular disease may improve CVD risk factor management, but its impact on lifestyle change remains unclear. This study aims to determine whether providing visualisation and pictorial representation of structural vascular disease (abdominal aortic calcification (AAC)) can result in healthful diet and lifestyle change.Methods and analysisThis study, including men and women aged 60–80 years, is a 12-week, two-arm, multisite randomised controlled trial. At baseline, all participants will have AAC assessed from a lateral spine image captured using a bone densitometer. Participants will then be randomised to receive their AAC results at baseline (intervention group) or a usual care control group that will receive their results at 12 weeks. All participants will receive information about routinely assessed CVD risk factors and standardised (video) diet and lifestyle advice with three simple goals: (1) increase fruit and vegetable (FV) intake by at least one serve per day, (2) improve other aspects of the diet and (3) reduce sitting time and increase physical activity. Clinical assessments will be performed at baseline and 12 weeks.OutcomesThe primary outcome is a change in serum carotenoid concentrations as an objective measure of FV intake. The study design, procedures and treatment of data will adhere to Standard Protocol Items for Randomized Trials guidelines.Ethics and disseminationEthics approval for this study has been granted by the Edith Cowan University and the Deakin University Human Research Ethics Committees (Project Numbers: 20513 HODGSON and 2019-220, respectively). Results of this study will be published in peer-reviewed academic journals and presented in scientific meetings and conferences. Information regarding consent, confidentiality, access to data, ancillary and post-trial care and dissemination policy has been disclosed in the participant information form.Trial registration numberAustralian New Zealand Clinical Trial Registry (ACTRN12618001087246).
Ankita Bhatt, Pratham Arora, Sanjeev Kumar Prajapati
Published: 11 November 2020
Frontiers in Microbiology, Volume 11; https://doi.org/10.3389/fmicb.2020.596374

Abstract:
Microalgae are defined as photosynthetic and unicellular organisms that demonstrate a wide range of adaptability to adverse environmental conditions like temperature extremes, photooxidation, high or low salinity, and osmotic stress (Holzinger and Karsten, 2013; Singh et al., 2019). Alternatively, macroalgae or seaweed includes multicellular, macroscopic, and marine algae belonging mostly to two phyla, namely, Rhodophyta and Phaeophyta (Peng et al., 2015). The micro/macro-algae have recently emerged as a source of various bioactive compounds like phycocyanin, lutein, vitamin E, B12 and K1, polyunsaturated fatty acids, polysaccharides and phenolics (Peng et al., 2015; Costa et al., 2020). These secondary metabolites have been studied for their anti-microbial, anti-inflammatory, immunosuppressive, anti-cancer and other pharmacologically important activities (Sathasivam et al., 2019). Thus, the algal metabolites find wide applicability in a vast array of biotechnological and pharmaceutical fields. In view of the ongoing COVID-19 pandemic caused by a novel coronavirus, designated as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), increased efforts are being made for developing efficient treatment options to tackle the disease. The SARS-CoV-2 has been identified as a single-stranded, positive-sense RNA virus belonging to the Betacoronavirus family (Yeo et al., 2020). Further, various structural (spike glycoprotein), non-structural (3-chymotrypsin-like protease, helicase, papain-like protease, and RNA-dependent RNA polymerase), and accessory proteins are encoded by SARS-CoV-2 genome (Li and De Clercq, 2020). The spike glycoprotein has been considered to be involved in the interaction between viruses and receptors present on the host cell (Li and De Clercq, 2020). Since this glycoprotein is an essential requirement for the entry of virus in host cells, many recent studies are focused on this structural protein (Zumla et al., 2016). It has been further concluded that the above mentioned five proteins also emerged as attractive targets for antiviral studies against SARS (Severe Acute Respiratory Syndrome) and MERS (Middle East respiratory syndrome) (Zumla et al., 2016). Considering all the facts related to exploration of algae for bioactive molecules, the present study provides an insight into the utilization of micro/macro-algal metabolites as therapeutic compounds against SARS-CoV-2 and like viruses. The key antiviral metabolites, namely, phycocyanobilins, lectins, and, sulphated polysaccharides have been discussed. Phycocyanobilins (PCBs) are tetrapyrrole chromophores present in certain cyanobacteria, rhodophytes, and are classified as blue phycobilinis (Figure 1) (Guedes et al., 2019). These light-capturing pigments are now widely studied for their antioxidative, antiviral (Hirata et al., 2000; Ramakrishnan, 2013) and NADPH-oxidase inhibitory activity (McCarty, 2007). Recently, Pendyala and Patras (2020) discussed the possible utilization of PCBs (source—Spirulina sp.) as inhibitors for the SARS-CoV-2 infection. The study involved in-silico screening (by the COVID-19 Docking Server) of the bioactive compounds for their activity against SARS-CoV-2. It was observed that the phycocyanobilin demonstrates a high binding affinity toward the potential targets, namely, the Main protease (Mpro) and RNA-dependent RNA polymerase (RdRp). The Main protease is involved in the processing of polyproteins (translated from SARS-CoV-2 RNA) while the replication of viral RNA is catalyzed by the polymerase. High binding energy of −8.6 kcal/mol was observed for PCB-Mpro while −9.3 kcal/mol for PCB-RdRp. Noteworthy, the PCB demonstrated a superior binding to target enzymes as compared to antiviral drugs like remdesivir (−8.1 kcal/mol for Mpro, −9.0 kcal/mol for RdRp), lopinavir (−7.9 kcal/mol) and nelfinavir (−7.9 kcal/mol for Mpro, −9.3 kcal/mol for RdRp). Thus, the study highlighted the significant potential of PCB as antiviral. However, as recommended by Pendyala and Patras (2020), further in-vitro and/or in-vivo studies will be crucially needed to support the obtained docking results and unravel the underlying potential of PCB as therapeutic for COVID-19. Additionally, the purified allophycocyanin obtained from Spirulina platensis has been demonstrated to exhibit significant activity against enterovirus 71 (Singh et al., 2020). It was observed that the cytopathic effects of the viral infection were neutralized and the viral RNA synthesis was delayed by the microalgal pigment allophycocyanin. Likewise, results of an in-silico study reported that the PCB expressed by Arthrospira sp. could serve as a potent antiviral against SARS-CoV-2 (Petit et al., 2020). The study evaluated the interaction between the Arthrospira sp. PCB and the receptor binding domain (RBD) of SARS-CoV-2 spike glycoprotein. It was observed that five Van der Waals interactions (involving residues ARG403, TYR453, LEU492, GLN493, and ASN501) contributed to the PCB/Spike RBD complex. The five π-alkyl bonds between the PCB and spike RBD involved the residues TYR449, TYR495, PHE497, and TYR505 with a hydrogen-bond on TYR449. The other residues involving the hydrogen-bond were SER494, GLY496, and GLN498 with the GLY496 linked to PCB by a π-donor hydrogen bond. Finally, a competitive binding energy (−7.2 kcal/mol) demonstrated the possibility to employ PCB as a potential antiviral agent (Petit et al., 2020). A recent study also reported the probability to utilize phycocyanobilin containing cyanobacteria like Spirulina sp. to control the RNA virus infections (Nikhra, 2020). A decrease in mortality rate in influenza-infected mice has been observed when administered orally with phycocyanin rich cold-water Spirulina sp. extracts in animal experimentation studies. The cold-water extract was well-tolerated even at high concentrations of 3,000 mg/kg/day in animal models for a period of 14 days (Chen et al., 2016). The PCB extracts thus demonstrated a substantial reduction in the survival of zoonotic RNA viruses by enhancing the type 1 interferon response of host immune system (Nikhra, 2020). Hence, it is likely possible that PCB producing microalgae may demonstrate substantial activity against SARS-CoV-2 as well (Cascella et al., 2020; Zhou et al., 2020). Moreover, further research along with in-vivo studies is necessary to understand the specific bioactivity of PCBs for the development of therapeutic strategies against human pathogenic viruses, including SARS-CoV-2. Figure 1. Algal antiviral metabolites (a) phycocyanobilin; (b) lectin; (c) fucoidan (a representative SP); (d) SPs-mediated humoral activation; (e) activation of host cellular immune response by SPs; (f) SPs-driven inhibition of virus entry/attachment to the host cell receptor. The macroalgae are rich in certain carbohydrate-binding proteins called lectins that demonstrate high specificity for sugar groups of other molecules like the oligosaccharide chains of the viral glycoproteins (Figure 1). Thus, lectins have been widely employed in various pharmacological and medical applications (Breitenbach Barroso Coelho et al., 2018). The mannose-binding lectins (MBL) are the predominant proteins to be studied in the viral infection pathways (Mitchell et al., 2017). The self-assembly of viruses during replication is interrupted by MBLs (Liu et al., 2015); thus, they have also emerged as a potential therapy against Ebola (Michelow et al., 2011). The red algae-derived lectins were initially brought to the limelight when griffithsin was discovered by Watson and Waaland (1983) from Griffithsia sp. Since then, it has been widely studied for various applications (Mori et al., 2004). It has been observed to possess high specificity for mannose residues present on viral glycoproteins. Some studies have demonstrated its antiviral activity against HIV-1 (Lusvarghi et al., 2016), Hepatitis C (Meuleman et al., 2011), and SARS-CoV glycoprotein (Zumla et al., 2016). A recent study analyzed the anti MERS-CoV activity of griffithsin and concluded that the lectin inhibits the entry of the virus while imparting negligible cellular toxicity (Millet et al., 2016). The inhibitory effect of griffithsin at the binding step during virus infection was assayed by time-course experiments. Thus, the study by Millet et al. (2016) demonstrated the griffithsin-mediated inhibition of MERS-CoV infectivity in-vitro. Additionally, various studies have reported the in-vivo antiviral activity of griffithsin against Japanese encephalitis virus (Ishag et al., 2013), herpes simplex virus 2 (Nixon et al., 2013) and human papillomavirus (Levendosky et al., 2015). For instance, the impact of an anti-HIV griffithsin containing microbicide on the rectal microbiome was assessed in the non-human primates (Rhesus macaques) (Girard et al., 2018). It was observed that 0.1% of griffithsin gel did not negatively impact the rectal mucosal proteome or microbiome. Further, O'Keefe et al. (2010) reported a 100% survival of model mice infected with a high dose of SARS-CoV upon providing a griffithsin dose of 10 mg/kg(b.w.)/day. Based on griffithsin activity against SARS-CoV, it may be investigated as a therapeutic option for SARS-CoV-2. Likewise, a novel D-mannose-binding lectin was identified from the red macroalgae Grateloupia chianggi and designated as GCL (Grateloupia chianggi lectin) (Hwang et al., 2020). The study focussed on GCL purification, its molecular and functional characterization, and subsequent analysis of its antiviral activity against influenza virus, herpes simplex virus and HIV. A quantity of 1–20 nM GCL was required for effective inhibition of HSV. Thus, it may be concluded that GCL also holds the potential to be utilized in virology and biomedical research. It is significant to note here that the SARS-CoV-2 is similar to the influenza virus as both are characterized as enveloped RNA viruses (Noda, 2012; Yeo et al., 2020). Based on the activity of GCL against the influenza virus, its activity may be explored against SARS-CoV-2 as well. Various researchers have demonstrated the beneficial effects of algal sulphated polysaccharides (SPs) under defined in-vitro and/or in-vivo conditions. Both the cellular and/or the humoral response of the immune system can be activated by these compounds (de Paniagua-Michel et al., 2014) (Figure 1). A recent study emphasized on the purification and structural characterization of two fucoidans from the brown macroalgae Sargassum henslowianum (Sun et al., 2020). These fucoidans designated as SHAP-1 and SHAP-2 were studied for their activity against two strains of herpes simplex virus, i.e., HSV-1 and HSV-2. It was observed that both compounds possessed significant anti-HSV activity with the IC50 value estimated to be 0.89 and 0.82 μg/mL for SHAP-1 and SHAP-2, respectively, against HSV-1 strain. Surprisingly, the IC50 values for both polysaccharides against HSV-2 were very low, i.e., 0.48 μg/mL. Also, time-of-addition experiments revealed that more efficient anti-HSV activities were obtained when fucoidans were added during the infection stage, thereby signifying their role at the early stages of viral infection. The adsorption and penetration assays further demonstrated that the fucoidans were involved in interruption of HSV adsorption to the host cell. Hence, it may be concluded that fucoidans could serve as promising candidates for inhibition of HSV-2 viruses and may be successfully utilized for various clinical applications. Similarly, a sulphated polysaccharide was isolated from the green macroalgae Monostroma nitidum (Wang et al., 2020). The compound isolated from M. nitidum was identified as a water-soluble sulphated glucuronorhamnan and thus designated as MWS. Various cytotoxicity and antiviral assays were performed to estimate the activity of MWS against EV71, a strain of human pathogenic enterovirus. It was observed that MWS was not toxic to the used cell lines and demonstrated a broad-spectrum of antiviral activity, especially against EV71 under defined in-vitro conditions. Further, it was concluded that MWS inhibits the EV71 infection by either targeting the host signaling pathway (down-regulation of host phosphoinositide 3-kinase/protein kinase B signaling pathway) in EV71 early life cycle and/or interrupting adsorption of virus to the host cell. The former mechanism has been concerned with the suppression of viral infection. The study also involved animal experiments, and a significant reduction in the viral titers was observed upon intramuscular administration of MWS in EV71 infected mice (Wang et al., 2020). Additionally, the SPs obtained from macroalgae Cladosiphon okamuranus and Ulva clathrata were also observed to demonstrate significant antiviral activity against the Newcastle disease virus under defined in-vitro conditions (Aguilar-Briseño et al., 2015). Another study elaborated the antiviral activity of SPs obtained from Ulva pertusa, Grateloupia filicina, and Sargassum qingdaoense against the avian influenza virus under in-vitro and in-vivo conditions (Song et al., 2016). A recent review highlighted the possibility of utilizing the SPs obtained from Porphyridium sp. (red microalga) as a potential therapeutic to combat COVID-19 disease (Gaikwad et al., 2020). Based on the antiviral activity of Porphyridium polysaccharides against a wide range of viruses including HSV (Huheihel et al., 2002), varicella zoster virus (Raposo et al., 2013), hepatitis B virus, vaccinia virus (Radonić et al., 2010) and retroviruses (Xiao and Zheng, 2016), this microalga has been considered to hold immense potential for the development of an antiviral pharmaceutical composition against SARS-CoV-2 as well (Gaikwad et al., 2020). Also, the effective inhibition (in-vitro) of SARS-CoV-2 by SPs (fucoidans) obtained from macroalgae Saccharina japonica was reported by Kwon et al. (2020). The fucoidans labeled as RPI-27 and RPI-28 demonstrated significant activity against SARS-CoV-2 with RPI-27 being more potent than the antiviral drug remdesivir. These highly branched fucoidans were observed to interfere with the binding of viral S protein to the heparan sulfate co-receptor of the host cells, thereby, inhibiting the viral infection. Thus, the study suggested the possibility of utilizing fucoidans alone or in combination with other antivirals as a promising therapeutic strategy against SARS-CoV-2 infection (Kwon et al., 2020). These studies indicate the potential therapeutic role of algal sulphated polysaccharides. There has been a substantial increase in evidence that reveals the antiviral activity of various microalgal and macroalgal metabolites like lectins, sulphated polysaccharides, and phycocyanobilins. Recent studies have reported that these compounds demonstrate substantial activity against a wide array of DNA and RNA viruses, including the influenza virus known to be associated with respiratory illnesses. As discussed, the bioactive molecules could serve as a novel therapeutic option to tackle SARS-CoV-2 and alike viruses. Considering the dire need for the development of therapeutics against SARS-CoV-2, there is a necessity to screen through the myriad of algae-derived potential antivirals which demands further evaluation and research. AB: conceptualization, data curation, visualization, and writing - original draft. PA: validation, writing - review & editing. SP: conceptualization, writing - review & editing, and supervision. All authors contributed to the article and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Aguilar-Briseño, J., Cruz-Suarez, L., Sassi, J. F., Ricque-Marie, D., Zapata-Benavides, P., Mendoza-Gamboa, E., et al. (2015). Sulphated polysaccharides from Ulva clathrata and Cladosiphon okamuranus seaweeds both inhibit viral attachment/entry and cell-cell fusion, in NDV infection. Mar. Drugs 13, 697–712. doi: 10.3390/md13020697 PubMed Abstract | CrossRef Full Text | Google Scholar Breitenbach Barroso Coelho, L. C., Marcelino dos Santos Silva, P., Felix de Oliveira, W., de Moura, M. C., Viana Pontual, E., Soares Gomes, F., et al. (2018). Lectins as antimicrobial agents. J. Appl. Microbiol. 125, 1238–1252. doi: 10.1111/jam.14055 PubMed Abstract | CrossRef Full Text | Google Scholar Cascella, M., Rajnik, M., Cuomo, A., Dulebohn, S. C., and Di Napoli, R. (2020). Features, Evaluation and Treatment Coronavirus (COVID-19). Florida: StatPearls Publishing. Google Scholar Chen, Y. H., Chang, G. K., Kuo, S. M., Huang, S. Y., Hu, I. C., Lo, Y. L., et al. (2016). Well-tolerated Spirulina extract inhibits influenza virus replication and reduces virus-induced mortality. Sci. Rep. 6, 1–11. doi: 10.1038/srep24253 PubMed Abstract | CrossRef Full Text | Google Scholar Costa, J. A. V., Moreira, J. B., Fanka, L. S., da Kosinski, R. C., and de Morais, M. G. (2020). “Microalgal biotechnology applied in biomedicine,” in Handbook of Algal Science, Technology and Medicine, ed O. Konur (Rio Grande, TX: Elsevier), 429–439. Google Scholar de Paniagua-Michel, J. J., Olmos-Soto, J., and Morales-Guerrero, E. R. (2014). “Algal and microbial exopolysaccharides: new insights as biosurfactants and bioemulsifiers,” in Advances in Food and Nutrition Research, ed S.-K. Kim (Baja California: Academic Press Inc.), 221–257. PubMed Abstract Gaikwad, M., Pawar, Y., Nagle, V., and Dasgupta, S. (2020). Marine Red Alga Porphyridium sp. as a Source of Sulfated Polysaccharides (SPs) for Combating Against COVID-19. Available online at: www.preprints.org (accessed August 18, 2020). Google Scholar Girard, L., Birse, K., Holm, J. B., Gajer, P., Humphrys, M. S., Garber, D., et al. (2018). Impact of the griffithsin anti-HIV microbicide and placebo gels on the rectal mucosal proteome and microbiome in non-human primates. Sci. Rep. 8, 1–13. doi: 10.1038/s41598-018-26313-8 PubMed Abstract | CrossRef Full Text | Google Scholar Guedes, A. C., Amaro, H. M., Sousa-Pinto, I., and Malcata, F. X. (2019). “Algal spent biomass—A pool of applications,” in Biofuels From Algae, eds A. Pandey, J.-S. Chang, C. R. Soccol, D.-J. Lee, and Y. Chisti (Porto: Elsevier), 397–433. Google Scholar Hirata, T., Tanaka, M., Ooike, M., Tsunomura, T., and Sakaguchi, M. (2000). Antioxidant activities of phycocyanobilin prepared from Spirulina platensis. J. Appl. Phycol. 12, 435–439. doi: 10.1023/A:1008175217194 CrossRef Full Text | Google Scholar Holzinger, A., and Karsten, U. (2013). Desiccation stress and tolerance in green algae: consequences for ultrastructure, physiological, and molecular mechanisms. Front. Plant Sci. 4:327. doi: 10.3389/fpls.2013.00327 PubMed Abstract | CrossRef Full Text | Google Scholar Huheihel, M., Ishanu, V., Tal, J., and Arad, S. (2002). Activity of Porphyridium sp. polysaccharide against herpes simplex viruses in vitro and in vivo. J. Biochem. Biophys. Methods 50, 189–200. doi: 10.1016/S0165-022X(01)00186-5 PubMed Abstract | CrossRef Full Text | Google Scholar Hwang, H. J., Han, J. W., Jeon, H., Cho, K., Kim, J., Lee, D. S., et al. (2020). Characterization of a novel mannose-binding lectin with antiviral activities from red alga, Grateloupia chiangii. Biomolecules 10:333. doi: 10.3390/biom10020333 PubMed Abstract | CrossRef Full Text | Google Scholar Ishag, H. Z. A., Li, C., Huang, L., Sun, M., Wang, F., Ni, B., et al. (2013). Griffithsin inhibits Japanese encephalitis virus infection in vitro and in vivo. Arch. Virol. 158, 349–358. doi: 10.1007/s00705-012-1489-2 PubMed Abstract | CrossRef Full Text | Google Scholar Kwon, P. S., Oh, H., Kwon, S. J., Jin, W., Zhang, F., Fraser, K., et al. (2020). Sulfated polysaccharides effectively inhibit SARS-CoV-2 in vitro. Cell Discov. 6:50. doi: 10.1038/s41421-020-00192-8 PubMed Abstract | CrossRef Full Text | Google Scholar Levendosky, K., Mizenina, O., Martinelli, E., Jean-Pierre, N., Kizima, L., Rodriguez, A., et al. (2015). Griffithsin and carrageenan combination to target herpes simplex virus 2 and human papillomavirus. Antimicrob. Agents Chemother. 59, 7290–7298. doi: 10.1128/AAC.01816-15 PubMed Abstract | CrossRef Full Text | Google Scholar Li, G., and De Clercq, E. (2020). Therapeutic options for the 2019 novel coronavirus (2019-nCoV). Nat. Rev. Drug Discov. 19, 149–150. doi: 10.1038/d41573-020-00016-0 PubMed Abstract | CrossRef Full Text | Google Scholar Liu, Y., Liu, J., Pang, X., Liu, T., Ning, Z., and Cheng, G. (2015). The roles of direct recognition by animal lectins in antiviral immunity and viral pathogenesis. Molecules 20, 2272–2295. doi: 10.3390/molecules20022272 PubMed Abstract | CrossRef Full Text | Google Scholar Lusvarghi, S., Bewley, C. A., and O'keefe, B. R. (2016). Griffithsin: an antiviral lectin with outstanding therapeutic potential. Viruses 8:296. doi: 10.3390/v8100296 PubMed Abstract | CrossRef Full Text | Google Scholar McCarty, M. F. (2007). Clinical potential of Spirulina as a source of phycocyanobilin. J. Med. Food 10, 566–570. doi: 10.1089/jmf.2007.621 PubMed Abstract | CrossRef Full Text | Google Scholar Meuleman, P., Albecka, A., Belouzard, S., Vercauteren, K., Verhoye, L., Wychowski, C., et al. (2011). Griffithsin has antiviral activity against hepatitis C virus. Antimicrob. Agents Chemother. 55, 5159–5167. doi: 10.1128/AAC.00633-11 PubMed Abstract | CrossRef Full Text | Google Scholar Michelow, I., Lear, C., Scully, C., Prugar, L. I., Longley, C. B., Yantosca, L. M., et al. (2011). High-Dose Mannose-Binding Lectin Therapy for Ebola Virus Infection. Available online at: https://academic.oup.com/jid/article-abstract/203/2/175/906751 (accessed April 12, 2020). PubMed Abstract | Google Scholar Millet, J. K., Séron, K., Labitt, R. N., Danneels, A., Palmer, K. E., Whittaker, G. R., et al. (2016). Middle East respiratory syndrome coronavirus infection is inhibited by griffithsin. Antiviral Res. 133, 1–8. doi: 10.1016/j.antiviral.2016.07.011 PubMed Abstract | CrossRef Full Text | Google Scholar Mitchell, C. A., Ramessar, K., and O'Keefe, B. R. (2017). Antiviral lectins: selective inhibitors of viral entry. Antiviral Res. 142, 37–54. doi: 10.1016/j.antiviral.2017.03.007 PubMed Abstract | CrossRef Full Text | Google Scholar Mori, T., O'keefe, B. R., Sowder, R. C., Bringans, S., Gardella, R., Berg, S., et al. (2004). Isolation and characterization of griffithsin, a novel HIV-inactivating protein, from the red alga Griffithsia sp. J. Biol. Chem. 280, 9345–9353. doi: 10.1074/jbc.M411122200 PubMed Abstract | CrossRef Full Text | Google Scholar Nikhra, V. (2020). The Trans-Zoonotic Virome Interface: Measures to Balance, Control and Treat Epidemics. New Delhi: Annals of Biomedical Engineering; Springer. Google Scholar Nixon, B., Stefanidou, M., Mesquita, P. M. M., Fakioglu, E., Segarra, T., Rohan, L., et al. (2013). Griffithsin protects mice from genital herpes by preventing cell-to-cell spread. J. Virol. 87, 6257–6269. doi: 10.1128/JVI.00012-13 PubMed Abstract | CrossRef Full Text | Google Scholar Noda, T. (2012). Native morphology of influenza virions. Front. Microbiol. 2:269. doi: 10.3389/fmicb.2011.00269 PubMed Abstract | CrossRef Full Text | Google Scholar O'Keefe, B. R., Giomarelli, B., Barnard, D. L., Shenoy, S. R., Chan, P. K. S., McMahon, J. B., et al. (2010). Broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae. J. Virol. 84, 2511–2521. doi: 10.1128/JVI.02322-09 PubMed Abstract | CrossRef Full Text | Google Scholar Pendyala, B., and Patras, A. (2020). In silico screening of food bioactive compounds to predict potential inhibitors of COVID-19 Main protease (Mpro) and RNA-dependent RNA polymerase (RdRp). ChemRxiv [Preprints]. doi: 10.26434/chemrxiv.12051927.v2 CrossRef Full Text | Google Scholar Peng, Y., Hu, J., Yang, B., Lin, X. P., Zhou, X. F., Yang, X. W., et al. (2015). “Chemical composition of seaweeds,” in Seaweed Sustainability: Food and Non-Food Applications, eds B. K. Tiwari and D. J. Troy (Zhanjiang; Guangzhou: Elsevier Inc.), 79–124. doi: 10.1016/B978-0-12-418697-2.00005-2 CrossRef Full Text | Google Scholar Petit, L., Vernes, L., and Cadoret, J. P. (2020). Docking and In Silico Toxicity Assessment of Arthrospira Compounds as Potential Antiviral Agents Against SARS-CoV-2. Paris: Research Square. Google Scholar Radonić, A., Thulke, S., Achenbach, J., Kurth, A., Vreemann, A., König, T., et al. (2010). Anionic polysaccharides from phototrophic microorganisms exhibit antiviral activities to vaccinia virus. J. Antivir. Antiretrovir. 2, 51–55. doi: 10.4172/jaa.1000023 CrossRef Full Text | Google Scholar Ramakrishnan, R. (2013). Antiviral properties of Cyanobacterium, Spirulina platensis-a review. Int. J. Med. Pharm. Sci. 3, 1–10. Google Scholar Raposo, M., De Morais, R., and Bernardo de Morais, A. (2013). Bioactivity and applications of sulphated polysaccharides from marine microalgae. Mar. Drugs 11, 233–252. doi: 10.3390/md11010233 PubMed Abstract | CrossRef Full Text | Google Scholar Sathasivam, R., Radhakrishnan, R., Hashem, A., and Abd Allah, E. F. (2019). Microalgae metabolites: a rich source for food and medicine. Saudi J. Biol. Sci. 26, 709–722. doi: 10.1016/j.sjbs.2017.11.003 PubMed Abstract | CrossRef Full Text | Google Scholar Singh, S., Dwivedi, V., Sanyal, D., and Dasgupta, S. (2020). Therapeutic and nutritional potential of Spirulina in combating COVID-19 infection. AIJR [Preprints]. doi: 10.21467/preprints.49 CrossRef Full Text | Google Scholar Singh, S. K., Kaur, R., Bansal, A., Kapur, S., and Sundaram, S. (2019). “Biotechnological exploitation of cyanobacteria and microalgae for bioactive compounds,” in Biotechnological Production of Bioactive Compounds, eds M. L. Verma, and A. K. Chandel (Prayagraj; Jalandhar; Telangana: Elsevier), 221−259. doi: 10.1016/B978-0-444-64323-0.00008-4 CrossRef Full Text | Google Scholar Song, L., Chen, X., Liu, X., Zhang, F., Hu, L., Yue, Y., et al. (2016). Characterization and comparison of the structural features, immune-modulatory and anti-avian influenza virus activities conferred by three algal sulfated polysaccharides. Mar. Drugs 14:4. doi: 10.3390/md14010004 CrossRef Full Text | Google Scholar Sun, Q. L., Li, Y., Ni, L. Q., Li, Y. X., Cui, Y. S., Jiang, S. L., et al. (2020). Structural characterization and antiviral activity of two fucoidans from the brown algae Sargassum henslowianum. Carbohydr. Polym. 229:115487. doi: 10.1016/j.carbpol.2019.115487 PubMed Abstract | CrossRef Full Text | Google Scholar Wang, S., Wang, W., Hou, L., Qin, L., He, M., Li, W., et al. (2020). A sulfated glucuronorhamnan from the green seaweed Monostroma nitidum: characteristics of its structure and antiviral activity. Carbohydr. Polym. 227:115280. doi: 10.1016/j.carbpol.2019.115280 PubMed Abstract | CrossRef Full Text | Google Scholar Watson, B. A., and Waaland, S. D. (1983). Partial purification and characterization of a glycoprotein cell fusion hormone from Griffithsia pacifica, a red alga. Plant Physiol. 71, 327–332. doi: 10.1104/pp.71.2.327 PubMed Abstract | CrossRef Full Text | Google Scholar Xiao, R., and Zheng, Y. (2016). Overview of microalgal extracellular polymeric substances (EPS) and their applications. Biotechnol. Adv. 34, 1225–1244. doi: 10.1016/j.biotechadv.2016.08.004 PubMed Abstract | CrossRef Full Text | Google Scholar Yeo, C., Kaushal, S., and Yeo, D. (2020). Enteric involvement of coronaviruses: is faecal–oral transmission of SARS-CoV-2 possible? Lancet Gastroenterol. Hepatol. 5, 335–337. doi: 10.1016/S2468-1253(20)30048-0 PubMed Abstract | CrossRef Full Text | Google Scholar Zhou, P., Yang, X. L., Wang, X. G., Hu, B., Zhang, L., Zhang, W., et al. (2020). A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273. doi: 10.1038/s41586-020-2012-7 PubMed Abstract | CrossRef Full Text | Google Scholar Zumla, A., W., Chan, J. F., Azhar, E. I., C., Hui, D. S., et al. (2016). Coronaviruses — drug discovery and therapeutic options. Nat. Rev. Drug Discov. 15, 327–347. doi: 10.1038/nrd.2015.37 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: microalgae, seaweed, antiviral, COVID-19, sulphated polysaccharides Citation: Bhatt A, Arora P and Prajapati SK (2020) Can Algal Derived Bioactive Metabolites Serve as Potential Therapeutics for the Treatment of SARS-CoV-2 Like Viral Infection? Front. Microbiol. 11:596374. doi: 10.3389/fmicb.2020.596374 Received: 19 August 2020; Accepted: 05 October 2020; Published: 11 November 2020. Edited by: Reviewed by: Copyright © 2020 Bhatt, Arora and Prajapati. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Sanjeev Kumar Prajapati, [email protected]; [email protected]
, Nicholas A. Young, Grégoire Mignot, Jean-Marie Bach
Published: 30 October 2020
Frontiers in Genetics, Volume 11; https://doi.org/10.3389/fgene.2020.578335

Abstract:
Immune and inflammatory diseases arise from a complex combination of genetic and environmental factors (David et al., 2018; Surace and Hedrich, 2019). MicroRNA are a class of non-coding single-stranded RNA molecules of 19–23 nucleotides in length. In response to environmental triggers, microRNA mediate epigenetic cell fate decisions critical in immune homeostasis by driving cellular activation, polarization, and immunological memory cell development (Mehta and Baltimore, 2016; Curtale et al., 2019). Pattern recognition receptors (PRR) recognize conserved molecular components of pathogens and respond by secreting reactive oxygen species and cytokines that alert the immune system about infection (Medzhitov et al., 1997). They can also interact with various endogenous ligands i.e., lipids, glycans, proteins, and nucleic acids, when released under sterile conditions of cellular stress, tissue injury, and transplantation. As activators of PRR-signaling, endogenous ligands initiate immune cell recruitment and tissue repair. However, sustained PRR-signaling may result in an exacerbated inflammatory response, which can have lethal effects or lead to autoimmunity (reviewed in Yu et al., 2010). In addition to their well-documented canonical function regulating gene expression through RNA interference in the cytoplasm (Bartel, 2004), specific GU-rich microRNA sequences can activate pro-inflammatory signaling pathways by direct interaction with the ribonucleic-acid binding Toll-like receptor 7/8 (TLR-7/8) of innate immunity located in cellular endosomes (Heil et al., 2004). Extracellular vesicles are a heterogeneous population of membrane vesicles naturally secreted by living cells that facilitate intercellular exchanges (Valadi et al., 2007; Raposo and Stoorvogel, 2013). Exported inside extracellular vesicles, Toll-like receptor-binding microRNA released by cells from injured or stressed tissues can reach the endosomal compartment and propagate inflammatory signals in distant recipient cells (Figure 1). The contributions of a dozen of TLR-7/8-binding microRNA (let-7b/c, miR-7a, miR-21, miR-29a/b, miR-34a, miR-122, miR-133a, miR-142, miR-145, miR-146a, miR-208a, and miR-210) to inflammation have been described to date in settings of cancer, sepsis, neurological, autoimmune, and graft-vs.-host diseases (Fabbri et al., 2012; Lehmann et al., 2012; He et al., 2014; Park et al., 2014; Salama et al., 2014; Liu et al., 2015; Yelamanchili et al., 2015; Kim et al., 2016; Coleman et al., 2017; Feng et al., 2017; Ranganathan et al., 2017; Young et al., 2017; Salvi et al., 2018; Xu et al., 2018; Wang et al., 2019). Using confocal microscopy co-localization, co-precipitation, and TLR inhibitors, these studies demonstrate direct binding of these microRNA to TLR-7 in mouse and TLR-8 in human, independently of RNA interference. Furthermore, transgenic TLR-7−/− mice are protected against the degenerative and inflammation-related effects of TLR-binding microRNA (Fabbri et al., 2012; Lehmann et al., 2012; Yelamanchili et al., 2015; Liang et al., 2019). Since their discovery in 2012, the significance of microRNA as endogenous ligands of innate immunity in health and disease is still a matter of debate (Chen et al., 2013; Fabbri et al., 2013; He, X. et al., 2014; Bayraktar et al., 2019). As part of the dynamic continuum of the endocytic intercellular communication pathway, TLR-binding microRNA transported via extracellular vesicles likely serve both adaptive and maladaptive stress responses in cells expressing TLR-7/8. Figure 1. As part of the intercellular endocytic communication pathway, TLR-binding microRNA transmitted via extracellular vesicles serve adaptive and maladaptive stress responses. Environmental stress (1) promotes secretion of extracellular vesicles and microRNA, (self-) antigen and danger-associated molecule release (2). After uptake by innate immune cells, specific GU-rich extracellular vesicle-encapsulated microRNA sequences can stimulate TLR-7/8 signaling in the endosome of recipient cells. Subsequent activation of the NF-κB pathway exacerbates inflammation through cytokine secretion, expression of co-stimulatory molecules (3) and self-induction of TLR-binding microRNA expression and extracellular vesicle secretion (4). So far, unconventional TLR-binding activity has been observed solely for extracellular microRNA and, out of 14 studies, 11 ascertain transfer in association with extracellular vesicles. The effects of danger-associated molecular patterns depend on their detection, a truism applicable to TLR-7-binding microRNA: they can act as such if and only if they reach the endosomal compartment. Encapsulation within extracellular vesicles constitutes a means for microRNA to enter the endocytic pathway where they may directly engage TLR-7/8 signaling (Mulcahy et al., 2014). In contrast, for RNA-interference activity, internalized microRNA have to escape from the endosome (Montecalvo et al., 2012), a rate-limiting step identified in the delivery of therapeutic short interference RNA (Johannes and Lucchino, 2018) and viral infection (Staring et al., 2018). It is conceivable that TLR-binding microRNA are conducive to exerting RNA interference-mediated effects in donor cells and TLR-binding effects or combinations of both after transfer via extracellular vesicles in recipient immune cells, i.e., major sites of TLR-7/8 expression (Lin et al., 2020; Sun et al., 2020). The relative proportion of free and particulate microRNA in biofluids still raises controversy, which is in part linked to technical pitfalls in the proper assessment of RNA concentrations in extracellular vesicles and biofluids (Arroyo et al., 2011; Turchinovich et al., 2011; Gallo et al., 2012; Crossland et al., 2016; Jeppesen et al., 2019). While free soluble RNA are short-lived due to high physiological levels of ribonuclease activity, microRNA chaperone protein complexes, or extracellular vesicle microRNA have sufficiently low clearance to support autocrine and paracrine signaling loops (Mitchell et al., 2008). Interaction of extracellular vesicles with patrolling immune cells can further transmit local signals of inflammation to the level of the organism. Useful on one hand for systemic coordination, this transmission can prove detrimental in the case of self-sustaining inflammatory responses. Indeed, let-7b for example, whose production can be enhanced by NF-κb activation (Wang et al., 2012) is also a potent TLR-ligand and thus may enhance its own synthesis, a mechanism perpetuating the vicious circle of inflammation in rheumatoid arthritis (Kim et al., 2016). We have demonstrated previously that liposome-encapsulated miR-21 can induce enhanced extracellular secretion in hematopoietic cells through TLR-7/8 signaling (Chang, 2010; Yang et al., 2015; Young et al., 2017). Similarly, the activation of the type 1 interferon/NF-κb pathway has been shown to induce let-7e, miR-21 and miR-146a expression by a positive amplification loop (Chang, 2010; Yang et al., 2015). If the body produces endogenous ligands of innate immunity, then how does this influence immune homeostasis? Fabbri and colleagues suggested that it is “the type and amount of information that cells exchange that ultimately affect cancer phenotype” (Fabbri et al., 2013). Indeed, biological active cargo is exported within extracellular vesicles sometimes at higher concentrations than in the donor cells and enhanced vesicle release has been broadly associated with inflammation and degeneration in pathological settings (Valadi et al., 2007; Zomer et al., 2015; Robbins et al., 2016; Young et al., 2017; Giri et al., 2020). As detoxifying “garbage bags” (Vidal, 2019), the enhanced extracellular vesicle outflow is presumably beneficial for the donor cell by permitting material clearance, but might entail deleterious consequences for the organism as a whole. The largely overlapping data reported in biomarker studies have built consensus indicating that measurable changes in circulating microRNA do not directly mirror changes in the diseased tissue, but are indicative of a secondary non-specific inflammatory response (Chen et al., 2008; Witwer, 2015). Presumably not by coincidence, TLR-binding microRNA overexpression is recurrently observed in pathological settings for miR-21, miR-7 and members of the let-7 and miR-29 families opening the way for subsequent polyvalent stimulation of the immune system. Although seemingly a critical factor, the quantitative requirements to modulate functional cellular responses are not well-understood. The biological activity of extracellular vesicle-encapsulated microRNA in recipient cells was first demonstrated in microRNA overexpression reporter experiments in vitro or after the transfer of concentrated suspensions of purified vesicles (Kosaka et al., 2010; Montecalvo et al., 2012). However, the number of copies measured per vesicles of a given endogenous microRNA is very low, even for abundant microRNA in extracellular vesicles, which raises questions about the physiological relevance of cell-to-cell microRNA-based communication (Williams et al., 2013; Chevillet et al., 2014). For RNA interference–mediated effects, a threshold of 1,000 copies of microRNA has to be reached in the recipient cells to trigger measurable effects (Brown et al., 2007), which represents the successful delivery of an estimated ≈105 extracellular vesicles (Igaz, 2015). While these concentrations seem realistic for extracellular vesicles released from broadly distributed tissues such as blood, fat, or muscle (Sender et al., 2016), this seems unlikely for less abundant cell types. In contrast, in the attoliter (10−18 L) volume of the endosome, a single RNA molecule equates to a 3 μM concentration, which given the micromolar-affinity of the TLR-7 receptor for guanosine-uracil oligomers, might more easily elicit an immune response (Crozat and Beutler, 2004; Zhang et al., 2016). If present, distinct TLR-binding microRNA sequences could synergistically activate TLR-7/8. The absolute quantity and diversity of microRNA exported is highest in large apoptotic bodies and shedding microvesicles. Yet, the majority of evidence on TLR-binding microRNA activity has focused on small ~100 nm vesicles. Similarly, among extracellular vesicles released by serum-starved endothelial cells, only small exosome-like vesicles display immunogenic properties involving the activation of innate PRR by a specific repertoire of non-coding self-RNA (Hardy et al., 2019). In addition to being a consequence of exosome-focused research predominating the field in the past decade, this phenomenon may be explained by evidence of preferential sorting of GU-rich RNA and TLR-binding microRNA into small vesicles in situations of stress (Kouwaki et al., 2016; Fleshner and Crane, 2017; Hardy et al., 2019; Giri et al., 2020; Mensà et al., 2020). Differences in the mechanism and cellular targeting of extracellular vesicle uptake could further influence the impact of TLR-ligand microRNA on recipient cells. Studies on synthetic RNA-containing particles provide evidence that nanometric particles are selectively internalized by plasmacytoid dendritic cells leading to the production of large amounts of interferon-α whereas micrometric particles preferentially induce tumor necrosis factor-α secretion from monocytes (Rettig et al., 2010). The authors infer that, in addition to surface protein expression, nano- or micro-particle size discrimination per se allows the immune defense to adapt to viral or bacterial/fungal infection, respectively. In line with this hypothesis, small extracellular vesicles from systemic lupus erythematosus patients and apoptotic lymphoblasts readily stimulate interferon release form plasmacytoid dendritic cells via TLR-signaling (Schiller et al., 2012; Salvi et al., 2018). In contrast, extracellular vesicles derived from healthy tissue are essentially immune-silent (reviewed in Fleshner and Crane, 2017). For apoptotic bodies, the intrinsic tolerogenic properties rely on the expression of “find” and “eat-me” signals like phosphatidyl-serine that promote the production of anti-inflammatory mediators like the cytokine transforming growth factor-β and the prostaglandin E2 (Fadok et al., 1998; Pujol-Autonell et al., 2013). Equivalent signals may be absent, weak, or masked in microvesicles and exosomes in pathological settings. Indeed, encapsulated inside extracellular vesicles, microRNA are delivered as a bundle, along with many other immune active molecules i.e., lipids (Sagini et al., 2018), cytokines (Fitzgerald et al., 2018), prostaglandins (Lacy et al., 2019), auto-antigens, ATP or danger-associated molecules (Chalmin et al., 2010; Fleshner and Crane, 2017), which have been shown to concentrate in small extracellular vesicles in acute stress responses (Beninson et al., 2014). Evidence from kidney transplant recipients suggests that small exosome-like vesicles released from stressed or injured tissues create a permissive environment promoting the production of autoantibodies against formerly cryptic antigens (Dieudé et al., 2015; Cardinal et al., 2017). In concert with extracellular vesicle-independent co-stimulants, these factors may further shape the outcome of immune responses that rely on the combination of several activation signals. As part of the oldest arm of the immune system, TLR developed 1,350 million years ago to adapt to environmental changes by controlling the activation and differentiation of immune cells by epigenetic mechanisms (Nie et al., 2018). Recent drastic alterations in our environment have been linked to an imbalance in immunity and the spread of inflammatory diseases. As catalyst of inflammation, the physiological significance of extracellular vesicle-encapsulated microRNA binding to TLR-7/8 has probably been over-looked. Further experimental evidence is needed to establish the dominant endogenous activator(s) of the inflammatory response. In particular, we lack (i) studies correlating TLR-binding microRNA expression to disease activity (ii) side-by-side comparisons of the dichotomous function of a given microRNA in its soluble form or encapsulated within specific subpopulations of extracellular vesicles, and (iii) evaluation of extracellular vesicle self-antigen modulation of (auto-) immune responses. The use of animal models should be valuable to further explore thresholds of physiological consequences of TLR-7/8 microRNA activation and systemic interactions in an integrated fashion, in vivo. Ultimately, new medication antagonizing TLR-binding microRNA may present an opportunity to prevent excessive inflammatory responses. All authors conceptualized, wrote, edited, and approved the manuscript. SB designed the figure. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer FC declared a shared affiliation, with no collaboration, with one of the authors, NY, to the handling editor at the time of the review. Arroyo, J. D., Chevillet, J. R., Kroh, E. M., Ruf, I. K., Pritchard, C. C., Gibson, D. F., et al. (2011). Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc. Natl. Acad. Sci. U.S.A. 108, 5003–5008. doi: 10.1073/pnas.1019055108 PubMed Abstract | CrossRef Full Text | Google Scholar Bartel, D. P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297. doi: 10.1016/S0092-8674(04)00045-5 CrossRef Full Text | Google Scholar Bayraktar, R., Bertilaccio, M. T. S., and Calin, G. A. (2019). The interaction between two worlds: microRNAs and toll-like receptors. Front. Immunol. 10:1053. doi: 10.3389/fimmu.2019.01053 PubMed Abstract | CrossRef Full Text | Google Scholar Beninson, L. A., Brown, P. N., Loughridge, A. B., Saludes, J. P., Maslanik, T., Hills, A. K., et al. (2014). Acute stressor exposure modifies plasma exosome-associated heat shock protein 72 (Hsp72) and microRNA (miR-142-5p and miR-203). PLoS ONE 9:e108748. doi: 10.1371/journal.pone.0108748 CrossRef Full Text | Google Scholar Brown, B. D., Gentner, B., Cantore, A., Colleoni, S., Amendola, M., Zingale, A., et al. (2007). Endogenous microRNA can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state. Nat. Biotechnol. 25, 1457–1467. doi: 10.1038/nbt1372 PubMed Abstract | CrossRef Full Text | Google Scholar Cardinal, H., Dieudé, M., and Hébert, M. J. (2017). The emerging importance of non-HLA autoantibodies in kidney transplant complications. J. Am. Soc. Nephrol. 28, 400–406. doi: 10.1681/ASN.2016070756 PubMed Abstract | CrossRef Full Text | Google Scholar Chalmin, F., Ladoire, S., Mignot, G., Vincent, J., Bruchard, M., Remy-Martin, J. P., et al. (2010). Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells. J. Clin. Invest. 120, 457–471. doi: 10.1172/JCI40483 PubMed Abstract | CrossRef Full Text | Google Scholar Chang, Z. L. (2010). Important aspects of Toll-like receptors, ligands and their signaling pathways. Inflamm. Res. 59, 791–808. doi: 10.1007/s00011-010-0208-2 PubMed Abstract | CrossRef Full Text | Google Scholar Chen, X., Ba, Y., Ma, L. J., Cai, X., Yin, Y., Wang, K. H., et al. (2008). Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. 18, 997–1006. doi: 10.1038/cr.2008.282 PubMed Abstract | CrossRef Full Text | Google Scholar Chen, X., Liang, H. W., Zhang, J. F., Zen, K., and Zhang, C. Y. (2013). microRNAs are ligands of Toll-like receptors. RNA 19, 737–739. doi: 10.1261/rna.036319.112 PubMed Abstract | CrossRef Full Text | Google Scholar Chevillet, J. R., Kang, Q., Ruf, I. K., Briggs, H. A., Vojtech, L. N., Hughes, S. M., et al. (2014). Quantitative and stoichiometric analysis of the microRNA content of exosomes. Proc. Natl. Acad. Sci. U.S.A. 111, 14888–14893. doi: 10.1073/pnas.1408301111 PubMed Abstract | CrossRef Full Text | Google Scholar Coleman, L. G., Zou, J., and Crews, F. T. (2017). Microglial-derived miRNA let-7 and HMGB1 contribute to ethanol-induced neurotoxicity via TLR7. J. Neuroinflamm. 14:22. doi: 10.1186/s12974-017-0799-4 PubMed Abstract | CrossRef Full Text | Google Scholar Crossland, R. E., Norden, J., Bibby, L. A., Davis, J., and Dickinson, A. M. (2016). Evaluation of optimal extracellular vesicle small RNA isolation and qRT-PCR normalisation for serum and urine. J. Immunol. Methods 429, 39–49. doi: 10.1016/j.jim.2015.12.011 PubMed Abstract | CrossRef Full Text | Google Scholar Crozat, K., and Beutler, B. (2004). TLR7: a new sensor of viral infection. Proc. Natl. Acad. Sci. U.S.A. 101, 6835–6836. doi: 10.1073/pnas.0401347101 PubMed Abstract | CrossRef Full Text | Google Scholar Curtale, G., Rubino, M., and Locati, M. (2019). MicroRNAs as molecular switches in macrophage activation. Front. Immunol. 10:799. doi: 10.3389/fimmu.2019.00799 PubMed Abstract | CrossRef Full Text | Google Scholar David, T., Ling, S. F., and Barton, A. (2018). Genetics of immune-mediated inflammatory diseases. Clin. Exp. Immunol. 193, 3–12. doi: 10.1111/cei.13101 PubMed Abstract | CrossRef Full Text | Google Scholar Dieudé, M., Bell, C., Turgeon, J., Beillevaire, D., Pomerleau, L., Yang, B., et al. (2015). The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection. Sci. Transl. Med. 7:318ra200. doi: 10.1126/scitranslmed.aac9816 PubMed Abstract | CrossRef Full Text | Google Scholar Fabbri, M., Paone, A., Calore, F., Galli, R., and Croce, C. M. (2013). A new role for microRNAs, as ligands of Toll-like receptors. RNA Biol. 10, 169–174. doi: 10.4161/rna.23144 PubMed Abstract | CrossRef Full Text | Google Scholar Fabbri, M., Paone, A., Calore, F., Galli, R., Gaudio, E., Santhanam, R., et al. (2012). MicroRNAs bind to Toll-like receptors to induce prometastatic inflammatory response. Proc. Natl. Acad. Sci. U.S.A. 109, E2110–E2116. doi: 10.1073/pnas.1209414109 PubMed Abstract | CrossRef Full Text | Google Scholar Fadok, V. A., Bratton, D. L., Konowal, A., Freed, P. W., Westcott, J. Y., and Henson, P. M. (1998). Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF. J. Clin. Invest. 101, 890–898. doi: 10.1172/JCI1112 PubMed Abstract | CrossRef Full Text | Google Scholar Feng, Y., Zou, L., Yan, D., Chen, H., Xu, G., Jian, W., et al. (2017). Extracellular microRNAs induce potent innate immune responses via TLR7/MyD88-dependent mechanisms. J. Immunol. 199, 2106–2117. doi: 10.4049/jimmunol.1700730 PubMed Abstract | CrossRef Full Text | Google Scholar Fitzgerald, W., Freeman, M. L., Lederman, M. M., Vasilieva, E., Romero, R., and Margolis, L. (2018). A system of cytokines encapsulated in extracellular vesicles. Sci. Rep. 8:8973. doi: 10.1038/s41598-018-27190-x PubMed Abstract | CrossRef Full Text | Google Scholar Fleshner, M., and Crane, C. R. (2017). Exosomes, DAMPs and miRNA: features of stress physiology and immune homeostasis. Trends Immunol. 38, 768–776. doi: 10.1016/j.it.2017.08.002 PubMed Abstract | CrossRef Full Text | Google Scholar Gallo, A., Tandon, M., Alevizos, I., and Illei, G. G. (2012). The majority of microRNAs detectable in serum and saliva is concentrated in exosomes. PLoS ONE 7:e30679. doi: 10.1371/journal.pone.0030679 PubMed Abstract | CrossRef Full Text | Google Scholar Giri, K. R., de Beaurepaire, L., Jegou, D., Lavy, M., Mosser, M., Aurélien, D., et al. (2020). Molecular and functional diversity of distinct subpopulations of the stressed insulin-secreting cell's vesiculome. Front. Immunol. 11:1814. doi: 10.3389/fimmu.2020.01814 CrossRef Full Text | Google Scholar Hardy, M. P., Audemard, É., Migneault, F., Feghaly, A., Brochu, S., Gendron, P., et al. (2019). Apoptotic endothelial cells release small extracellular vesicles loaded with immunostimulatory viral-like RNAs. Sci. Rep. 9:7203. doi: 10.1038/s41598-019-43591-y PubMed Abstract | CrossRef Full Text | Google Scholar He, W. A., Calore, F., Londhe, P., Canella, A., Guttridge, D. C., and Croce, C. M. (2014). Microvesicles containing miRNAs promote muscle cell death in cancer cachexia via TLR7. Proc. Natl. Acad. Sci. U.S.A. 111, 4525–4529. doi: 10.1073/pnas.1402714111 PubMed Abstract | CrossRef Full Text | Google Scholar He, X., Jing, Z., and Cheng, G. (2014). MicroRNAs: new regulators of Toll-like receptor signalling pathways. Biomed Res. Int. 2014:945169. doi: 10.1155/2014/945169 PubMed Abstract | CrossRef Full Text | Google Scholar Heil, F., Hemmi, H., Hochrein, H., Ampenberger, F., Kirschning, C., Akira, S., et al. (2004). Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303, 1526–1529. doi: 10.1126/science.1093620 PubMed Abstract | CrossRef Full Text | Google Scholar Igaz, P. (2015). Circulating MicroRNAs in Disease Diagnostics and their Potential Biological Relevance. Basel: Springer. doi: 10.1007/978-3-0348-0955-9 CrossRef Full Text | Google Scholar Jeppesen, D. K., Fenix, A. M., Franklin, J. L., Higginbotham, J. N., Zhang, Q., Zimmerman, L. J., et al. (2019). Reassessment of exosome composition. Cell 177, 428–445.e418. doi: 10.1016/j.cell.2019.02.029 PubMed Abstract | CrossRef Full Text | Google Scholar Johannes, L., and Lucchino, M. (2018). Current challenges in delivery and cytosolic translocation of therapeutic RNAs. Nucl. Acid Ther. 28, 178–193. doi: 10.1089/nat.2017.0716 PubMed Abstract | CrossRef Full Text | Google Scholar Kim, S. J., Chen, Z., Essani, A. B., Elshabrawy, H. A., Volin, M. V., Volkov, S., et al. (2016). Identification of a novel toll-like receptor 7 endogenous ligand in rheumatoid arthritis synovial fluid that can provoke arthritic joint inflammation. Arthritis Rheumatol. 68, 1099–1110. doi: 10.1002/art.39544 PubMed Abstract | CrossRef Full Text | Google Scholar Kosaka, N., Iguchi, H., Yoshioka, Y., Takeshita, F., Matsuki, Y., and Ochiya, T. (2010). Secretory mechanisms and intercellular transfer of microRNAs in living cells. J. Biol. Chem. 285, 17442–17452. doi: 10.1074/jbc.M110.107821 PubMed Abstract | CrossRef Full Text | Google Scholar Kouwaki, T., Fukushima, Y., Daito, T., Sanada, T., Yamamoto, N., Mifsud, E. J., et al. (2016). Extracellular vesicles including exosomes regulate innate immune responses to Hepatitis B virus infection. Front. Immunol. 7:335. doi: 10.3389/fimmu.2016.00335 PubMed Abstract | CrossRef Full Text | Google Scholar Lacy, S. H., Woeller, C. F., Thatcher, T. H., Pollock, S. J., Small, E. M., Sime, P. J., et al. (2019). Activated human lung fibroblasts produce extracellular vesicles with antifibrotic prostaglandins. Am. J. Respir. Cell Mol. Biol. 60, 269–278. doi: 10.1165/rcmb.2017-0248OC PubMed Abstract | CrossRef Full Text | Google Scholar Lehmann, S. M., Krueger, C., Park, B., Derkow, K., Rosenberger, K., Baumgart, J., et al. (2012). An unconventional role for miRNA: let-7 activates Toll-like receptor 7 and causes neurodegeneration. Nat. Neurosci. 15, 827–U844. doi: 10.1038/nn.3113 PubMed Abstract | CrossRef Full Text | Google Scholar Liang, H., Kidder, K., and Liu, Y. (2019). Extracellular microRNAs initiate immunostimulation via activating toll-like receptor signaling pathways. ExRNA 1:9. doi: 10.1186/s41544-019-0009-x CrossRef Full Text | Google Scholar Lin, F., Yin, H. B., Li, X. Y., Zhu, G. M., He, W. Y., and Gou, X. (2020). Bladder cancer cell-secreted exosomal miR-21 activates the PI3K/AKT pathway in macrophages to promote cancer progression. Int. J. Oncol. 56, 151–164. doi: 10.3892/ijo.2019.4933 PubMed Abstract | CrossRef Full Text | Google Scholar Liu, H. Y., Huang, C. M., Hung, Y. F., and Hsueh, Y. P. (2015). The microRNAs Let7c and miR21 are recognized by neuronal Toll-like receptor 7 to restrict dendritic growth of neurons. Exp. Neurol. 269, 202–212. doi: 10.1016/j.expneurol.2015.04.011 PubMed Abstract | CrossRef Full Text | Google Scholar Medzhitov, R., Preston-Hurlburt, P., and Janeway, C. A. (1997). A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388, 394–397. doi: 10.1038/41131 PubMed Abstract | CrossRef Full Text | Google Scholar Mehta, A., and Baltimore, D. (2016). MicroRNAs as regulatory elements in immune system logic. Nat. Rev. Immunol. 16, 279–294. doi: 10.1038/nri.2016.40 PubMed Abstract | CrossRef Full Text | Google Scholar Mensà, E., Guescini, M., Giuliani, A., Bacalini, M. G., Ramini, D., Corleone, G., et al. (2020). Small extracellular vesicles deliver miR-21 and miR-217 as pro-senescence effectors to endothelial cells. J. Extracell Vesicles 9:1725285. doi: 10.1080/20013078.2020.1725285 PubMed Abstract | CrossRef Full Text | Google Scholar Mitchell, P. S., Parkin, R. K., Kroh, E. M., Fritz, B. R., Wyman, S. K., Pogosova-Agadjanyan, E. L., et al. (2008). Circulating microRNAs as stable blood-based markers for cancer detection. Proc. Natl. Acad. Sci. U.S.A. 105, 10513–10518. doi: 10.1073/pnas.0804549105 PubMed Abstract | CrossRef Full Text | Google Scholar Montecalvo, A., Larregina, A. T., Shufesky, W. J., Stolz, D. B., Sullivan, M. L. G., Karlsson, J. M., et al. (2012). Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes. Blood 119, 756–766. doi: 10.1182/blood-2011-02-338004 PubMed Abstract | CrossRef Full Text | Google Scholar Mulcahy, L. A., Pink, R. C., and Carter, D. R. (2014). Routes and mechanisms of extracellular vesicle uptake. J Extracell Vesicles 3:24641. doi: 10.3402/jev.v3.24641 PubMed Abstract | CrossRef Full Text | Google Scholar Nie, L., Cai, S. Y., Shao, J. Z., and Chen, J. (2018). Toll-like receptors, associated biological roles, and signaling networks in non-mammals. Front. Immunol. 9:1523. doi: 10.3389/fimmu.2018.01523 PubMed Abstract | CrossRef Full Text | Google Scholar Park, C. K., Xu, Z. Z., Berta, T., Han, Q. J., Chen, G., Liu, X. J., et al. (2014). Extracellular microRNAs activate nociceptor neurons to elicit pain via TLR7 and TRPA1. Neuron 82, 47–54. doi: 10.1016/j.neuron.2014.02.011 PubMed Abstract | CrossRef Full Text | Google Scholar Pujol-Autonell, I., Ampudia, R. M., Planas, R., Marin-Gallen, S., Carrascal, J., Sanchez, A., et al. (2013). Efferocytosis promotes suppressive effects on dendritic cells through prostaglandin E2 production in the context of autoimmunity. PLoS ONE 8:e63296. doi: 10.1371/journal.pone.0063296 PubMed Abstract | CrossRef Full Text | Google Scholar Ranganathan, P., Ngankeu, A., Zitzer, N. C., Leoncini, P., Yu, X. Y., Casadei, L., et al. (2017). Serum miR-29a is upregulated in acute graft-versus-host disease and activates dendritic cells through TLR Binding. J. Immunol. 198, 2500–2512. doi: 10.4049/jimmunol.1601778 PubMed Abstract | CrossRef Full Text | Google Scholar Raposo, G., and Stoorvogel, W. (2013). Extracellular vesicles: exosomes, microvesicles, and friends. J. Cell Biol. 200, 373–383. doi: 10.1083/jcb.201211138 PubMed Abstract | CrossRef Full Text | Google Scholar Rettig, L., Haen, S. P., Bittermann, A. G., von Boehmer, L., Curioni, A., Krämer, S. D., et al. (2010). Particle size and activation threshold: a new dimension of danger signaling. Blood 115, 4533–4541. doi: 10.1182/blood-2009-11-247817 PubMed Abstract | CrossRef Full Text | Google Scholar Robbins, P. D., Dorronsoro, A., and Booker, C. N. (2016). Regulation of chronic inflammatory and immune processes by extracellular vesicles. J. Clin. Invest. 126, 1173–1180. doi: 10.1172/JCI81131 PubMed Abstract | CrossRef Full Text | Google Scholar Sagini, K., Costanzi, E., Emiliani, C., Buratta, S., and Urbanelli, L. (2018). Extracellular vesicles as conveyors of membrane-derived bioactive lipids in immune system. Int. J. Mol. Sci. 19:1227. doi: 10.3390/ijms19041227 PubMed Abstract | CrossRef Full Text | Google Scholar Salama, A., Fichou, N., Allard, M., Dubreil, L., de Beaurepaire, L., Viel, A., et al. (2014). MicroRNA-29b modulates innate and antigen-specific immune responses in mouse models of autoimmunity. PLoS ONE 9:e106153. doi: 10.1371/journal.pone.0106153 PubMed Abstract | CrossRef Full Text | Google Scholar Salvi, V., Gianello, V., Busatto, S., Bergese, P., Andreoli, L., D'Oro, U., et al. (2018). Exosome-delivered microRNAs promote IFN-alpha secretion by human plasmacytoid DCs via TLR7. JCI Insight 3:e98204. doi: 10.1172/jci.insight.98204 PubMed Abstract | CrossRef Full Text | Google Scholar Schiller, M., Parcina, M., Heyder, P., Foermer, S., Ostrop, J., Leo, A., et al. (2012). Induction of Type I IFN is a physiological immune reaction to apoptotic cell-derived membrane microparticles. J. Immunol. 189, 1747–1756. doi: 10.4049/jimmunol.1100631 PubMed Abstract | CrossRef Full Text | Google Scholar Sender, R., Fuchs, S., and Milo, R. (2016). Revised estimates for the number of human and bacteria cells in the body. PLoS Biol. 14:e1002533. doi: 10.1371/journal.pbio.1002533 PubMed Abstract | CrossRef Full Text | Google Scholar Staring, J., Raaben, M., and Brummelkamp, T. R. (2018). Viral escape from endosomes and host detection at a glance. J. Cell Sci. 131:jcs216259. doi: 10.1242/jcs.216259 PubMed Abstract | CrossRef Full Text | Google Scholar Sun, L. H., Tian, D., Yang, Z. C., and Li, J. L. (2020). Exosomal miR-21 promotes proliferation, invasion and therapy resistance of colon adenocarcinoma cells through its target PDCD4. Sci. Rep. 10:8271. doi: 10.1038/s41598-020-65207-6 PubMed Abstract | CrossRef Full Text | Google Scholar Surace, A. E. A., and Hedrich, C. M. (2019). The role of epigenetics in autoimmune/inflammatory disease. Front. Immunol. 10:1525. doi: 10.3389/fimmu.2019.01525 PubMed Abstract | CrossRef Full Text | Google Scholar Turchinovich, A., Weiz, L., Langheinz, A., and Burwinkel, B. (2011). Characterization of extracellular circulating microRNA. Nucl. Acids Res. 39, 7223–7233. doi: 10.1093/nar/gkr254 PubMed Abstract | CrossRef Full Text | Google Scholar Valadi, H., Ekstrom, K., Bossios, A., Sjostrand, M., Lee, J. J., and Lotvall, J. O. (2007). Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654–672. doi: 10.1038/ncb1596 PubMed Abstract | CrossRef Full Text | Google Scholar Vidal, M. (2019). Exosomes: revisiting their role as “garbage bags”. Traffic 20, 815–828. doi: 10.1111/tra.12687 PubMed Abstract | CrossRef Full Text | Google Scholar Wang, D. J., Legesse-Miller, A., Johnson, E. L., and Coller, H. A. (2012). Regulation of the let-7a-3 promoter by NF-κB. PLoS ONE 7:e31240. doi: 10.1371/journal.pone.0031240 PubMed Abstract | CrossRef Full Text | Google Scholar Wang, Y., Liang, H., Jin, F., Yan, X., Xu, G., Hu, H., et al. (2019). Injured liver-released miRNA-122 elicits acute pulmonary inflammation via activating alveolar macrophage TLR7 signaling pathway. Proc. Natl. Acad. Sci. U.S.A. 116, 6162–6171. doi: 10.1073/pnas.1814139116 PubMed Abstract | CrossRef Full Text | Google Scholar Williams, Z., Ben-Dov, I. Z., Elias, R., Mihailovic, A., Brown, M., Rosenwaks, Z., et al. (2013). Comprehensive profiling of circulating microRNA via small RNA sequencing of cDNA libraries reveals biomarker potential and limitations. Proc. Natl. Acad. Sci. U.S.A. 110, 4255–4260. doi: 10.1073/pnas.1214046110 PubMed Abstract | CrossRef Full Text | Google Scholar Witwer, K. W. (2015). Circulating microRNA biomarker studies: pitfalls and potential solutions. Clin. Chem. 61, 56–63. doi: 10.1373/clinchem.2014.221341 PubMed Abstract | CrossRef Full Text | Google Scholar Xu, J., Feng, Y., Jeyaram, A., Jay, S. M., Zou, L., and Chao, W. (2018). Circulating plasma extracellular vesicles from septic mice induce inflammation via microRNA- and TLR7-dependent mechanisms. J. Immunol. 201, 3392–3400. doi: 10.4049/jimmunol.1801008 PubMed Abstract | CrossRef Full Text | Google Scholar Yang, C. H., Li, K., Pfeffer, S. R., and Pfeffer, L. M. (2015). The Type I IFN-induced miRNA, miR-21. Pharmaceuticals 8, 836–847. doi: 10.3390/ph8040836 PubMed Abstract | CrossRef Full Text | Google Scholar Yelamanchili, S. V., Lamberty, B. G., Rennard, D. A., Morsey, B. M., Hochfelder, C. G., Meays, B. M., et al. (2015). MiR-21 in extracellular vesicles leads to neurotoxicity via TLR7 signaling in SIV neurological disease. PLoS Pathog. 11:e1005032. doi: 10.1371/journal.ppat.1005032 CrossRef Full Text | Google Scholar Young, N. A., Valiente, G. R., Hampton, J. M., Wu, L. C., Burd, C. J., Willis, W. L., et al. (2017). Estrogen-regulated STAT1 activation promotes TLR8 expression to facilitate signaling via microRNA-21 in systemic lupus erythematosus. Clin. Immunol. 176, 12–22. doi: 10.1016/j.clim.2016.12.005 PubMed Abstract | CrossRef Full Text | Google Scholar Yu, L., Wang, L., and Chen, S. (2010). Endogenous toll-like receptor ligands and their biological significance. J. Cell. Mol. Med. 14, 2592–2603. doi: 10.1111/j.1582-4934.2010.01127.x PubMed Abstract | CrossRef Full Text | Google Scholar Zhang, Z., Ohto, U., Shibata, T., Krayukhina, E., Taoka, M., Yamauchi, Y., et al. (2016). Structural analysis reveals that toll-like receptor 7 is a dual receptor for guanosine and single-stranded RNA. Immunity 45, 737–748. doi: 10.1016/j.immuni.2016.09.011 PubMed Abstract | CrossRef Full Text | Google Scholar Zomer, A., Maynard, C., Verweij, F. J., Kamermans, A., Schäfer, R., Beerling, E., et al. (2015). In vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior. Cell 161, 1046–1057. doi: 10.1016/j.cell.2015.04.042 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: biological relevance, extracellular vesicles, immune activation, epigenetic, innate immunity, Toll-like receptor, miRNA—microRNA Citation: Bosch S, Young NA, Mignot G and Bach J-M (2020) Epigenetic Mechanisms in Immune Disease: The Significance of Toll-Like Receptor-Binding Extracellular Vesicle-Encapsulated microRNA. Front. Genet. 11:578335. doi: 10.3389/fgene.2020.578335 Received: 07 July 2020; Accepted: 05 October 2020; Published: 30 October 2020. Edited by: Reviewed by: Copyright © 2020 Bosch, Young, Mignot and Bach. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Steffi Bosch, [email protected]
, Dana M. Barberio
Frontiers in Cellular and Infection Microbiology, Volume 10; https://doi.org/10.3389/fcimb.2020.567252

Abstract:
Lyme disease (LD), caused by the spirochetal bacterium Borrelia burgdorferi, is transmitted by the black-legged tick Ixodes scapularis (Hu, 2016). LD is the fastest growing global tick-borne disease and annually affects >300,000 people in the U.S. alone (Steere et al., 2016). The economic impact is a staggering $1.3 billion dollars per year (Adrion et al., 2015). LD can cause long-term, debilitating symptoms, including arthritis, carditis, and neurological complications (Hu, 2016; Steere et al., 2016). A longstanding question is why antibodies produced during primary infection are not able to completely clear spirochetes or confer protective immunity (Barbour et al., 2008). Antibody titers can remain for years in some LD patients while in others, they wane over time or never develop at all (Kalish et al., 2001). Herein, we describe animal studies that reveal mechanisms behind dysregulated development of adaptive immunity and provide insights that may be relevant to human immunity to B. burgdorferi infection. Antibodies produced through B and T cell interactions either within or outside germinal centers are termed T cell-dependent (TD) whereas those produced without the aid of T cells are T cell-independent (TI). One mechanism whereby B. burgdorferi attempt to evade adaptive immunity is by continuously changing the sequence of a unique surface protein called variable major protein-like sequence (VlsE) (Norris, 2015). Such sequence variation generates a large repertoire of antigenically-distinct spirochetes that become unrecognizable to the antibodies that are mounted against a previous version of this protein. This may allow B. burgdorferi to persist for months or years if not effectively cleared through innate immune response and/or early diagnosis and treatment with antibiotics. Another defensive strategy relies on switching which immunogenic proteins are surface expressed [e.g., Outer Surface Protein A (OspA) and OspC], as spirochetes transit from one environment to another. OspA and OspC are predominately expressed when spirochetes are in the tick vs. the mammalian host, respectively. Notably, whole-proteome microarray analysis revealed that while relatively few B. burgdorferi open reading frames (~15%) encode immunogens, those that do elicit the same detectable antibodies in naturally infected humans and wild white-footed mice (Peromyscus leucopus), the predominant maintenance reservoir for B. burgdorferi (Barbour et al., 2008). Interestingly, a spectrum of disease severity has been observed in different mouse strains, reflecting their unique genetic composition, which controls the magnitude of humoral responses during B. burgdorferi infection (Weis et al., 1999). Despite strong antibody responses in animals experimentally-infected with B. burgdorferi and in many LD patients, this does not translate to robust disease-resolving and long-term immunity (Barthold and Bockenstedt, 1993; Aguero-Rosenfeld et al., 1996). In order to explore the mechanisms through which B. burgdorferi infection impacts the immune system and gain an understanding of the role of B and T cells in LD pathogenesis, researchers have conducted studies in mice lacking either or both of these lymphocyte populations. Pathologies associated with B. burgdorferi infection of mice often spontaneously resolve, although animals may never completely clear spirochetes. In contrast, after infection with B. burgdorferi, severe combined immunodeficient (SCID) and recombination activating gene (RAG)-deficient mice, both of which lack B and T cells, developed severe, persistent arthritis that remained unresolved (Hastey et al., 2012). While B cells regulate disease progression and resolution in wild-type mice (McKisic and Barthold, 2000), adoptive transfer of CD4+ T cells into RAG-deficient mice prior to B. burgdorferi infection increased arthritis and carditis severity (unless B cells were co-transferred), and CD8+ T cell transfer increased arthritis severity (McKisic et al., 2000). Conversely, adoptive transfer of serum from immunocompetent B. burgdorferi-infected mice into SCID mice ameliorated both arthritis and carditis (Barthold et al., 1997; McKisic and Barthold, 2000). Transfer of immune serum into naive recipient mice either prior to or at the time of inoculation also prevented B. burgdorferi infection (Barthold et al., 1997). Immunization of mice with late-stage LD patient sera that demonstrated strong antibody reactivity to several B. burgdorferi proteins, including OspA and B, provided partial protection against B. burgdorferi challenge (Fikrig et al., 1994). These findings reveal that humoral immune responses generated in experimentally-infected mice and LD patients play an important role in the resolution of some of the most commonly reported clinical manifestations (arthritis and carditis), which are driven principally by activation of inflammatory T cells and release of potent inflammatory mediators. Researchers found unusually strong and persistent TD and TI IgM antibody production in lymph nodes during early infection and in bone marrow later on in the course of murine infection (Hastey et al., 2012; Richards et al., 2015). IgG-secreting plasma cells, on the other hand, accumulate slowly in the bone marrow. Only about 50% of the IgG response is clearly TD and, coupled with IgM, is thought to contribute to the reduction but not elimination of B. burgdorferi from tissues (Hodzic et al., 2003). This TD repertoire of IgG contributes minimally to long-term antibody-mediated immunity, unlike the typical humoral response to bacterial pathogens (Hastey et al., 2012; Tracy and Baumgarth, 2017). To dissect the mechanisms behind this dysregulated response, Hastey et al. (2012) elucidated distinct stages of altered immune response using a mouse model of LD. In the first phase of infection, B cells accumulated in lymph nodes and induced antibodies in a TI manner and in the absence of germinal centers. In other infectious diseases, such as mumps and HIV, swollen lymph nodes are a frequent early symptom of infection. Normally, the areas in which T and B cells are found in lymph nodes are well-defined. However, in B. burgdorferi-infected mice, this typical architecture was disrupted, with loss of organized B cell follicles and T cell zones (Tunev et al., 2011). Deterioration of B cell follicles, between days 5 and 10 post-infection occurred together with the presence of spirochetes within the lymph nodes (Hastey et al., 2014). In addition, B cells began to accumulate in large numbers, reaching over 70% in some instances and disrupting normal T/B cell ratios (Hastey et al., 2012). In the second phase, roughly 2–3 weeks later, short-lived germinal centers developed in lymph nodes. These germinal centers gave rise to relatively few antibody-producing plasma cells within bone marrow, leading to a third phase in which plasma cells only slowly accumulated. Lymph node germinal centers disappeared about 1 month after infection, despite the continued presence of bacteria at these sites. Curiously, B cell accumulation occurred after, not before, destructive changes in lymph node morphology. This suggests that the Borrelia infection, not B cell accumulation, somehow drives lymphoid tissue atrophy (Hastey et al., 2014). So, while B. burgdorferi infection prompts strong serum antibody levels, and titers increase over the course of infection, the antibody response is ultimately ineffective in completely eradicating spirochetes and/or establishing long-term immunity (Tunev et al., 2011; Hastey et al., 2012; Elsner et al., 2015). B. burgdorferi benefits from this maladaptive immune response. This premise is corroborated by a study of antibiotic-treated human LD patients, which included patients who had persistent symptomatology and those who had returned to health within 6 months after diagnosis and treatment (Blum et al., 2018). The researchers focused on plasmablasts, activated B cells that mature into plasma cells within germinal centers. They found that patients who ultimately recovered their health, as compared to those with persistent symptoms, had significantly more plasmablasts during early infection. The researchers determined this by comparing the percentage of plasmablasts as a total of all B cells at the initial clinic visit, during early infection (even before beginning Doxycycline therapy). In addition, patients who ultimately returned to health had significantly higher titers of antibodies to a diverse array of B. burgdorferi proteins (Blum et al., 2018). Taken together, this is evidence that B. burgdorferi infection redirects the adaptive immune system away from a long-term protective antibody response and toward a less efficacious, rapid and strong, though short-lived antibody response (Richards et al., 2015). Interestingly, Hastey et al. (2014) also provided evidence that Borrelia infection itself may have broader immunosuppressive effects. They tested this by using influenza virus vaccination as a tool to study TD antibody responses. Two groups of mice were influenza-vaccinated, with one group being infected with B. burgdorferi while the other was not. For the first 3 weeks, both groups of mice produced similar amounts of influenza-specific IgG. However, by 4 weeks, and until the study ended at 26 weeks, the B. burgdorferi-infected animals made significantly less influenza-specific IgG than the uninfected mice. By 9 weeks post-infection, there were far fewer influenza-specific antibody-secreting cells in the bone marrow of the Borrelia-infected animals compared to uninfected influenza-vaccinated mice (Elsner et al., 2015). This finding engenders an intriguing question about whether LD might negatively impact vaccination efficacy. There is more to be done in exploring these mechanisms of dysregulated antibody response in LD patients and there are clear implications for development of improved diagnostic tests. Physicians often follow the CDC-recommended two-tiered testing algorithm to detect B. burgdorferi-specific antibodies in patients suspected of having LD (Marques, 2015). The first-tier test is an enzyme-linked immunosorbent assay or ELISA, and if results are positive or borderline, a confirmatory second-tier test is done; a Western immunoblot analysis to detect IgM and IgG antibodies that are specific for B. burgdorferi proteins. In theory, this test determines if a B. burgdorferi infection is active (Marques, 2015). However, this CDC-recommended serodiagnosis may be misleading. In a small study of 79 patients who no longer had symptoms, but had a history of LD with and without arthritis 10–20 years ago, researchers examined antibody titers using the CDC two-tiered test (Kalish et al., 2001). They found that 10 individuals (13%) currently had IgM responses (reflecting initial exposure) to B. burgdorferi and 34 (43%) had IgG reactivity (reflecting longer term exposure) to B. burgdorferi. Antibody titers were even higher for patients who had LD with arthritis (but were currently asymptomatic), as six of 39 (15%) currently had IgM responses and 24 of 39 (62%) had IgG reactivity. This trend also is seen in infected mice, where IgM antibody levels do not wane but stay relatively high along with the increased IgG response (Hastey et al., 2012). Theoretically, it would be expected that all recovered patients would lack evidence of IgM and many or all would continue to have circulating IgG. The presence of IgM in 13% of patients would be cause for confusion for physicians as the presence of this class of antibody typically wanes with clearance of the pathogen and recovery from infection. Larger studies need to be done to confirm and to explain the reasons for the continued presence of IgM. Another consideration is that high antibody levels, as discussed, may only offer transient protection, with alterations in germinal center architecture and defective production of long-lived plasma cells and memory cells leading to poor immunoprotection in the long-term (Hastey et al., 2012; Elsner et al., 2015). Future efforts in Lyme disease diagnosis need to focus on distinguishing between active and inactive infection and improving sensitivity in detecting early disease while maintaining high specificity. Diagnosis would be greatly enhanced with the development and broad adoption of point-of-care testing, and simplified diagnosis. Addition of antigen targets expressed very early in LD (e.g., VlsE1 and pepC10) to current antibody-based diagnostic testing procedures have enhanced performance of the diagnostic assays (Porwancher et al., 2011; Marques, 2015). Additionally, direct detection of B. burgdorferi antigens or nucleic acid rather than antibody testing may eventually be possible with the development of advanced technologies (Branda et al., 2018). Not only might direct detection of spirochetal components be indicative of active infection, but the presence of nucleic acids and certain antigens coincides with the earliest stage of infection, when B. burgdorferi-specific antibodies have yet to be produced. Examples include B. burgdorferi DNA detected using PCR (Mosel et al., 2019) and antigens such as OspC (Ohnishi et al., 2001) or peptidoglycan (Jutras et al., 2019). OspC is expressed on spirochetes as they transit from tick to mammalian host (Ohnishi et al., 2001) while peptidoglycan has been shown to persist in patients long after antibiotic treatment has ceased and patients are theoretically cured of active infection (Jutras et al., 2019). The latter observation strongly supports the notion of persistence of B. burgdorferi after antibiotic treatment as peptidoglycan is only produced by metabolically-active spirochetes. There has been significant progress in deciphering the mechanistic foundation of B. burgdorferi's impact on the adaptive immune response, specifically the B cell response and antibody production. While B. burgdorferi initially elicits a strong immune response, the end result is a failure by the immune system to clear the infection. This could set the stage for B. burgdorferi's persistence (Tracy and Baumgarth, 2017), which may underlie chronic symptoms such as arthritis, carditis, and skin and neurological complications. The many animal studies conducted to date reveal that B. burgdorferi relies upon multiple strategies to evade and disrupt the normal functioning of the immune system. The end results are inhibition of effective B cell responses, disruption of the formation of stable germinal centers, and dampening the production of optimally protective antibodies and establishment of long-term memory cell populations. Importantly, many of these same evasion strategies appear to be employed by B. burgdorferi in LD patients, particularly those suffering from persistent or chronic disease. These intriguing observations provide an excellent foundation and springboard for further animal and human studies, with the goal of increased understanding of LD pathogenesis, better diagnostics, and ultimately novel and more effective therapeutic options for long-suffering patients. DB and TS contributed to the writing of this manuscript. All authors contributed to the article and approved the submitted version. Publication of this manuscript was financially supported by Global Lyme Alliance, Inc. DB is the owner of Edge Bioscience Communications, a freelance, contract scientific/medical writing and consulting company. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We would like to acknowledge Drs. Mayla Hsu and Nicole Baumgarth for thoughtful review of this manuscript prior to its submission for review. Adrion, E. R., Aucott, J., Lemke, K. W., and Weiner, J. P. (2015). Health care costs, utilization and patterns of care following Lyme disease. PLoS ONE 10:e0116767. doi: 10.1371/journal.pone.0116767 PubMed Abstract | CrossRef Full Text | Google Scholar Aguero-Rosenfeld, M. E., Nowakowski, J., Bittker, S., Cooper, D., Nadelman, R. B., and Wormser, G. P. (1996). Evolution of the serologic response to Borrelia burgdorferi in treated patients with culture-confirmed erythema migrans. J. Clin. Microbiol. 34, 1–9. PubMed Abstract | Google Scholar Barbour, A. G., Jasinskas, A., Kayala, M. A., Davies, D. H., Steere, A. C., Baldi, P., et al. (2008). A genome-wide proteome array reveals a limited set of immunogens in natural infections of humans and white-footed mice with Borrelia burgdorferi. Infect. Immun. 76, 3374–3389. doi: 10.1128/IAI.00048-08 PubMed Abstract | CrossRef Full Text | Google Scholar Barthold, S. W., and Bockenstedt, L. K. (1993). Passive immunizing activity of sera from mice infected with Borrelia burgdorferi. Infect. Immun. 61, 4696–4702. Google Scholar Barthold, S. W., Feng, S., Bockenstedt, L. K., Fikrig, E., and Feen, K. (1997). Protective and arthritis-resolving activity in sera of mice infected with Borrelia burgdorferi. Clin. Infect. Dis. 25, S9–S17. PubMed Abstract | Google Scholar Blum, L. K., Adamska, J. Z., Martin, D. S., Rebman, A. W., Elliott, S. E., Cao, R. R. L., et al. (2018). Robust B cell responses predict rapid resolution of Lyme disease. Front. Immunol. 9:1634. doi: 10.3389/fimmu.2018.01634 PubMed Abstract | CrossRef Full Text | Google Scholar Branda, J. A., Body, B. A., Boyle, J., Branson, B. M., Dattwyler, R. J., Fikrig, E., et al. (2018). Advances in serodiagnostic testing for Lyme disease are at hand. Clin. Infect. Dis. 66, 1133–1139. doi: 10.1093/cid/cix943 PubMed Abstract | CrossRef Full Text | Google Scholar Elsner, R. A., Hastey, C. J., Olsen, K. J., and Baumgarth, N. (2015). Suppression of long-lived humoral immunity following Borrelia burgdorferi infection. PLoS Pathog. 11:e1004976. doi: 10.1371/journal.ppat.1004976 PubMed Abstract | CrossRef Full Text | Google Scholar Fikrig, E., Bockenstedt, L. K., Barthold, S. W., Chen, M., Tao, H., Ali-Salaam, P., et al. (1994). Sera from patients with chronic Lyme disease protect mice from Lyme borreliosis. J. Infect. Dis. 169, 568–574. PubMed Abstract | Google Scholar Hastey, C. J., Elsner, R. A., Barthold, S. W., and Baumgarth, N. (2012). Delays and diversions mark the development of B cell responses to Borrelia burgdorferi infection. J. Immunol. 188, 5612–5622. doi: 10.4049/jimmunol.1103735 PubMed Abstract | CrossRef Full Text | Google Scholar Hastey, C. J., Ochoa, J., Olsen, K. J., Barthold, S. W., and Baumgarth, N. (2014). MyD88- and TRIF-independent induction of type I interferon drives naive B cell accumulation but not loss of lymph node architecture in Lyme disease. Infect. Immun. 82, 1548–1558. doi: 10.1128/IAI.00969-13 PubMed Abstract | CrossRef Full Text | Google Scholar Hodzic, E., Feng, S., Freet, K. J., and Barthold, S. W. (2003). Borrelia burgdorferi population dynamics and prototype gene expression during infection of immunocompetent and immunodeficient mice. Infect. Immun. 71, 5042–5055. doi: 10.1128/IAI.71.9.5042-5055.2003 PubMed Abstract | CrossRef Full Text | Google Scholar Hu, L. T. (2016). Lyme disease. Ann. Intern. Med. 165:677. doi: 10.7326/L16-0409 PubMed Abstract | CrossRef Full Text | Google Scholar Jutras, B. L., Lochhead, R. B., Kloos, Z. A., Biboy, J., Strle, K., Booth, C. J., et al. (2019). Borrelia burgdorferi peptidoglycan is a persistent antigen in patients with Lyme arthritis. Proc. Natl. Acad. Sci. U.S.A. 116, 13498–13507. doi: 10.1073/pnas.190417011 PubMed Abstract | CrossRef Full Text | Google Scholar Kalish, R. A., McHugh, G., Granquist, J., Shea, B., Ruthazer, R., Steere, A. C., et al. (2001). Persistence of immunoglobulin M or immunoglobulin G antibody responses to Borrelia burgdorferi 10-20 years after active Lyme disease. Clin. Infect. Dis. 33, 780–785. doi: 10.1086/322669 PubMed Abstract | CrossRef Full Text | Google Scholar Marques, A. R. (2015). Laboratory diagnosis of Lyme disease: advances and challenges. Infect. Dis. Clin. North Am. 29, 295–307. doi: 10.1016/j.idc.2015.02.005 PubMed Abstract | CrossRef Full Text | Google Scholar McKisic, M. D., and Barthold, S. W. (2000). T-cell-independent responses to Borrelia burgdorferi are critical for protective immunity and resolution of lyme disease. Infect. Immun. 68, 5190–5197. PubMed Abstract | Google Scholar McKisic, M. D., Redmond, W. L., and Barthold, S. W. (2000). Cutting edge: T cell-mediated pathology in murine Lyme borreliosis. J. Immunol. 164, 6096–6099. doi: 10.4049/jimmunol.164.12.6096 PubMed Abstract | CrossRef Full Text | Google Scholar Mosel, M. R., Carolan, H. E., Rebman, A. W., Castro, S., Massire, C., Ecker, D. J., et al. (2019). Molecular testing of serial blood specimens from patients with early Lyme disease during treatment reveals changing coinfection with mixtures of Borrelia burgdorferi genotypes. Antimicrob. Agents Chemother. 63:e00237-19. doi: 10.1128/AAC.00237-19 PubMed Abstract | CrossRef Full Text | Google Scholar Norris S. (2015). “vls antigenic variation systems of lyme disease Borrelia: eluding host immunity through both random, segmental gene conversion and framework heterogeneity,” in Mobile DNA III, eds N. Craig, M. Chandler, M. Gellert, A. Lambowitz, P. Rice, and S. Sandmeyer (Washington, DC: ASM Press), 471–489. doi: 10.1128/microbiolspec.MDNA3-0038-2014 PubMed Abstract | CrossRef Full Text | Google Scholar Ohnishi, J., Piesman, J., and de Silva, A. M. (2001). Antigenic and genetic heterogeneity of Borrelia burgdorferi populations transmitted by ticks. Proc. Natl. Acad. Sci. U.S.A. 98, 670–675. doi: 10.1073/pnas.98.2.670 PubMed Abstract | CrossRef Full Text | Google Scholar Porwancher, R. B., Hagerty, C. G., Fan, J., Landsberg, L., Johnson, B. J., Kopnitsky, M., et al. (2011). Multiplex immunoassay for Lyme disease using VlsE1-IgG and pepC10-IgM antibodies: improving test performance through bioinformatics. Clin. Vaccine Immunol. 18, 851–859. doi: 10.1128/CVI.00409-10 PubMed Abstract | CrossRef Full Text | Google Scholar Richards, K. A., Chaves, F. A., and Sant, A. J. (2015). CD4+ T cells promote antibody production but not sustained affinity maturation during Borrelia burgdorferi infection. Infect. Immun. 83, 48–56. doi: 10.1128/IAI.02471-14 PubMed Abstract | CrossRef Full Text | Google Scholar Steere, A. C., Strle, F., Wormser, G. P., Hu, L. T., Branda, J. A., Hovius, J. W. R., et al. (2016). Lyme borreliosis. Nat. Rev. Dis. Primers 2:16090. doi: 10.1038/nrdp.2016.90 CrossRef Full Text | Google Scholar Tracy, K. E., and Baumgarth, N. (2017). Borrelia burgdorferi manipulates innate and adaptive immunity to establish persistence in rodent reservoir hosts. Front. Immunol. 8:116. doi: 10.3389/fimmu.2017.00116 PubMed Abstract | CrossRef Full Text | Google Scholar Tunev, S. S., Hastey, C. J., Hodzic, E., Feng, S., Barthold, S. W., and Baumgarth, N. (2011). Lymphoadenopathy during lyme borreliosis is caused by spirochete migration-induced specific B cell activation. PLoS Pathog. 7:e1002066. doi: 10.1371/journal.ppat.1002066 PubMed Abstract | CrossRef Full Text | Google Scholar Weis, J. J., McCracken, B. A., Ma, Y., Fairbairn, D., Roper, R. J., Morrison, T. B., et al. (1999). Identification of quantitative trait loci governing arthritis severity and humoral responses in the murine model of Lyme disease. J. Immunol. 162, 948–956. PubMed Abstract | Google Scholar Keywords: Lyme disease, Borrelia burgdorferi, antigen-presenting cell, B cell, antibody Citation: Sellati TJ and Barberio DM (2020) Mechanisms of Dysregulated Antibody Response in Lyme Disease. Front. Cell. Infect. Microbiol. 10:567252. doi: 10.3389/fcimb.2020.567252 Received: 29 May 2020; Accepted: 02 September 2020; Published: 07 October 2020. Edited by: Reviewed by: Copyright © 2020 Sellati and Barberio. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Timothy J. Sellati, [email protected]
, Patrícia Poeta, Bo Peng
Frontiers in Cellular and Infection Microbiology, Volume 10; https://doi.org/10.3389/fcimb.2020.586460

Abstract:
Editorial on the Research TopicThe Molecular Mechanisms of Antibiotic Resistance in Aquatic Pathogens Bacterial antibiotic resistance has become a formidable problem in the treatment of infectious diseases worldwide. The molecular mechanisms of antibiotic resistance remain elusive, especially for aquatic pathogens. Antibiotic resistance and virulence determinants in environmental/aquatic bacterial pathogens are the subject of five research articles published in this Research Topic. Mishra et al. have investigated the influence of outer membrane proteins (OMPs) on the antibiotic resistance profile and virulence factors of Enterobacter isolated from aquatic environments and clinical settings. The environmental Enterobacter isolates lack OmpC, are unable to invade host cells, and induce low amounts of reactive oxygen species (ROS) production by neutrophils. In contrast, a clinical Enterobacter isolate described possesses OmpF, has better invasive and adhesive capabilities, and stimulates a higher amount of ROS production. Furthermore, some clinical Enterobacter isolates which have the ompA gene also have the strong capacity to form biofilms. Taking a different angle, Abdelhamed et al. and Opazo-Capurro et al. have sequenced the whole genomes of the Edwardsiella piscicida MS 18-99 strain possessing a 117.4 kb conjugative plasmid pEPMS-1899, and Acinetobacter radioresistens A154 harboring several plasmids, respectively. The genome analysis revealed that several antimicrobial resistance genes/elements confer resistance to various antibiotics, such as tetA&R, strA&B, sul2, and floR in MS 18-99, arsA and arsD on pEPMS-1899, and tetB, strA, strB, aph(3)-Vla, aac(3)-IIa, blaSCO-1, blaTEM-1B, and blaPER-2 in A. radioresistens. A study by Montso et al. describes the presence of genes such as shiga toxins (stx) and bundle-forming pili (bfpA) that are related to virulence determinants and antibiotic resistance in atypical enteropathogenic Escherichia coli (aEPEC) O177, the first reported occurrence of the aEPEC O177 strain in South African cattle. Tekedar et al. have characterized genome sequences of Aeromonas veronii MS 17-88 strains isolated from channel catfish to uncover the mechanism of multidrug resistance. This strain has several antimicrobial resistance genes/elements such as tet(34), tet(35), tet(E), tetR, mcr-7.1, and floR. These five studies have all contributed further evidence of the abundance and prevalence of antibiotic resistance genes in different bacterial genomes. Proteins are the fundamental and vital material of life. Ribosome rescue is a mechanism to maintain protein synthesis when mistakes or stalling occur. Quality control of protein synthesis is part of this process, which requires transfer-messenger RNA (tmRNA) and small protein B (SmpB). In this Research Topic, there are three articles from Prof. Liu's group describing the involvement of tmRNA and SmpB in the antibiotic resistance and persistence development of A. veronii. The transcriptomic data presented reveal the downregulation of protein secretion systems, such as the type VI secretion system (T6SS) and type IV pilus assembly, in the SmpB deficient strain. This downregulation attenuated the production of virulence factors and the colonization ability of A. veronii. However, the alternative rescue factor A (AvrA) acts as a substitute for virulence compensation. The decreased level of avrA expression increases the expression level of iron-sulfur protein activator iscR, resulting in an increase in bacterial antioxidant ability through enhancement of the synthesis of the iron-sulfur protein and assembly of iron-sulfur clusters (Santos et al., 2014). SmpB deficiency showed attenuated virulence and enhanced tolerance to oxidative stress. In another study, the deletion of the smpB gene in A. veronii enhanced the expression of enzymes related to adenosine metabolism, which leads to increases in the products such as adenosine AMP, cAMP and deoxyadenosine. By comparison, the deletion of ssrA (tmRNA) upregulated the expression of guanosine metabolism and hypoxanthine synthesis, eventually increasing the levels of the metabolic product xanthine, which in turn downregulated the genes related to the efflux pump such as acrA and acrB. With the ssrA and smpB genes deleted, the minimal inhibitory concentration of trimethoprim against A. veronii was lower. Therefore, this study demonstrates the effect of SmpB and tmRNA on different branches of purine metabolism and tolerance to trimethoprim (Wang et al.). A study by Yu et al. showed that the deletion of ssrA (tmRNA) increased the capacity of A. veronii to form persister cells. Moreover, ΔssrA significantly raised the intercellular levels of N-acetylglucosamine production, promoting NaCl osmotic stress tolerance. To confirm this, the authors exogenously added N-acetylglucosamine to the medium and observed resistance to osmotic pressure and higher persistence to cefotaxime in A. veronii. Overall, these three research articles suggest that the trans-translation system might be an effective target in the treatment of A. veronii and other multidrug resistant aquatic bacterial pathogens. Enhancing the efficiency of traditional antibiotics is a vital strategy for combatting the antibiotic resistance in bacterial pathogens. A study by Sun et al. has revealed that indole and its derivative 2,5-methylindole potentiate the killing efficacy of tobramycin against persister cells of Staphylococcus aureus. Furthermore, combinations of indole and its derivatives with aminoglycoside antibiotics were effective in inhibiting the growth of many Gram-positive bacterial pathogens such as Streptococcus pyogenes and Enterococcus faecalis. Finally, Zhou et al. have studied the role of the type III secretion system (T3SS) on the pathogenicity of Vibrio alginolyticus. Swarming motility is inhibited in the T3SS mutant strain tyeA and its virulence toward zebrafish is also reduced. Further, the tyeA mutant strain is extremely susceptible to cefoperazone, minocycline, amikacin, and gentamicin. The effects of a live attenuated vaccine made from the mutant strain on the survival of zebrafish was tested. The data presented show that the survival of zebrafish infected with mutant was enhanced compared to the control infected with the wild-type strain. Both of these approaches may pave the way for the development of an effective treatment strategy to combat antibiotic resistance in bacterial pathogens. XL and PP wrote the manuscript. BP revised and approved the final version of the manuscript. All authors conceived the outline of the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This work was sponsored by grants from NSFC projects (Nos. 31670129). We thank Dr. Xiaopeng Xiong for suggesting on the manuscript. Santos, J. A., Alonso-García, N., Macedo-Ribeiro, S., and Pereira, P. J.B. (2014). The unique regulation of iron-sulfur cluster biogenesis in a Gram-positive bacterium. Proc. Natl. Acad. Sci. U.S.A. 111, 2251–2260. doi: 10.1073/pnas.1322728111 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: aquatic pathogens, antibiotic resistance, mechanisms, multidrug resistance, prevention strategies Citation: Lin X, Poeta P and Peng B (2020) Editorial: The Molecular Mechanisms of Antibiotic Resistance in Aquatic Pathogens. Front. Cell. Infect. Microbiol. 10:586460. doi: 10.3389/fcimb.2020.586460 Received: 23 July 2020; Accepted: 20 August 2020; Published: 24 September 2020. Edited and reviewed by: John S. Gunn, The Research Institute at Nationwide Children's Hospital, United States Copyright © 2020 Lin, Poeta and Peng. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Xiangmin Lin, [email protected]
Published: 16 September 2020
Frontiers in Pharmacology, Volume 11; https://doi.org/10.3389/fphar.2020.580163

Abstract:
A Commentary onRecommendation of Antimicrobial Dosing Optimization During Continuous Renal Replacement Therapyby Li L., Li X., Xia Y., Chu Y., Zhong H., Li J. et al. (2020). Front. Pharmacol. 11:786 doi: 10.3389/fphar.2020.00786 Facing challenges of appropriate antimicrobial dosing during Continuous Renal Replacement Therapy (CRRT), Li et al. recently published a review to optimize efficacy and to limit toxicity (Li et al., 2020). Although it is helpful for clinician at patient bedside to choose dosing in the absence of pharmacokinetic individual data, some limitations should be highlighted. One dose does not fit all and antimicrobial optimization in critically ill patients requires a holistic approach applying different pharmacokinetic-pharmacodynamic (PK-PD) principles not only based on CRRT modalities. Existing literature presents many limitations. As noticed by Li et al., CRRT practices are heterogeneous leading to subsequent diversity in antimicrobial dosing (Churchwell and Mueller, 2009; Lewis and Mueller, 2014). Other authors demonstrated low quality of CRRT pharmacokinetic studies and lack of key information in the literature needed to define dosing regimens, particularly the delivered CRRT dose (Li et al., 2009; Vaara et al., 2012). The studies often only include a few patients, unrepresentative of the population with single or multi-compartmental models. Critical care populations variety may jeopardize the extrapolation of the pharmacokinetic results (Ulldemolins et al., 2014). Pathophysiology of the diseases and patients severity are different leading to wide pharmacokinetics variations, as described in the DALI study reporting that beta-lactams concentrations could vary by a factor of 100 from patient to patient in Intensive Care Unit (ICU) (Roberts et al., 2014). Therefore, pharmacokinetic parameters of healthy volunteers cannot be used. Extreme inter/intra-individual pharmacokinetic variability in ICU and potential PK-PD uncertainties necessitate dosing simulations in different clinical contexts to suggest appropriate dosing regimens at individual level (Dhaese et al., 2019). As noticed by Li, his recommendations may be not sufficient for patients with residual renal function. Dynamic change of patient’s clinical status with subsequent modifications of pharmacokinetics should be considered for dosing adaptation (Choi et al., 2009; Guilhaumou et al., 2019). Underdosing is over-prevalent during the initial septic phase and antimicrobial initial doses should consider the increase of volume of distribution (De Waele and Carlier, 2014; Wong et al., 2015). However, this requires sophisticated PK-PD models integrating patients and population data. Subsequent doses should be decided according to total clearance. Therefore, fixed doses do not appear appropriate during CRRT. As noted by Li, drug clearance depends on CRRT modalities. Potential drug infusion incompatibilities and interactions with the whole circuit should also be consider, including tubing, especially when polyvinylchloride (PVC) is used (Preston et al., 2007; Shekar et al., 2015). Lack of CRRT standardization leads to high pharmacokinetics differences requiring in vitro data to study the impact of CRRT modalities. Other pharmacokinetic modifications than CRRT may impact antimicrobial concentrations, as fluid balance variability, protein binding modifications and hepatic function. This may impact the PK-PD target attainment (Ulldemolins et al., 2011; Lewis and Mueller, 2014; Vanstraelen et al., 2014; Kurland et al., 2019). Some studies consider total antimicrobial concentration instead of active unbound concentration without possible interpretation of the target attainment (Roberts et al., 2013). Precaution should therefore be taken to define dosing regimens. Li did not clearly describe PK-PD targets selected to define antimicrobial dosing. Lack of consensus for some antimicrobials as beta-lactams leads to different dosing regimens recommendations, as reported for meropenem (PK-PD target from 40% T > MIC to 5 x 100% T > MIC) (Kawano et al., 2015; Ulldemolins et al., 2015). PK-PD targets and subsequent dosing depend on bacteria sensibility and should be consider for all antimicrobials. Infusion mode is crucial to guide dosing regimens, optimizing the probability of PK-PD target attainment, limiting potential toxicity and the emergence of bacterial resistance. The randomized controlled BLISS study demonstrated higher clinical cure rates in septic patients with continuous infusions of beta-lactams than with intermittent infusions for the same daily dose, and higher PK-PD target attainment rates at 100% fT > MIC (Abdul-Aziz et al., 2016). However, extended and continuous infusions should not be used without protocolization to assure stability, especially for carbapenems and amoxicillin+/-clavulanic acid. Continuous/extended infusions including a loading dose were therefore recommended for beta-lactams in some clinically contexts or in case of severity (Guilhaumou et al., 2019). Moreover, the same intermittent infusions dosing cannot be applied to continuous and extended infusions to obtain a defined target concentration. When PK-PD target is attained, continuous/extended infusions also allow a daily dose reduction permitting to limit toxicity (Guilhaumou et al., 2019). Regarding inter/intra-individual variability, individualized PK-PD targets and dosing are required (Goldstein and Nolin, 2014; Roberts and Roberts, 2014; Shaw et al., 2016). Some European learnt societies recommended TDM as a standard of care for most antimicrobials in ICU, especially for patients treated by CRRT, and Li suggested it to optimize therapy (Guilhaumou et al., 2019; Abdul-Aziz et al., 2020). TDM allows a higher probability of PK-PD target attainment and to limit over and underdosing, especially when Monte Carlo simulations are not available in the population of interest. To assure better accuracy of dose adaptations, a modeling population pharmacokinetic approach should be used, permitting to consider inter/intraindividual variations (Fuchs, 2015; Penetrat-Roger, 2018). Bayesian approach, established on conditional probabilities, permits to predict individual antibiotic concentrations integrating data from population models and individual pharmacokinetic parameters, bacterial ecology and previous concentrations observed. Many factors are required to determine appropriated dosing regimens for patients treated by CRRT. Dosing data from the literature can be applied under the strict similar conditions of the studies (Choi et al., 2009). Facing pharmacokinetic variability in critically ill patients, CRRT practices heterogeneity and the bacterial local ecology, TDM and Bayesian modelling represent the best options to optimize antimicrobial dosing and their development should be a priority to individualize dosing. However, in the absence of sophisticated pharmacokinetic tools, Li’s recommendations contribute to give an order of magnitude of CRRT impact and may help to avoid underdoses/overdoses on the basis of the available data, with a possible integration in an antibiotic stewardship program considering other PK-PD considerations. The author confirms being the sole contributor of this work and has approved it for publication. The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. CRRT, Continuous Renal Replacement Therapy; ICU, Intensive Care Unit; PK-PD, Pharmacokinetic-Pharmacodynamic; TDM, Therapeutic Drug Monitoring. Abdul-Aziz, M. H., Sulaiman, H., Mat-Nor, M.-B., Rai, V., Wong, K. K., Hasan, M. S., et al. (2016). Beta-Lactam Infusion in Severe Sepsis (BLISS): a prospective, two-centre, open-labelled randomised controlled trial of continuous versus intermittent beta-lactam infusion in critically ill patients with severe sepsis. Intensive Care Med. 42 (10), 1535−45. doi: 10.1007/s00134-015-4188-0 CrossRef Full Text | Google Scholar Abdul-Aziz, M. H., Alffenaar, J.-W. C., Bassetti, M., Bracht, H., Dimopoulos, G., Marriott, D., et al. (2020). Antimicrobial therapeutic drug monitoring in critically ill adult patients: a Position Paper. Intensive Care Med. 46 (6), 1127−53. doi: 10.1007/s00134-020-06050-1 CrossRef Full Text | Google Scholar Choi, G., Gomersall, C. D., Tian, Q., Joynt, G. M., Freebairn, R., Lipman, J. (2009). Principles of antibacterial dosing in continuous renal replacement therapy. Crit. Care Med. 37 (7), 2268−82. doi: 10.1097/CCM.0b013e3181aab3d0 PubMed Abstract | CrossRef Full Text | Google Scholar Churchwell, M. D., Mueller, B. A. (2009). Drug dosing during continuous renal replacement therapy. Semin. Dial. 22 (2), 185−8. doi: 10.1111/j.1525-139X.2008.00541.x PubMed Abstract | CrossRef Full Text | Google Scholar De Waele, J. J., Carlier, M. (2014). Beta-lactam antibiotic dosing during continuous renal replacement therapy: how can we optimize therapy? Crit. Care 18 (3), 158. doi: 10.1186/cc13945 PubMed Abstract | CrossRef Full Text | Google Scholar Dhaese, S. A. M., Farkas, A., Colin, P., Lipman, J., Stove, V., Verstraete, A. G., et al. (2019). Population pharmacokinetics and evaluation of the predictive performance of pharmacokinetic models in critically ill patients receiving continuous infusion meropenem: a comparison of eight pharmacokinetic models. J. Antimicrob. Chemother. 74 (2), 432−41. doi: 10.1093/jac/dky434 CrossRef Full Text | Google Scholar Fuchs, A. (2015). Implementation of Bayesian Therapeutic Drug Monitoring In Modern Patient Care. [Ph.D.’s thesis] (Lausanne: Université de Lausanne). Available at: https://serval.unil.ch/resource/serval:BIB_5A2ED1C8C297.P001/REF.pdf (Accessed May 30, 2020). Google Scholar Goldstein, S. L., Nolin, T. D. (2014). Lack of drug dosing guidelines for critically ill patients receiving continuous renal replacement therapy. Clin. Pharmacol. Ther. 96 (2), 159−61. doi: 10.1038/clpt.2014.102 PubMed Abstract | CrossRef Full Text | Google Scholar Guilhaumou, R., Benaboud, S., Bennis, Y., Dahyot-Fizelier, C., Dailly, E., Gandia, P., et al. (2019). Optimization of the treatment with beta-lactam antibiotics in critically ill patients-guidelines from the French Society of Pharmacology and Therapeutics (Société Française de Pharmacologie et Thérapeutique-SFPT) and the French Society of Anaesthesia and Intensive Care Medicine (Société Française d’Anesthésie et Réanimation-SFAR). Crit. Care Med. 23 (1), 104. doi: 10.1186/s13054-019-2378-9 CrossRef Full Text | Google Scholar Kawano, S., Matsumoto, K., Hara, R., Kuroda, Y., Ikawa, K., Morikawa, N., et al. (2015). Pharmacokinetics and dosing estimation of meropenem in Japanese patients receiving continuous venovenous hemodialysis. J. Infect. Chemother. 21 (6), 476−8. doi: 10.1016/j.jiac.2015.02.011 PubMed Abstract | CrossRef Full Text | Google Scholar Kurland, S., Furebring, M., Löwdin, E., Eliasson, E., Nielsen, E. I., Sjölin, J. (2019). Pharmacokinetics of Caspofungin in Critically Ill Patients in Relation to Liver Dysfunction: Differential Impact of Plasma Albumin and Bilirubin Levels. Antimicrob. Agents Chemother. 63 (6), e02466–e02418. doi: 10.1128/AAC.02466-18 PubMed Abstract | CrossRef Full Text | Google Scholar Lewis, S. J., Mueller, B. A. (2014). Antibiotic dosing in critically ill patients receiving CRRT: underdosing is overprevalent. Semin. Dial. 27 (5), 441−5. doi: 10.1111/sdi.12203 PubMed Abstract | CrossRef Full Text | Google Scholar Li, A. M. M. Y., Gomersall, C. D., Choi, G., Tian, Q., Joynt, G. M., Lipman, J. (2009). A systematic review of antibiotic dosing regimens for septic patients receiving continuous renal replacement therapy: do current studies supply sufficient data? J. Antimicrob. Chemother. 64 (5), 929−37. doi: 10.1093/jac/dkp302 PubMed Abstract | CrossRef Full Text | Google Scholar Li, L., Li, X., Xia, Y., Chu, Y., Zhong, H., Li, J., et al. (2020). Recommendation of Antimicrobial Dosing Optimization During Continuous Renal Replacement Therapy. Front. Pharmacol. 11:786. doi: 10.3389/fphar.2020.00786 PubMed Abstract | CrossRef Full Text | Google Scholar Penetrat-Roger, C. (2018). Impact du sepsis et de l"épuration extra-rénale sur la pharmacocinétique des antibiotiques. [Ph.D. thesis] (Montpellier: Université de Montpellier). Available at: https://tel.archives-ouvertes.fr/tel-01690753/document (Accessed May 30, 2020). Google Scholar Preston, T. J., Hodge, A. B., Riley, J. B., Leib-Sargel, C., Nicol, K. K. (2007). In Vitro Drug Adsorption and Plasma Free Hemoglobin Levels Associated With Hollow Fiber Oxygenators in the Extracorporeal Life Support (ECLS) Circuit. J. Extra Corpor. Technol. 39 (4), 234−7. PubMed Abstract | Google Scholar Roberts, J. A., Roberts, D. M. (2014). Antibiotic dosing in critically ill patients with septic shock and on continuous renal replacement therapy: can we resolve this problem with pharmacokinetic studies and dosing guidelines? Crit. Care 18 (3), 156. doi: 10.1186/cc13939 PubMed Abstract | CrossRef Full Text | Google Scholar Roberts, J. A., Pea, F., Lipman, J. (2013). The clinical relevance of plasma protein binding changes. Clin. Pharmacokinet. 52 (1), 1−8. doi: 10.1007/s40262-012-0018-5 PubMed Abstract | CrossRef Full Text | Google Scholar Roberts, J. A., Paul, S. K., Akova, M., Bassetti, M., De Waele, J. J., Dimopoulos, G., et al. (2014). DALI: defining antibiotic levels in intensive care unit patients: are current β-lactam antibiotic doses sufficient for critically ill patients? Clin. Infect. Dis. 58 (8), 1072−83. doi: 10.1093/cid/ciu027 PubMed Abstract | CrossRef Full Text | Google Scholar Shaw, A. R., Chaijamorn, W., Mueller, B. A. (2016). We Underdose Antibiotics in Patients on CRRT. Semin. Dial. 29 (4), 278−80. doi: 10.1111/sdi.12496 PubMed Abstract | CrossRef Full Text | Google Scholar Shekar, K., Roberts, J. A., Mcdonald, C. I., Ghassabian, S., Anstey, C., Wallis, S. C., et al. (2015). Protein-bound drugs are prone to sequestration in the extracorporeal membrane oxygenation circuit: results from an ex vivo study. Crit. Care 19 (1), 164. doi: 10.1186/s13054-015-0891-z PubMed Abstract | CrossRef Full Text | Google Scholar Ulldemolins, M., Roberts, J. A., Rello, J., Paterson, D. L., Lipman, J. (2011). The effects of hypoalbuminaemia on optimizing antibacterial dosing in critically ill patients. Clin. Pharmacokinet. 50 (2), 99−110. doi: 10.2165/11539220-000000000-00000 PubMed Abstract | CrossRef Full Text | Google Scholar Ulldemolins, M., Vaquer, S., Llauradó-Serra, M., Pontes, C., Calvo, G., Soy, D., et al. (2014). Beta-lactam dosing in critically ill patients with septic shock and continuous renal replacement therapy. Crit. Care 18 (3), 227. doi: 10.1186/cc13938 PubMed Abstract | CrossRef Full Text | Google Scholar Ulldemolins, M., Soy, D., Llaurado-Serra, M., Vaquer, S., Castro, P., Rodríguez, A. H., et al. (2015). Meropenem population pharmacokinetics in critically ill patients with septic shock and continuous renal replacement therapy: influence of residual diuresis on dose requirements. Antimicrob. Agents Chemother. 59 (9), 5520−8. doi: 10.1128/AAC.00712-15 PubMed Abstract | CrossRef Full Text | Google Scholar Vaara, S., Pettila, V., Kaukonen, K.-M. (2012). Quality of pharmacokinetic studies in critically ill patients receiving continuous renal replacement therapy. Acta Anaesthesiol. Scand. 56 (2), 147−57. doi: 10.1111/j.1399-6576.2011.02571.x PubMed Abstract | CrossRef Full Text | Google Scholar Vanstraelen, K., Wauters, J., Vercammen, I., De Loor, H., Maertens, J., Lagrou, K., et al. (2014). Impact of hypoalbuminemia on voriconazole pharmacokinetics in critically ill adult patients. Antimicrob. Agents Chemother. 58 (11), 6782–6789. doi: 10.1128/AAC.03641-14 PubMed Abstract | CrossRef Full Text | Google Scholar Wong, W., Choi, G., Gomersall, C. D., Lipman, J. (2015). To increase or decrease dosage of antimicrobials in septic patients during continuous renal replacement therapy: the eternal doubt. Curr. Opin. Pharmacol. 24, 68−78. doi: 10.1016/j.coph.2015.07.003 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: anti-infective agents, antimicrobials, pharmacokinetics, pharmacodynamics, critical care, continuous renal replacement therapy, therapeutic drug monitoring, dosing optimization Citation: Matusik E (2020) Commentary: Recommendation of Antimicrobial Dosing Optimization During Continuous Renal Replacement Therapy. Front. Pharmacol. 11:580163. doi: 10.3389/fphar.2020.580163 Received: 04 July 2020; Accepted: 28 August 2020; Published: 16 September 2020. Edited by: Reviewed by: Copyright © 2020 Matusik. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Elodie Matusik, [email protected]
Arun Suria Karnan Mahendran, Yin Sze Lim, Chee-Mun Fang, Hwei-San Loh,
Published: 15 September 2020
Frontiers in Pharmacology, Volume 11; https://doi.org/10.3389/fphar.2020.575444

Abstract:
To date, more than 17 million cases of coronavirus disease 2019 (COVID-19) and 900,000 deaths have been reported globally (World Health Organization, 2020). Major collaborative efforts focusing on the development of anti-SARS-CoV-2 vaccines and antiviral therapeutics are accelerated at an unprecedented rate. Various therapeutic agents quickly taken into clinical trials are largely based on existing drugs with non-specific antiviral activities or compounds pharmacologically speculated to be effective in enhancing the overall clinical outcome of COVID-19 patients. To name a few, these include the nucleoside inhibitor prodrug remdesivir (GS-5734), the antiparasitic drug ivermectin, the HIV protease inhibitor nelfinavir, the anti-inflammatory drug cepharanthine, the antimalarial drug hydroxychloroquine, the general steroid dexamethasone, and others (Caly et al., 2020; Ledford, 2020; Ohashi et al., 2020; Vanden Eynde, 2020; Wang Y. et al., 2020). However, only dexamethasone and remdesivir appear to confer promising treatment outcomes by reducing deaths or time to clinical improvement in the severe COVID-19 patients (Grein et al., 2020; Ledford, 2020; Wang Y. et al., 2020). The world is extremely desperate for a cure or prophylaxis against SARS-CoV-2 with the hope of saving more lives. Nevertheless, little has been described for antiviral peptides (AVPs) or alternatively known as antimicrobial peptides (AMPs) that possess antiviral activities. AVPs are a class of short (8–40 amino acids in length) polycationic antivirals with potent broad spectrum antiviral activities (Chang and Yang, 2013; Skalickova et al., 2015; Ahmed et al., 2019; Nyanguile, 2019; Sala et al., 2019; Vilas Boas et al., 2019). Interestingly, there are AVPs demonstrated to exert prophylactic and therapeutic effects against coronaviruses (CoVs). In this opinion paper, we describe the potential use of AVPs against COVID-19 based on the documented evidence against SARS-CoV2, SARS-CoV, MERS-CoV, SARS-related CoVs, and other respiratory viruses that shall warrant further development of this class of compounds in the face of the current pandemic threat. SARS-CoV-2 is an enveloped virus with the characteristic spike (S) glycoprotein to mediate viral entry via the cell surface receptor angiotensin-converting enzyme-2 (ACE2) (Zhou et al., 2020). The S1 subunit of S protein is the receptor-binding domain responsible for ACE2 binding (Xia et al., 2020b). Subsequently, a cellular serine protease TMPRSS2 is required for S protein priming, which induces proteolytic cleavage of S protein at S1/S2 and S2’ sites (Hoffmann et al., 2020). Following cleavage, both heptapeptide repeat 1 (HR1) and heptapeptide repeat 2 (HR2) regions of the S2 subunit of S protein interact to form the 6-helix bundle (6HB) fusion core (Liu et al., 2004; Kang et al., 2020). The formation of 6HB is critical in facilitating the viral membrane fusion process for viral entry into the host cell via endocytosis. During the late endosomal stage, endosomal acidification would induce virus-endosome membrane fusion, thereby leading to viral uncoating (viral RNA release) for the initiation of viral replication and infection (Du et al., 2009; Das et al., 2010; Letko et al., 2020). Notably, the S1 subunit of SARS-CoV-2 showed a 10- to 20-fold higher ACE2 binding affinity comparing to SARS-CoV, which may explain the higher transmissibility and infectivity of COVID-19 (Wrapp et al., 2020). Mucroporin-M1 (LFRLIKSLIKRLVSAFK) is a peptide analog designed with four residual mutations at positions G3R, P6K, G10K, and G11R from the parent peptide mucroporin (LFGLIPSLIGGLVSAFK) isolated from the venom of the scorpion Lychas mucronatus (Dai et al., 2008; Li et al., 2011). Such increase in cationicity enhanced the dipolar characteristic of the peptide helix, which contributed to the drastic increment in antiviral activity against SARS-CoV (50% virus infection, EC50 of 14.46 µg/ml), influenza H5N1 (EC50 of 2.10 µg/ml), and measles (EC50 of 7.15 µg/ml) pseudoviruses (Li et al., 2011). Based on the observations that mucroporin-M1 directly interacted with measles viral particles and a greater antiviral effect with pre-treatment, mucroporin-M1 was proposed to serve the role of a molecular blocker, which must find its target before viral attachment to the host cells (Figure 1). Following peptide-virus binding, the strong electrostatic affinity of mucroporin-M1 could allow interaction and disruption of the viral envelope, thereby exerting a direct virucidal effect against SARS-CoV, MERS-CoV, and influenza H5N1 viruses (Li et al., 2011). Figure 1 Mechanism of actions of AVPs with potential anti-SARS-CoV-2 activities. Several AVPs target the key structural components of the virus to exert antiviral effects: mucroporin-M1 acts by disrupting viral envelope, HR2P-M2 targets the viral S protein-mediated fusion, EK1 and EK1C4 block the HR1 domain of viral S2 subunit, and P9 peptide inhibits late endosomal acidification and thus preventing viral RNA release. There are AVPs which confer antiviral protection to the host: RTD-1 is a potent antiviral immunomodulator to trigger protective immunity, and HD5 binds to and shields ACE2 from viral recognition and binding. EK1 (SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL) is a pan-CoV fusion inhibitor designed by Xia and colleagues. This 36-residue peptide showed high cross-reactivity against all SARS-CoV, MERS-CoV, and three SARS-related CoVs from bats tested (Xia et al., 2019). As shown in Figure 1, EK1 acts by blocking the HR1 domain to disrupt the formation of the 6HB core, which causes inhibition of viral fusion entry into the host cell (Xia et al., 2020b). Intranasal application of EK1 further protected mice from pre- and post-challenges by the HCoV-OC43 alphacoronavirus and MERS-CoV challenges. Such dual therapeutic and prophylactic effects displayed by a single compound against various closely related CoVs are highly desirable. The broad spectrum anti-CoV activity strongly suggests that EK1 could be useful against SARS-CoV-2 infection. Recently, a highly potent lipopeptide variant EK1C4 was generated by C-terminally conjugating EK1 with a cholesterol moiety using a glycine/serine linker and a polyethylene glycol (PEG) spacer (-EK1-GSGSG-PEG4-chol) (Xia et al., 2019). The results were exciting, where SARS-CoV-2 cell-cell fusion was severely impaired at 50% inhibitory concentration (IC50) of 1.3nM, while anti-SARS-CoV-2 pseudoviruses infection activity was recorded at 15.8 nM. Notably, the SARS-CoV-2 HR1 protein binding affinity of EK1C4 was drastically enhanced by 226-fold, which also explained the observed 149-fold higher in antiviral activity of EK1C4 as compared to EK1. It was hypothesized that the conjugated cholesterol might have a profound role in facilitating EK1C4 binding toward the HR1 protein. Indeed, cholesterol conjugation enhancement of antiviral activity had previously been reported with the HIV-1 fusion inhibitor C34 (Hollmann et al., 2013). Besides EK1C4, HR2P-M2 was a peptide designed based on the 6HB core of MERS-CoV HR1 and HR2 domains (Channappanavar et al., 2015). This peptide acted by blocking the S protein-mediated membrane fusion mechanism of MERS-CoV. Of note, intranasal administration of HR2P-M2 was found to significantly reduce lung viral titers by >1,000-fold and protected the mice from MERS-CoV infection (Channappanavar et al., 2015). However, the narrow specificity of HR2P-M2 against MERS-CoV but not SARS-CoV may indirectly suggest an insufficient cross-reactivity with HR2P-M2 against other betacoronaviruses. The mouse β-defensins-4 derived P9 (NGAICWGPCPTAFRQIGNCGHFKVRCCKIR) was shown to bind to the MERS-CoV S2 subunit and remained co-localized with the viruses without inhibiting viral entry into the host cell via endocytosis (Zhao et al., 2016). While in the endosomes, the polycationic property of P9 induced a basic microenvironment to prevent endosomal acidification of the late endosomes. Without endosomal acidification, the pH-dependent activation of viral fusion proteins to initiate viral-host endosomal membrane fusion failed to occur and thus the critical step in viral uncoating prior to viral RNA release (Figure 1). This mechanism of action was indeed highly effective, broadly inhibiting SARS-CoV, MERS-CoV, and a diverse panel of influenza viruses H1N1, H3N2, H5N1, H7N7, and H7N9. Such target specificity nature of P9 also partly explained the low cytotoxic property of P9 (IC50 of 380 µg/ml) against the mammalian Madin-Darby canine kidney cells tested. The broad therapeutic windows of P9 would be a significant advantage in the future development of P9 as an antiviral agent. Notably, P9 displayed both prophylactic and therapeutic effects as observed in an in vivo SARS-CoV mouse model of infection (Zhao et al., 2016). ACE2 is well-known as the primary receptor for CoV at the first step of viral infection. It was found that HD5 (ATCYCRTGRCATRESLSGVCEISGRLYRLCCR), a natural lectin-like human defensins-5 (HD5) peptide secreted by the Paneth cells in the crypts of Lieberkuhn, could interact with glycosylated proteins and lipid components (Wang C. et al., 2020). Based on the structural and biochemical properties, HD5 was initially hypothesized to recognize and inhibit SARS-CoV-2 S protein, host cell ACE2 or both of these target components (Wang C. et al., 2020). However, it was later confirmed that HD5 competitively blocked ACE2 receptors on the host cells instead of targeting the viral S1 subunit (Figure 1). The high-affinity binding between HD5 and the ligand-binding domain of ACE2 through the formation of multiple hydrogen bonds effectively shielded (protected) the host cells from viral recognition and infection. The cyclic peptide RTD-1 (GFCRCLCRRGVCRCICTR) from rhesus macaque leukocytes was reported to decrease disease pathogenesis of SARS-CoV infection in mice, as observed with a substantial reduction in perivascular infiltrate and necrotizing bronchiolitis (Wohlford-Lenane et al., 2009). Interestingly, neither did RTD-1 inhibit the virus or interact with the host cell receptors to exert an antiviral effect. In contrast, it was noticed that the virus titers and lung tissue nucleocapsid (N) gene antigen expression were similar to the untreated control mice. Together with an increase in cytokine levels of interleukin-6, keratinocyte chemoattractant, and granulocyte colony-stimulating factor in lung cell homogenates, RTD-1 was suggested to act as an immunomodulatory effector molecule via a blunted proinflammatory cytokine response in eliminating SARS-CoV (Wohlford-Lenane et al., 2009). Prioritization and emergency preparedness in responding to the emerging infectious diseases associated with outbreaks and pandemics are of utmost urgency. In search of an effective antiviral against COVID-19, we believe AVPs could represent one of the potential classes of new antiviral agents against SARS-CoV-2. It is fascinating to discover how AVPs, composed principally of short and simple amino acid sequences, could interact with and specifically target the different viral components to achieve potent antiviral effects. Here, we would like to bring the attention to a number of AVPs with highly promising anti-CoV activities: mucroporin-M1 disrupts viral envelope, HR2P-M2 targets the viral S protein-mediated fusion mechanism, EK1 and EK1C4 block the HR1 domain of viral S2 subunit, and P9 peptide inhibits late endosomal acidification and thus preventing viral RNA release. Besides, there are AVPs that confer protection to the host: RTD-1 is an antiviral immunomodulator that triggers protective immunity; HD5 binds to and shields host ACE2 receptor to prevent viral recognition and attachment. Recent findings have gathered evidence on the potential role of a host serine protease TMPRSS2 in facilitating the S protein priming process of SARS-CoV-2 that is crucial for the subsequent viral membrane fusion events (Hoffmann et al., 2020). Furthermore, the newly discovered S protein furin-like cleavage site and the novel CD147-mediated viral entry pathway could have important implications in SARS-CoV-2 viral pathogenicity (Coutard et al., 2020; Wang K. et al., 2020; Xia et al., 2020a). These key viral components could represent valuable targets in the development of novel antiviral agents. Cocktail therapy using a selected combination of AVPs or as supplemental therapeutics in combination with other classes of antiviral agents could be a promising treatment strategy that would worth further clinical investigations (Vilas Boas et al., 2019). Such optimism is not unfounded, as enfuvirtide, a 36-amino acid fusion inhibitor AVP, was approved by the US Food and Drug Administration in 2003 for treatment against human immunodeficiency virus in combination with other antiretroviral drugs (Lalezari et al., 2003; Fung and Guo, 2004; Poveda et al., 2005). It acts by blocking the HR1 domain of the viral envelope glycoprotein 41. Sifuvertide, an AVP with enhanced potency and a lower threshold for resistance than enfuvirtide, is currently under phase III trials in China (He et al., 2008; Wang et al., 2009; Su et al., 2015; Yu et al., 2018). Although clinical trials of AVPs against CoVs have not been reported thus far, the dire need for an effective anti-SARS-CoV-2 agent could potentially reshape the drug discovery landscape as with the initiation of Operation Warp Speed by the US government in April 2020 for COVID-19 vaccine, therapeutic, and diagnostic development (Cohen, 2020). In conclusion, AVPs are structurally and functionally versatile owing to its simple primary structure and could serve as the molecular templates for the generation of fast-track therapeutic candidates in the face of COVID-19 or emerging outbreaks posing severe public health threats in the unpredictable future. Given its unique and promising antiviral activity, the potential use of AVPs in clinical treatment and as prophylaxis against COVID-19 should be further explored. AM, YL, C-MF, H-SL, and CL conceived the idea. AM and CL wrote the article. YL, C-MF, and H-SL provided critical comments on the article. AM, YL, C-MF, H-SL, and CL revised the article. All authors contributed to the article and approved the submitted version. The current work was supported by the Fundamental Research Grant Scheme (FRGS), Ministry of Education Malaysia (MOE), grant number FRGS/1/2018/STG05/UNIM/03/1. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ahmed, A., Siman-Tov, G., Hall, G., Bhalla, N., Narayanan, A. (2019). Human antimicrobial peptides as therapeutics for viral infections. Viruses 11, 704. doi: 10.3390/v11080704 CrossRef Full Text | Google Scholar Caly, L., Druce, J. D., Catton, M. G., Jans, D. A., Wagstaff, K. M. (2020). The FDA-approved drug ivermectin inhibits the replication of SARS-CoV-2 in vitro. Antiviral Res. 178, 1–4. doi: 10.1016/j.antiviral.2020.104787 CrossRef Full Text | Google Scholar Chang, K. Y., Yang, J. R. (2013). Analysis and Prediction of Highly Effective Antiviral Peptides Based on Random Forests. PLoS One. 11, 704. doi: 10.1371/journal.pone.0070166 CrossRef Full Text | Google Scholar Channappanavar, R., Lu, L., Xia, S., Du, L., Meyerholz, D. K., Perlman, S., et al. (2015). Protective effect of intranasal regimens containing peptidic middle east respiratory syndrome coronavirus fusion inhibitor against MERS-CoV infection. J. Infect. Dis. 212, 1894–1903. doi: 10.1093/infdis/jiv325 PubMed Abstract | CrossRef Full Text | Google Scholar Cohen, J. (2020). Unveiling ‘Warp Speed,’ the White House’s America-first push for a coronavirus vaccine. Sci. (80-. ). doi: 10.1126/science.abc7056 CrossRef Full Text | Google Scholar Coutard, B., Valle, C., de Lamballerie, X., Canard, B., Seidah, N. G., Decroly, E. (2020). The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade. Antiviral Res. 176, 1–5. doi: 10.1016/j.antiviral.2020.104742 CrossRef Full Text | Google Scholar Dai, C., Ma, Y., Zhao, Z., Zhao, R., Wang, Q., Wu, Y., et al. (2008). Mucroporin, the first cationic host defense peptide from the venom of Lychas mucronatus. Antimicrob. Agents Chemother. 52, 3967–3972. doi: 10.1128/AAC.00542-08 PubMed Abstract | CrossRef Full Text | Google Scholar Das, K., Aramini, J. M., Ma, L. C., Krug, R. M., Arnold, E. (2010). Structures of influenza A proteins and insights into antiviral drug targets. Nat. Struct. Mol. Biol. 17, 530–538. doi: 10.1038/nsmb.1779 PubMed Abstract | CrossRef Full Text | Google Scholar Du, L., He, Y., Zhou, Y., Liu, S., Zheng, B. J., Jiang, S. (2009). The spike protein of SARS-CoV - A target for vaccine and therapeutic development. Nat. Rev. Microbiol. 7, 226–236. doi: 10.1038/nrmicro2090 PubMed Abstract | CrossRef Full Text | Google Scholar Fung, H. B., Guo, Y. (2004). Enfuvirtide: A fusion inhibitor for the treatment of HIV infection. Clin. Ther. 26, 352–378. doi: 10.1016/S0149-2918(04)90032-X PubMed Abstract | CrossRef Full Text | Google Scholar Grein, J., Ohmagari, N., Shin, D., Diaz, G., Asperges, E., Castagna, A., et al. (2020). Compassionate use of remdesivir for patients with severe Covid-19. N. Engl. J. Med. 382, 2327–2336. doi: 10.1056/NEJMoa2007016 PubMed Abstract | CrossRef Full Text | Google Scholar He, Y., Xiao, Y., Song, H., Liang, Q., Ju, D., Chen, X., et al. (2008). Design and evaluation of sifuvirtide, a novel HIV-1 fusion inhibitor. J. Biol. Chem. 283, 11126–11134. doi: 10.1074/jbc.M800200200 PubMed Abstract | CrossRef Full Text | Google Scholar Hoffmann, M., Kleine-Weber, H., Schroeder, S., Krüger, N., Herrler, T., Erichsen, S., et al. (2020). SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181, 271–280. doi: 10.1016/j.cell.2020.02.052 PubMed Abstract | CrossRef Full Text | Google Scholar Hollmann, A., Matos, P. M., Augusto, M. T., Castanho, M. A. R. B., Santos, N. C. (2013). Conjugation of Cholesterol to HIV-1 Fusion Inhibitor C34 Increases Peptide-Membrane Interactions Potentiating Its Action. PLoS One. 8, e60302. doi: 10.1371/journal.pone.0060302 PubMed Abstract | CrossRef Full Text | Google Scholar Kang, S., Peng, W., Zhu, Y., Lu, S., Zhou, M., Lin, W., et al. (2020). Recent progress in understanding 2019 novel coronavirus (SARS-CoV-2) associated with human respiratory disease: detection, mechanisms and treatment. Int. J. Antimicrob. Agents. 55, 1059. doi: 10.1016/j.ijantimicag.2020.105950 CrossRef Full Text | Google Scholar Lalezari, J. P., Henry, K., O’Hearn, M., Montaner, J. S. G., Piliero, P. J., Trottier, B., et al. (2003). Enfuvirtide, an HIV-1 fusion inhibitor, for drug-resistant HIV infection in North and South America. N. Engl. J. Med. 348, 2175–2185. doi: 10.1056/NEJMoa035026 PubMed Abstract | CrossRef Full Text | Google Scholar Ledford, H. (2020). Coronavirus breakthrough: dexamethasone is first drug shown to save lives. Nature 582, 469. doi: 10.1038/d41586-020-01824-5 PubMed Abstract | CrossRef Full Text | Google Scholar Letko, M., Marzi, A., Munster, V. (2020). Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat. Microbiol. 5, 562–569. doi: 10.1038/s41564-020-0688-y PubMed Abstract | CrossRef Full Text | Google Scholar Li, Q., Zhao, Z., Zhou, D., Chen, Y., Hong, W., Cao, L., et al. (2011). Virucidal activity of a scorpion venom peptide variant mucroporin-M1 against measles, SARS-CoV and influenza H5N1 viruses. Peptides 32, 1518–1525. doi: 10.1016/j.peptides.2011.05.015 PubMed Abstract | CrossRef Full Text | Google Scholar Liu, S., Xiao, G., Chen, Y., He, Y., Niu, J., Escalante, C. R., et al. (2004). Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: Implications for virus fusogenic mechanism and identification of fusion inhibitors. Lancet 363, 938–947. doi: 10.1016/S0140-6736(04)15788-7 PubMed Abstract | CrossRef Full Text | Google Scholar Nyanguile, O. (2019). Peptide antiviral strategies as an alternative to treat lower respiratory viral infections. Front. Immunol. 10, 1366. doi: 10.3389/fimmu.2019.01366 PubMed Abstract | CrossRef Full Text | Google Scholar Ohashi, H., Watashi, K., Saso, W., Shionoya, K., Iwanami, S., Hirokawa, T., et al. (2020). Multidrug treatment with nelfinavir and cepharanthine against COVID-19. bioRxiv. doi: 10.1101/2020.04.14.039925 CrossRef Full Text | Google Scholar Poveda, E., Briz, V., Soriano, V. (2005). Enfuvirtide, the first fusion inhibitor to treat HIV infection. AIDS Rev. 7, 139–147. doi: 10.1517/14656566.6.3.453 PubMed Abstract | CrossRef Full Text | Google Scholar Sala, A., Ardizzoni, A., Ciociola, T., Magliani, W., Conti, S., Blasi, E., et al. (2019). Antiviral Activity of Synthetic Peptides Derived from Physiological Proteins. Intervirology 61, 166–173. doi: 10.1159/000494354 CrossRef Full Text | Google Scholar Skalickova, S., Heger, Z., Krejcova, L., Pekarik, V., Bastl, K., Janda, J., et al. (2015). Perspective of use of antiviral peptides against influenza virus. Viruses 7, 5428–5442. doi: 10.3390/v7102883 PubMed Abstract | CrossRef Full Text | Google Scholar Su, Y., Chong, H., Qiu, Z., Xiong, S., He, Y. (2015). Mechanism of HIV-1 Resistance to Short-Peptide Fusion Inhibitors Targeting the Gp41 Pocket. J. Virol. 89, 5801–5811. doi: 10.1128/jvi.00373-15 PubMed Abstract | CrossRef Full Text | Google Scholar Vanden Eynde, J. J. (2020). Covid-19: A brief overview of the discovery clinical trial. Pharmaceuticals 13, 65. doi: 10.3390/ph13040065 CrossRef Full Text | Google Scholar Vilas Boas, L. C. P., Campos, M. L., Berlanda, R. L. A., de Carvalho Neves, N., Franco, O. L. (2019). Antiviral peptides as promising therapeutic drugs. Cell. Mol. Life Sci. 76, 3525–3542. doi: 10.1007/s00018-019-03138-w PubMed Abstract | CrossRef Full Text | Google Scholar Wang, R. R., Yang, L. M., Wang, Y. H., Pang, W., Tam, S. C., Tien, P., et al. (2009). Sifuvirtide, a potent HIV fusion inhibitor peptide. Biochem. Biophys. Res. Commun. 382, 540–544. doi: 10.1016/j.bbrc.2009.03.057 PubMed Abstract | CrossRef Full Text | Google Scholar Wang, C., Wang, S., Li, D., Zhao, X., Han, S., Wang, T., et al. (2020). Lectin-like Intestinal Defensin Inhibits 2019-nCoV Spike binding to ACE2. bioRxiv. doi: 10.1101/2020.03.29.013490 CrossRef Full Text | Google Scholar Wang, K., Chen, W., Zhou, Y.-S., Lian, J.-Q., Zhang, Z., Du, P., et al. (2020). SARS-CoV-2 invades host cells via a novel route: CD147-spike protein. bioRxiv. doi: 10.1101/2020.03.14.988345 CrossRef Full Text | Google Scholar Wang, Y., Zhang, D., Du, G., Du, R., Zhao, J., Jin, Y., et al. (2020). Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trial. Lancet 395, 1569–1578. doi: 10.1016/S0140-6736(20)31022-9 PubMed Abstract | CrossRef Full Text | Google Scholar Wohlford-Lenane, C. L., Meyerholz, D. K., Perlman, S., Zhou, H., Tran, D., Selsted, M. E., et al. (2009). Rhesus Theta-Defensin Prevents Death in a Mouse Model of Severe Acute Respiratory Syndrome Coronavirus Pulmonary Disease. J. Virol. 83, 11385–11390. doi: 10.1128/jvi.01363-09 PubMed Abstract | CrossRef Full Text | Google Scholar World Health Organization (2020). Coronavirus Dis. Situat. Reports. Available at: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports (Accessed July 2, 2020). Google Scholar Wrapp, D., Wang, N., Corbett, K. S., Goldsmith, J. A., Hsieh, C. L., Abiona, O., et al. (2020). Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science (80-. ). 367, 1260–1263. doi: 10.1126/science.aax0902 CrossRef Full Text | Google Scholar Xia, S., Yan, L., Xu, W., Agrawal, A. S., Algaissi, A., Tseng, C. T. K., et al. (2019). A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike. Sci. Adv. 5, eaav4580. doi: 10.1126/sciadv.aav4580 PubMed Abstract | CrossRef Full Text | Google Scholar Xia, S., Liu, M., Wang, C., Xu, W., Lan, Q., Feng, S., et al. (2020a). Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion. Cell Res. 30, 343–355. doi: 10.1038/s41422-020-0305-x PubMed Abstract | CrossRef Full Text | Google Scholar Xia, S., Zhu, Y., Liu, M., Lan, Q., Xu, W., Wu, Y., et al. (2020b). Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein. Cell. Mol. Immunol. 17, 765–767. doi: 10.1038/s41423-020-0374-2 PubMed Abstract | CrossRef Full Text | Google Scholar Yu, D., Ding, X., Liu, Z., Wu, X., Zhu, Y., Wei, H., et al. (2018). Molecular mechanism of HIV-1 resistance to sifuvirtide, a clinical trial–approved membrane fusion inhibitor. J. Biol. Chem. 293, 12703–12718. doi: 10.1074/jbc.RA118.003538 PubMed Abstract | CrossRef Full Text | Google Scholar Zhao, H., Zhou, J., Zhang, K., Chu, H., Liu, D., Poon, V. K. M., et al. (2016). A novel peptide with potent and broad-spectrum antiviral activities against multiple respiratory viruses. Sci. Rep. 6, 1–13. doi: 10.1038/srep22008 PubMed Abstract | CrossRef Full Text | Google Scholar Zhou, P., Yang, X.-L., Wang, X. G., Hu, B., Zhang, L., Zhang, W., et al. (2020). A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273. doi: 10.1038/s41586-020-2012-7 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: antiviral peptides, antimicrobial peptides, antiviral prophylaxis, antiviral agents, COVID-19, SARS-CoV-2, coronaviruses, infectious disease Citation: Mahendran ASK, Lim YS, Fang C-M, Loh H-S and Le CF (2020) The Potential of Antiviral Peptides as COVID-19 Therapeutics. Front. Pharmacol. 11:575444. doi: 10.3389/fphar.2020.575444 Received: 06 July 2020; Accepted: 28 August 2020; Published: 15 September 2020. Edited by: Reviewed by: Copyright © 2020 Mahendran, Lim, Fang, Loh and Le. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Cheng Foh Le, [email protected]
Published: 9 September 2020
Frontiers in Neurology, Volume 11; https://doi.org/10.3389/fneur.2020.00960

Abstract:
The ability of MOG antibody (MOG-Ab) to induce autoimmune disease in animals has been known for decades (1), but it is only recently since the cell-based assay for MOG-Ab IgG1 has been developed and commercialized, that it became possible to characterize clinical syndromes associated with MOG-Ab in humans. Early reports of MOG Associated Disease (MOGAD) emphasized its similarity to Neuromyeliits Optica Spectrum Disorder (NMOSD) (2–4). Indeed, a minority of patients with Aquaporin-4 antibody (AQ4-ab)-seronegative NMOSD−42% in one series–test positive for MOG-Ab (5). However, because the spectrum of MOGAD encompasses many NMOSD-atypical presentations, and because of differences in pathophysiology–AQ4-ab-positive NMOSD being an astrocytopathy and MOGAD being an oligodendrocytopathy—there is an increasing tendency to recognize AQ4-Ab-positive NMOSD and MOGAD as distinct entities (6–10). In this review, we organize the clinical presentations of MOGAD by neuroanatomic compartments, while emphasizing the wide range of reported presentations. While this organization is useful for didactic purposes, it should be borne in mind that MOGAD may involve multiple regions of the CNS simultaneously– much more often than other CNS inflammatory diseases, and that half of MOGAD patients have active lesions in more than one location at the time of initial presentation (11–14). While no phenotype is restricted to any specific age group, some generalizations about clinical presentations of MOGAD in children and adults are possible. In children under the age of 11, ADEM-like phenotypes (encephalopathy, multifocal neurologic deficits and “fluffy” supratentorial cerebral lesions in a bilateral distribution) predominate, while in adolescents and adults, focal syndromes of optic neuritis or longitudinally extensive myelitis are more common (11, 15, 16). Unlike Multiple Sclerosis (MS), where relapse rates are higher in children and decline with older age, in MOGAD the majority of children are not prone to frequent relapses, with 80% of having a monophasic course (17). However, the high rate of monophasic disease may be an overestimate due short follow up (right censoring) as recent case reports documented disease reemergence years and even decades after the initial episode in childhood (18, 19). Given the important differences in pediatric and adult MOGAD, we will qualify discussion of specific syndromes with reference to the respective age group (with the caveat that the clinical distinctions across age groups are only generalizations). Optic neuritis (ON) is the most common initial presentation of MOGAD in adolescence and adulthood, and a frequent presentation in pediatric patients (11, 16, 20). It is associated with a higher risk of subsequent relapse compared to other clinical presentations (11–13, 18). At the onset, vision loss is often severe and up to 80% of patients have bilateral optic nerve involvement, which is highly unusual in MS (12, 14, 21–24). Despite the severity of vision loss in the acute phase, recovery is usually good, especially in children: 89–98% of children had visual acuity to 20/25 or better at 6 months (14, 25). In adults, 6–14% of patients had permanent loss of vision (≤ 20/200) in the affected eye (11, 13, 24). Optic disc edema is rare in MS or NMOSD but is present in up to 86% of patients with MOGAD-ON (13, 21, 22, 24, 26, 27). Rarely, bilateral ON with disc edema can be mistaken for idiopathic intracranial hypertension especially if the patient also complains of headache and has elevated opening pressure on lumbar puncture; however lymphocytic pleocytosis in CSF and enhancement of optic nerve on orbital MRI point toward an inflammatory etiology and should prompt testing for MOG-Ab (28). Fulminant disc edema with peripapillary hemorrhages and “macular star” have been described in MOGAD-ON (29–31). Both of these findings are considered highly atypical for other inflammatory-demyelinating diseases and are more often associated with infectious and ischemic etiologies (29, 30). Up to 50% of adults with MOG-ON have a recurrence of optic neuritis (11–13, 18), which may be the only manifestations of MOGAD. Two rare previously described phenotypes, chronic relapsing inflammatory optic neuropathy (CRION)– a rare condition characterized by relapsing, steroid-dependent optic neuritis (32), and relapsing isolated optic neuritis (RION), have been associated with MOG-Ab in some cases (33, 34). MRI of the orbits during acute MOG-ON typically shows longitudinally extensive optic nerve enhancement with a predilection for the anterior portion of optic nerves; the chiasm and optic tracts are less frequently affected (21, 31). “Optic perineuritis,” characterized by inflammation of the optic nerve sheath and surrounding structures on MRI (35), is seen in up to 50% of cases of MOGAD-ON (Figure 1A) (13, 21, 25, 36, 37). Perineural enhancement is a feature that can help differentiate MOGAD from NMOSD or MS (13, 21, 25, 36, 37). Isolated cases of MOGAD perineuritis, involving the nerve sheath and surrounding structures but not the optic nerve, have also been reported (38, 39). Rarely, uveitis and keratitis can occur simultaneously or subsequently to MOG-ON (38). Figure 1. (A) MRI brain T1 coronal post gadolinium contrast showing contrast enhancement of bilateral optic nerves and right optic nerve sheath consistent with perioptic neuritis. (B) MRI spine sagittal STIR showing longitudinal extensive patchy lesion spaning from cervical to thoracic cord. (C) MRI spine sagittal T2 showing hyperintense longitudinally extensive “pseudo-dilation” of central canal. (D) MRI spine sagittal T1 post gadolinium contrast showing patchy enhancement of the conus medullaris. (E) MRI brain axial FLAIR showing large subcortical and septal white matter lesions in a pediatric patient presenting with ADEM. (F) MRI brain axial T2 with hyperintense “H” sign outlining the central gray matter of the upper cervical cord in a teenager with myelitis. (G) MRI brain axial T2 with “fluffy” hyperintense lesion of gray and white matter of the left caudate and left occipital parietal regions in a pediatric patient who presenting with ADEM. (H) MRI brain axial T2 showing unilateral FLAIR hyperintensity and edema of right mesial frontal cortex in a patient with FLAMES syndrome. (I) MRI brain axial T1 post gadolinium contrast showing leptomeningeal enhancement of the midrain and right mesial temporal lobe. (J) MRI brain axial T1 post gadolinium contrast showing a lesion adjacent to the cerebellar vermis and dorsal medulla in a patient with brainstem syndrome and no other lesions. MOG-Ab associated acute transverse myelitis is a relatively common presentation of MOGAD in adults, and can be seen in children as well (11). In some cases of MOG-TM, there is an antecedent history of infection or vaccination, but in most patients, no such history can be elicited (11, 18, 40). While MOG-TM is typically steroid-responsive with favorable long-term recovery, around 9% of patients have poor recovery (11). Recurrent myelitis, without any other syndromes of MOGAD, is reported in up to 5% of patients (41). MOG-TM can affect any segments of the spinal cord but has a greater predilection for conus medullaris–reported in 11–41% patients–than other CNS inflammatory-demyelinating diseases (11, 18, 40, 42). The involvement of the conus (Figure 1D) may explain the high incidence of neurogenic bowel and bladder symptoms (83%), and erectile dysfunction (54%) during acute phase (40), as well as in the long-term (11). There are also reports of a steroid-dependent myeloradiculitis in MOGAD with a longitudinally extensive transverse lesion from T12 to the conus with sacral nerve root enhancement (43). Radiographically, MOG-TM is usually associated with a longitudinally extensive lesion spanning 3-4 vertebral segments (Figure 1B) (2, 18, 40, 44). In this respect, MOG-TM is similar to NMO-TM, but there are several radiographic differences between the two diseases. First, cord lesion of MOG-TM during the acute phase are much less likely to demonstrate gadolinium enhancement than in NMOSD: only 26% of MOG patients show enhancement vs. 78% of AQ4-ab-seropositive NMOSD (40). Secondly, spinal cord lesions in MOGAD can be multifocal: 62% of patients had ≥2 non-contiguous spinal cord lesions (40). The radiographic multifocality is in line with the notion that MOGAD has a tendency to affect multiple areas of CNS simultaneously. MOG-TM affects both gray and white matter of the cord. The involvement of gray matter can manifest as linear hyperintensity of the central spinal canal (“pseudo-dilation,” Figure 1C) (44), or as H-shaped T2-hyperintensity that outlines the anterior and posterior horns (“H-sign,” Figure 1F) (2, 18, 40). The “H-sign” is suggestive, but not specific for MOGAD, reported in 29% of patients with MOG-TM and 8% of patients with NMO-TM (40). The predilection for the gray matter may explain why MOG-TM sometimes presents as acute flaccid paralysis (AFM) (45): in one series 10 out of 47 MOGAD patients (21%) met clinical criteria for AFM (40). In young children, MOGAD frequently presents as ADEM or an ADEM-like syndrome (ADEM with optic neuritis, multiphasic disseminated encephalomyelitis) (16, 46–49). MRI of the brain typically shows large, ill-defined bilateral lesions frequently involving cortical and deep gray matter structures (Figure 1G) (50). Lesions may also involve subcortical white matter and corpus callosum as seen in Figure 1E. Optic nerves and spinal cord may be involved concurrently with brain (51). Recurrent ADEM or ADEM associated with recurrent optic neuritis (52, 53) are especially suggestive of MOGAD. Importantly, in children with clinical syndrome of encephalitis, MOGAD diagnosis is possible even when MRI findings are not compatible with ADEM—for example, exclusive cortical or symmetric thalamic/basal ganglia involvement, or even normal MRI (54). Cerebral involvement in adults is both less common and more restricted than in children, though there are exceptions (55). Syndrome of encephalitis with steroid-responsive seizures, also termed FLAMES (FLAIR-hyperintense Lesions and Anti-MOG-associated Encephalitis with Seizures), appears to be specific to MOGAD (20, 56–58). FLAMES patients present with focal-onset, tonic-clonic seizures, and have unilateral FLAIR hyperintensities with edema on MRI (Figure 1H). A review by Budhram et al. found 20 cases of FLAMES in the literature. The most common symptoms were seizures (85%), headache (70%), and fever (55%). CSF pleocytosis and cortical leptomeningeal enhancement (Figure 1I) were present in a minority of patients (57). All patients with FLAMES responded to high dose steroids with resolution of FLAIR changes. Of note, a number of patients developed ON either before or after seizures (56, 58, 59). Thus, the emergence of seizures in the context of ON or focal brain inflammatory lesions should prompt testing for MOG-Ab (52). Isolated seizures may rarely be an index event in MOGAD. In one case, an adult patient presented with aphasic status epilepticus with initial MRI showing no abnormalities. Six months later the patient developed a tumefactive demyelinating lesion, with MOG-Ab testing positive several months later (60). A similar presentation has been described in four pediatric patients who presented with isolated seizures and normal brain MRI and developed MRI brain lesions months, and in one case years, later (61). Several studies document an association between MOGAD and autoimmune encephalitis with NMDA-antibody (62–64). In a retrospective case review by Titulaer et al., 12 of 691 with NMDAR encephalitis patients (1.6%) tested positive for MOG-Ab. Some patients presented with MOGAD syndrome followed by encephalitis, others with encephalitis followed by MOGAD, and in some NMDA encephalitis and MOGAD were diagnosed concurrently. Three patients with NMDAR encephalitis and no clinical or MRI features to suggest MOGAD also tested positive for MOG-Ab (62). Finally, mention should be made of rare cases when MOG-Ab was found in patients with pathologically-proven CNS vasculitis (65, 66). Two patients presented with fever, headache, confusion, and focal neurologic deficits (66), and the third had 9 months of progressive cognitive and behavioral decline (65). MRI showed multifocal lesions in both the gray and white matter in two cases, one of whom also had open-ring contrast-enhancing lesions. The third case had findings of focal cortical encephalitis with gyriform FLAIR hyperintensities with edema, similar to findings seen in FLAMES. All three cases underwent brain biopsy, which showed small vessel perivascular inflammation, consistent with CNS vasculitis. However, fibrinoid necrosis, a pathologic requirement for small vessel CNS vasculitis, was absent in two of the cases (66, 67). Whether vasculitis should be regarded as a primary or secondary manifestation of MOGAD, or MOG-Ab is unrelated to vasculitis diagnosis, is difficult to determine given rarity of the association. Brainstem involvement is seen in 30% of MOGAD patients, and is a risk factor for a higher disability at long-term follow-up and more active disease (68). In one large series brainstem inflammation occurred concomitantly with inflammation in optic nerves in 40% of cases, spinal cord in 89% cases and cerebrum in 66% of cases (68). However, there are reports of isolated brainstem inflammation as well (Figure 1J) (68). Any part of the brainstem can be affected, medulla being the most common (11, 68). Brainstem lesions are usually associated with disabling symptoms—weakness, cranial nerve deficits, ataxia, hypoventilation syndrome, impaired consciousness and, and, exceptionally, a fatal outcome (68). Area postrema syndrome (APS), one of the core syndromes of NMOSD, has also been described in MOGAD (11, 68–70). MOGAD can mimic infective rhomboencephalitis when a patient presents with fever, CSF leukocytosis, brainstem enhancing lesions and leptomeningeal enhancement (44, 68), or Chronic Lymphocytic Inflammation with Pontine Perivascular Enhancement Responsive to Steroids (CLIPPERS), when MRI shows punctate, curvilinear enhancement in the pons (71–73). Whether CLIPPERS is a form of MOGAD or elicits an immune response to MOG-Ab is uncertain (73). Since the first reports of MOG-Ab associated neurologic diseases appeared just a few years ago (4), the floodgates of case reporting have been opened and our understanding of MOGAD has grown exponentially. We now recognize certain clinical and radiologic features that help to differentiate MOG-ON and MOG-TM from NMOSD syndromes; that pediatric ADEM is frequently associated with MOG-Ab, especially if followed by episodes of ADEM or ON; that in adults, MOG can be associated with seizures and focal cerebral edema (“FLAMES syndrome,” which appears to be unique to MOGAD); that brainstem inflammation is seen in a significant minority of MOGAD patients and may be an isolated finding; that MOG Ab is a common mimicker of infectious encephalitis (54) that MOG antibody is exceptionally rare in MS or AQ4 Ab positive NMOSD, but may co-exist with NMDA and other autoimmune encephalidites (64, 74). But many important questions remain. We need to determine sensitivity, specificity, positive and negative predictive value of MOG-Ab in the various neurologic syndromes; whether MOG-Ab shoud be tested in CSF, if it is negative in serum (75); whether various ultrarare presentions, such as isolated seizures without brain lesions, CLIPPERS, and a MOG-Ab-associated CNS vasculitis-type syndrome should be subsumed under MOGAD rubric. Most importantly, we need to better stratify risk of disease recurrence after the first or second episode and determine best treatments to prevent recurrence. With the rapid pace of progress, we can expect to answer these and other questions, and, no doubt, find new surprises along the way. EP and IK wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version. IK served on advisory boards for Biogen and Genentech and received consulting fees from Roche and research support for investigator-initiated grants from Sanofi Genzyme, Biogen, EMD Serono, National MS Society, and Guthy Jackson Charitable Foundation. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 1. Lebar R, Baudrimont M, Vincent C. Chronic experimental autoimmune encephalomyelitis in the guinea pig. presence of anti-M2 antibodies in central nervous system tissue and the possible role of M2 autoantigen in the induction of the disease. J Autoimmunity. (1989) 2:115–32. doi: 10.1016/0896-8411(89)90149-2 PubMed Abstract | CrossRef Full Text | Google Scholar 2. Kitley J, Woodhall M, Waters P, Leite MI, Devenney E, Craig J, et al. Myelin-oligodendrocyte glycoprotein antibodies in adults with a neuromyelitis optica phenotype. Neurology. (2012) 79:1273–7. doi: 10.1212/WNL.0b013e31826aac4e PubMed Abstract | CrossRef Full Text | Google Scholar 3. Mader S, Gredler V, Schanda K, Rostasy K, Dujmovic I, Pfaller K, et al. Complement activating antibodies to myelin oligodendrocyte glycoprotein in neuromyelitis optica and related disorders. J Neuroinflamm. (2011) 8:184. doi: 10.1186/1742-2094-8-184 PubMed Abstract | CrossRef Full Text | Google Scholar 4. Rostasy K, Mader S, Hennes EM, Schanda K, Gredler V, Guenther A, et al. Persisting myelin oligodendrocyte glycoprotein antibodies in aquaporin-4 antibody negative pediatric neuromyelitis optica. Mult Sclero J. (2013) 19:1052–9. doi: 10.1177/1352458512470310 PubMed Abstract | CrossRef Full Text | Google Scholar 5. Hamid SH, Whittam D, Mutch K, Linaker S, Solomon T, Das K, et al. What proportion of AQP4-IgG-negative NMO spectrum disorder patients are MOG-IgG positive? A cross sectional study of 132 patients. J Neurol. (2017) 264:2088–94. doi: 10.1007/s00415-017-8596-7 PubMed Abstract | CrossRef Full Text | Google Scholar 6. Dos Passos GR, Oliveira LM, da Costa BK, Apostolos-Pereira SL, Callegaro D, Fujihara K, et al. MOG-IgG-associated optic neuritis, encephalitis, and myelitis: lessons learned from neuromyelitis optica spectrum disorder. Front Neurol. (2018) 9:217. doi: 10.3389/fneur.2018.00217 PubMed Abstract | CrossRef Full Text | Google Scholar 7. You Y, Zhu L, Zhang T, Shen T, Fontes A, Yiannikas C, et al. Evidence of müller glial dysfunction in patients with aquaporin-4 immunoglobulin g-positive neuromyelitis optica spectrum disorder. Ophthalmology. (2019) 126:801–10. doi: 10.1016/j.ophtha.2019.01.016 PubMed Abstract | CrossRef Full Text | Google Scholar 8. Lucchinetti CF, Guo Y, Popescu BF, Fujihara K, Itoyama Y, Misu T. The pathology of an autoimmune astrocytopathy: lessons learned from neuromyelitis optica. Brain Pathol. (2014) 24:83–97. doi: 10.1111/bpa.12099 PubMed Abstract | CrossRef Full Text | Google Scholar 9. Fujihara K, Misu T, Nakashima I, Takahashi T, Bradl M, Lassmann H, et al. Neuromyelitis optica should be classified as an astrocytopathic disease rather than a demyelinating disease. Clin Experimental Neuroimmunol. (2012) 3:58–73. doi: 10.1111/j.1759-1961.2012.00030.x PubMed Abstract | CrossRef Full Text | Google Scholar 10. Zamvil SS, Slavin AJ. Does MOG Ig-positive AQP4-seronegative opticospinal inflammatory disease justify a diagnosis of NMO spectrum disorder? Neurol Neuroimmunol Neuroinflamm. (2015) 2:e62. doi: 10.1212/NXI.0000000000000062 PubMed Abstract | CrossRef Full Text | Google Scholar 11. Jurynczyk M, Messina S, Woodhall MR, Raza N, Everett R, Roca-Fernandez A, et al. Clinical presentation and prognosis in MOG-antibody disease: a UK study. Brain. (2017) 140:3128–38. doi: 10.1093/brain/awx276 PubMed Abstract | CrossRef Full Text | Google Scholar 12. de Mol CL, Wong Y, van Pelt ED, Wokke B, Siepman T, Neuteboom RF, et al. The clinical spectrum and incidence of anti-MOG-associated acquired demyelinating syndromes in children and adults. Mult Scler. (2020) 26:806–14. doi: 10.1177/1352458519845112 PubMed Abstract | CrossRef Full Text | Google Scholar 13. Chen JJ, Flanagan EP, Jitprapaikulsan J, López-Chiriboga AS, Fryer JP, Leavitt JA, et al. Myelin oligodendrocyte glycoprotein antibody–positive optic neuritis: clinical characteristics, radiologic clues, and outcome. Am J Ophthalmol. (2018) 195:8–15. doi: 10.1016/j.ajo.2018.07.020 PubMed Abstract | CrossRef Full Text | Google Scholar 14. Chen Q, Zhao G, Huang Y, Li Z, Sun X, Lu P, et al. Clinical characteristics of pediatric optic neuritis with myelin oligodendrocyte glycoprotein seropositive: a cohort study. Pediatric Neurol. (2018) 83:42–9. doi: 10.1016/j.pediatrneurol.2018.03.003 PubMed Abstract | CrossRef Full Text | Google Scholar 15. Cobo-Calvo Á, Ruiz A, D'Indy H, Poulat AL, Carneiro M, Philippe N, et al. MOG antibody-related disorders: common features and uncommon presentations. J Neurol. (2017) 264:1945–55. doi: 10.1007/s00415-017-8583-z PubMed Abstract | CrossRef Full Text | Google Scholar 16. Hennes EM, Baumann M, Schanda K, Anlar B, Bajer-Kornek B, Blaschek A, et al. Prognostic relevance of MOG antibodies in children with an acquired demyelinating syndrome. Neurology. (2017) 89:900–8. doi: 10.1212/WNL.0000000000004312 PubMed Abstract | CrossRef Full Text | Google Scholar 17. Waters P, Fadda G, Woodhall M, O'Mahony J, Brown RA, Castro DA, et al. Serial anti–myelin oligodendrocyte glycoprotein antibody analyses and outcomes in children with demyelinating syndromes. JAMA Neurol. (2019) 77:82–93. doi: 10.1001/jamaneurol.2019.2940 PubMed Abstract | CrossRef Full Text | Google Scholar 18. Jarius S, Ruprecht K, Kleiter I, Borisow N, Asgari N, Pitarokoili K, et al. MOG-IgG in NMO and related disorders: a multicenter study of 50 patients. Part 2: epidemiology, clinical presentation, radiological and laboratory features, treatment responses, and long-term outcome. J Neuroinflamm. (2016) 13:280. doi: 10.1186/s12974-016-0718-0 PubMed Abstract | CrossRef Full Text | Google Scholar 19. Numa S, Kasai T, Kondo T, Kushimura Y, Kimura A, Takahashi H, et al. An adult case of anti-myelin oligodendrocyte glycoprotein (MOG) antibody-associated multiphasic acute disseminated encephalomyelitis at 33-year intervals. Internal Med. (2016) 55:699–702. doi: 10.2169/internalmedicine.55.5727 PubMed Abstract | CrossRef Full Text | Google Scholar 20. Cobo-Calvo A, Ruiz A, Maillart E, Audoin B, Zephir H, Bourre B, et al. Clinical spectrum and prognostic value of CNS MOG autoimmunity in adults: the MOGADOR study. Neurology. (2018) 90:e1858–e1869. doi: 10.1212/WNL.0000000000005560 PubMed Abstract | CrossRef Full Text | Google Scholar 21. Ramanathan S, Prelog K, Barnes EH, Tantsis EM, Reddel SW, Henderson AP, et al. Radiological differentiation of optic neuritis with myelin oligodendrocyte glycoprotein antibodies, aquaporin-4 antibodies, and multiple sclerosis. Mult Sclero J. (2016) 22:470–82. doi: 10.1177/1352458515593406 PubMed Abstract | CrossRef Full Text | Google Scholar 22. Ramanathan S, Reddel SW, Henderson A, Parratt JD, Barnett M, Gatt PN, et al. Antibodies to myelin oligodendrocyte glycoprotein in bilateral and recurrent optic neuritis. Neurol Neuroimmunol Neuroinflamm. (2014) 1:e40. doi: 10.1212/NXI.0000000000000040 PubMed Abstract | CrossRef Full Text | Google Scholar 23. Lock JH, Newman NJ, Biousse V, Peragallo JH. Update on Pediatric Optic Neuritis. Curr Opinion Ophthalmol. (2019) 30:418–25. doi: 10.1097/ICU.0000000000000607 PubMed Abstract | CrossRef Full Text | Google Scholar 24. Zhao Y, Tan S, Chan TC, Xu Q, Zhao J, Teng D, et al. Clinical features of demyelinating optic neuritis with seropositive myelin oligodendrocyte glycoprotein antibody in Chinese patients. British J Ophthalmol. (2018) 102:1372–7. doi: 10.1136/bjophthalmol-2017-311177 PubMed Abstract | CrossRef Full Text | Google Scholar 25. Song H, Zhou H, Yang M, Tan S, Wang J, Xu Q, et al. Clinical characteristics and prognosis of myelin oligodendrocyte glycoprotein antibody-seropositive paediatric optic neuritis in China. British J Ophthalmol. (2019) 103:831–6. doi: 10.1136/bjophthalmol-2018-312399 PubMed Abstract | CrossRef Full Text | Google Scholar 26. Jarius S, Paul F, Aktas O, Asgari N, Dale RC, de Seze J, et al. MOG encephalomyelitis: international recommendations on diagnosis and antibody testing. J Neuroinflammation. (2018) 15:134. doi: 10.1186/s12974-018-1144-2 PubMed Abstract | CrossRef Full Text | Google Scholar 27. Ramanathan S, Mohammad S, Tantsis E, Nguyen TK, Merheb V, Fung VSC, et al. Clinical course, therapeutic responses and outcomes in relapsing MOG antibody-associated demyelination. J Neurol Neurosurg Psychiatry. (2018) 89:127–37. doi: 10.1136/jnnp-2017-316880 PubMed Abstract | CrossRef Full Text | Google Scholar 28. Narayan RN, Wang C, Sguigna P, Husari K, Greenberg B. Atypical Anti-MOG syndrome with aseptic meningoencephalitis and pseudotumor cerebri-like presentations. Mult Sclero Relat Disorde. (2019) 27:30–3. doi: 10.1016/j.msard.2018.10.003 PubMed Abstract | CrossRef Full Text | Google Scholar 29. Chen JJ, Bhatti MT. Clinical phenotype, radiological features, and treatment of myelin oligodendrocyte glycoprotein-immunoglobulin G (MOG-IgG) optic neuritis. Curr Opin Neurol. (2020) 33:47–54. doi: 10.1097/WCO.0000000000000766 PubMed Abstract | CrossRef Full Text | Google Scholar 30. Moura FC, Sato DK, Rimkus CM, Apóstolos-Pereira SL, de Oliveira LM, Leite CC, et al. Anti-MOG (Myelin Oligodendrocyte Glycoprotein)–positive severe optic neuritis with optic disc ischaemia and macular star. Neuro-Ophthalmology. (2015) 39:285–8. doi: 10.3109/01658107.2015.1084332 PubMed Abstract | CrossRef Full Text | Google Scholar 31. Biotti D, Bonneville F, Tournaire E, Ayrignac X, Dallière CC, Mahieu L, et al. Optic neuritis in patients with anti-MOG antibodies spectrum disorder: MRI and clinical features from a large multicentric cohort in France. J Neurol. (2017) 264:2173–5. doi: 10.1007/s00415-017-8615-8 PubMed Abstract | CrossRef Full Text | Google Scholar 32. Kidd D. Chronic relapsing inflammatory optic neuropathy (CRION). Brain. (2003) 126:276–84. doi: 10.1093/brain/awg045 PubMed Abstract | CrossRef Full Text | Google Scholar 33. Petzold A, Woodhall M, Khaleeli Z, Tobin WO, Pittock SJ, Weinshenker BG, et al. Aquaporin-4 and myelin oligodendrocyte glycoprotein antibodies in immune-mediated optic neuritis at long-term follow-up. J Neurol Neurosurg Psychiatry. (2019) 90:1021–6. doi: 10.1136/jnnp-2019-320493 PubMed Abstract | CrossRef Full Text | Google Scholar 34. Lee HJ, Kim B, Waters P, Woodhall M, Irani S, Ahn S, et al. Chronic relapsing inflammatory optic neuropathy (CRION): a manifestation of myelin oligodendrocyte glycoprotein antibodies. J Neuroinflamm. (2018) 15:302. doi: 10.1186/s12974-018-1335-x PubMed Abstract | CrossRef Full Text | Google Scholar 35. Purvin V. Optic perineuritis: clinical and radiographic features. Arch Ophthalmol. (2001) 119:1299. doi: 10.1001/archopht.119.9.1299 PubMed Abstract | CrossRef Full Text | Google Scholar 36. Zhou L, Huang Y, Li H, Fan J, Zhangbao J, Yu H, et al. MOG-antibody associated demyelinating disease of the CNS: a clinical and pathological study in Chinese Han patients. J Neuroimmunol. 305: 19–28. doi: 10.1016/j.jneuroim.2017.01.007 PubMed Abstract | CrossRef Full Text | Google Scholar 37. Akaishi T, Sato DK, Nakashima I, Takeshita T, Takahashi T, Doi H, et al. MRI and retinal abnormalities in isolated optic neuritis with myelin oligodendrocyte glycoprotein and aquaporin-4 antibodies: a comparative study. J Neurol Neurosurg Psychiatry. (2016) 87:446–8. doi: 10.1136/jnnp-2014-310206 PubMed Abstract | CrossRef Full Text | Google Scholar 38. Ramanathan S, Fraser C, Curnow SR, Ghaly M, Leventer RJ, Lechner-Scott J, et al. Uveitis and optic perineuritis in the context of myelin oligodendrocyte glycoprotein antibody seropositivity. Eur J Neurol. (2019) 26:1137. doi: 10.1111/ene.13932 PubMed Abstract | CrossRef Full Text | Google Scholar 39. Yanagidaira M, Hattori T, Emoto H, Kiyosawa M, Yokota T. Optic perineuritis with anti-myelin oligodendrocyte glycoprotein antibody. Mult Sclero Relat Disorde. (2020) 38:101444. doi: 10.1016/j.msard.2019.101444 PubMed Abstract | CrossRef Full Text | Google Scholar 40. Dubey D, Pittock SJ, Krecke KN, Morris PP, Sechi E, Zalewski NL, et al. Clinical, radiologic, and prognostic features of myelitis associated with myelin oligodendrocyte glycoprotein autoantibody. JAMA Neurol. (2019) 76:301. doi: 10.1001/jamaneurol.2018.4053 PubMed Abstract | CrossRef Full Text | Google Scholar 41. Anis S, Regev K, Pittock SJ, Flanagan EP, Alcalay Y, Gadoth A. Isolated recurrent myelitis in a persistent MOG positive patient. Mult Sclero Relat Disorde. (2019) 30:163–4. doi: 10.1016/j.msard.2019.02.016 PubMed Abstract | CrossRef Full Text | Google Scholar 42. Salama S, Khan M, Levy M, Izbudak I. Radiological characteristics of myelin oligodendrocyte glycoprotein antibody disease. Mult Sclero Relat Disorde. (2019) 29:15–22. doi: 10.1016/j.msard.2019.01.021 PubMed Abstract | CrossRef Full Text | Google Scholar 43. Sundaram S, Nair SS, Jaganmohan D, Unnikrishnan G, Nair M. Relapsing lumbosacral myeloradiculitis: an unusual presentation of mog antibody disease. Mult Sclero J. (2019) 26:509–11. doi: 10.1177/1352458519840747 PubMed Abstract | CrossRef Full Text | Google Scholar 44. Denève M, Biotti D, Patsoura S, Ferrier M, Meluchova Z, Mahieu L, et al. MRI features of demyelinating disease associated with anti-MOG antibodies in adults. J Neuroradiol. (2019) 46:312–8. doi: 10.1016/j.neurad.2019.06.001 PubMed Abstract | CrossRef Full Text | Google Scholar 45. Wang C, Narayan R, Greenberg B. Anti-myelin oligodendrocyte glycoprotein antibody associated with gray matter predominant transverse myelitis mimicking acute flaccid myelitis: a presentation of two cases. Pediatric Neurol. (2018) 86:42–5. doi: 10.1016/j.pediatrneurol.2018.06.003 PubMed Abstract | CrossRef Full Text | Google Scholar 46. Pröbstel AK, Dornmair K, Bittner R, Sperl P, Jenne D, Magalhaes S, et al. Antibodies to MOG are transient in childhood acute disseminated encephalomyelitis. Neurology. (2011) 77:580–8. doi: 10.1212/WNL.0b013e318228c0b1 PubMed Abstract | CrossRef Full Text | Google Scholar 47. Di Pauli F, Mader S, Rostasy K, Schanda K, Bajer-Kornek B, Ehling R, et al. Temporal dynamics of anti-MOG antibodies in CNS demyelinating diseases. Clin Immunol. (2011) 138:247–54. doi: 10.1016/j.clim.2010.11.013 PubMed Abstract | CrossRef Full Text | Google Scholar 48. Baumann M, Sahin K, Lechner C, Hennes EM, Schanda K, Mader S, et al. Clinical and neuroradiological differences of paediatric acute disseminating encephalomyelitis with and without antibodies to the myelin oligodendrocyte glycoprotein. J Neurol Neurosurg Psychiatry. (2015) 86:265–72. doi: 10.1136/jnnp-2014-308346 PubMed Abstract | CrossRef Full Text | Google Scholar 49. Hacohen Y, Mankad K, Chong WK, Barkhof F, Vincent A, Lim M, et al. Diagnostic algorithm for relapsing acquired demyelinating syndromes in children. Neurology. (2017) 89:269–78. doi: 10.1212/WNL.0000000000004117 PubMed Abstract | CrossRef Full Text | Google Scholar 50. Wegener-Panzer A, Cleaveland R, Wendel EM, Baumann M, Bertolini A, Häusler M, et al. Clinical and imaging features of children with autoimmune encephalitis and MOG antibodies. Neurol Neuroimmunol Neuroinflamm. (2020) 7:e731. doi: 10.1212/NXI.0000000000000731 PubMed Abstract | CrossRef Full Text | Google Scholar 51. Baumann M, Hennes EM, Schanda K, Karenfort M, Kornek B, Seidl R, et al. Children with multiphasic disseminated encephalomyelitis and antibodies to the myelin oligodendrocyte glycoprotein (MOG): extending the spectrum of MOG antibody positive diseases. Mult Sclero J. (2016) 22:1821–9. doi: 10.1177/1352458516631038 PubMed Abstract | CrossRef Full Text | Google Scholar 52. Gutman JM, Kupersmith M, Galetta S, Kister I. Anti-myelin oligodendrocyte glycoprotein (MOG) antibodies in patients with optic neuritis and seizures. J Neurol Sci. (2018) 387:170–3. doi: 10.1016/j.jns.2018.01.042 PubMed Abstract | CrossRef Full Text | Google Scholar 53. Huppke P, Rostasy K, Karenfort M, Huppke B, Seidl R, Leiz S, et al. Acute disseminated encephalomyelitis followed by recurrent or monophasic optic neuritis in pediatric patients. Mult Sclero J. (2013) 19:941–6. doi: 10.1177/1352458512466317 CrossRef Full Text | Google Scholar 54. Armangue T, Olivé-Cirera G, Martínez-Hernandez E, Sepulveda M, Ruiz-Garcia R, Muñoz-Batista M, et al. Associations of paediatric demyelinating and encephalitic syndromes with myelin oligodendrocyte glycoprotein antibodies: a multicentre observational study. Lancet Neurol. (2020) 19:234–46. doi: 10.1016/S1474-4422(19)30488-0 PubMed Abstract | CrossRef Full Text | Google Scholar 55. Uchigami H, Arai N, Sekiguchi M, Ogawa A, Yasuda T, Seto A, et al. Anti-myelin oligodendrocyte glycoprotein antibody-positive acute disseminated encephalomyelitis mimicking limbic encephalitis: a case report. Mult Scler Relat Disord. (2020) 38:101500. doi: 10.1016/j.msard.2019.101500 PubMed Abstract | CrossRef Full Text | Google Scholar 56. Ogawa R, Nakashima I, Takahashi T, Kaneko K, Akaishi T, Takai Y, et al. MOG antibody–positive, benign, unilateral, cerebral cortical encephalitis with epilepsy. Neurol Neuroimmunol Neuroinflamm. (2017) 4:e322. doi: 10.1212/NXI.0000000000000322 PubMed Abstract | CrossRef Full Text | Google Scholar 57. Budhram A, Mirian A, Le C, Hosseini-Moghaddam SM, Sharma M, Nicolle MW. Unilateral cortical FLAIR-hyperintense lesions in anti-MOG-associated encephalitis with seizures (FLAMES): characterization of a distinct clinico-radiographic syndrome. J Neurol. (2019) 266:2481–7. doi: 10.1007/s00415-019-09440-8 PubMed Abstract | CrossRef Full Text | Google Scholar 58. Ikeda T, Yamada K, Ogawa R, Takai Y, Kaneko K, Misu T, et al. The pathological features of MOG antibody-positive cerebral cortical encephalitis as a new spectrum associated with MOG antibodies: a case report. J Neurol Sci. (2018) 392:113–5. doi: 10.1016/j.jns.2018.06.028 PubMed Abstract | CrossRef Full Text | Google Scholar 59. Taraschenko O, Zabad R. Overlapping demyelinating syndrome and anti-n-methyl-d-aspartate receptor encephalitis with seizures. Epilepsy Behav Rep. (2019) 12:100338. doi: 10.1016/j.ebr.2019.100338 PubMed Abstract | CrossRef Full Text | Google Scholar 60. Katsuse K, Kurihara M, Sugiyama Y, Kodama S, Takahashi M, Momose T, et al. Aphasic status epilepticus preceding tumefactive left hemisphere lesion in anti-MOG antibody associated disease. Mult Sclero Relat Disorde. (2019) 27:91–4. doi: 10.1016/j.msard.2018.10.012 PubMed Abstract | CrossRef Full Text | Google Scholar 61. Ramanathan S, O'grady GL, Malone S, Spooner CG, Brown DA, Gill D, et al. Isolated seizures during the first episode of relapsing myelin oligodendrocyte glycoprotein antibody-associated demyelination in children. Dev Med Child Neurol. (2019) 61:610–4. doi: 10.1111/dmcn.14032 PubMed Abstract | CrossRef Full Text | Google Scholar 62. Titulaer MJ, Höftberger R, Iizuka T, Leypoldt F, McCracken L, Cellucci T, et al. Overlapping demyelinating syndromes and anti-N-Methyl-D-aspartate receptor encephalitis: anti-NMDAR encephalitis. Ann Neurol. (2014) 75:411–28. doi: 10.1002/ana.24117 PubMed Abstract | CrossRef Full Text | Google Scholar 63. Carroll E, Wallach A, Kurzweil A, Frucht S, Berk T, Boffa M, et al. Care report: seizure, fever, hallucinations & vision loss, a circuitous route to dual diagnoses. Pract Neurol. (2019). Available online at: https://practicalneurology.com/articles/2019-nov-dec/case-report-seizure-fever-hallucinations-vision-loss 64. Martinez-Hernandez E, Guasp M, García-Serra A, Maudes E, Ariño H, Sepulveda M, et al. Clinical significance of anti-NMDAR concurrent with glial or neuronal surface antibodies. Neurology. (2020) 94:e2302–e2310. doi: 10.1212/WNL.0000000000009239 CrossRef Full Text | Google Scholar 65. Baba T, Shinoda K, Watanabe M, Sadashima S, Matsuse D, Isobe N, et al. MOG antibody disease manifesting as progressive cognitive deterioration and behavioral changes with primary central nervous system vasculitis. Mult Sclero Relat Disorde. (2019) 30:48–50. doi: 10.1016/j.msard.2019.01.053 PubMed Abstract | CrossRef Full Text | Google Scholar 66. Patterson K, Iglesias E, Nasrallah M, González-Álvarez V, Suñol M, Anton J, et al. Anti-MOG encephalitis mimicking small vessel CNS vasculitis. Neurol Neuroimmunol Neuroinflamm. (2019) 6:e538. doi: 10.1212/NXI.0000000000000538 PubMed Abstract | CrossRef Full Text | Google Scholar 67. Giannini C, Salvarani C, Hunder G, Brown RD. Primary central nervous system vasculitis: pathology and mechanisms. Acta Neuropathol. (2012) 123:759–72. doi: 10.1007/s00401-012-0973-9 PubMed Abstract | CrossRef Full Text | Google Scholar 68. Jarius S, Kleiter I, Ruprecht K, Asgari N, Pitarokoili K, Borisow N, et al. MOG-IgG in NMO and related disorders: a multicenter study of 50 patients. Part 3: brainstem involvement - frequency, presentation and outcome. J Neuroinflamm. (2016) 13:281. doi: 10.1186/s12974-016-0719-z PubMed Abstract | CrossRef Full Text | Google Scholar 69. Jurynczyk M, Geraldes R, Probert F, Woodhall MR, Waters P, Tackley G, et al. Distinct brain imaging characteristics of autoantibody-mediated CNS conditions and multiple sclerosis. Brain. (2017) 140:617–27. doi: 10.1093/brain/aww350 PubMed Abstract | CrossRef Full Text | Google Scholar 70. Wingerchuk DM, Banwell B, Bennett JL, Cabre P, Carroll W, Chitnis T, et al. International consensus diagnostic criteria for neuromyelitis optica spectrum disorders. Neurology. (2015) 85:177–89. doi: 10.1212/WNL.0000000000001729 PubMed Abstract | CrossRef Full Text | Google Scholar 71. Berzero G, Taieb G, Marignier R, Younan N, Savatovsky J, Leclercq D, et al. CLIPPERS mimickers: relapsing brainstem encephalitis associated with anti-MOG antibodies. Eur J Neurol. (2018) 25:e16–e17. doi: 10.1111/ene.13483 PubMed Abstract | CrossRef Full Text | Google Scholar 72. Symmonds M, Waters PJ, Küker W, Leite MI, Schulz UG. Anti-MOG antibodies with longitudinally extensive transverse myelitis preceded by CLIPPERS. Neurology. (2015) 84:1177–9. doi: 10.1212/WNL.0000000000001370 PubMed Abstract | CrossRef Full Text | Google Scholar 73. Taieb G, Pierre L. ANti-MOG antibodies with longitudinally extensive transverse myelitis preceded by CLIPPERS. Neurology. (2015) 85:2. doi: 10.1212/01.wnl.0000472753.44905.98 PubMed Abstract | CrossRef Full Text | Google Scholar 74. Kunchok A, Chen JJ, McKeon A, Mills JR, Flanagan EP, Pittock SJ. Coexistence of myelin oligodendrocyte glycoprotein and aquaporin-4 antibodies in adult and pediatric patients. JAMA Neurol. (2019) 77:257–9. doi: 10.1001/jamaneurol.2019.3656 PubMed Abstract | CrossRef Full Text | Google Scholar 75. Mariotto S, Gajofatto A, Batzu L, Delogu R, Sechi G, Leoni S, et al. Relevance of antibodies to myelin oligodendrocyte glycoprotein in CSF of seronegative cases. Neurology. (2019) 93:e1867–e1872. doi: 10.1212/WNL.0000000000008479 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: MOG (myelin oligodendrocyte glyco protein), MOG antibody disease, ADEM, myelitis, optic neuritis, CRION, brainstem encephalitis Citation: Parrotta E and Kister I (2020) The Expanding Clinical Spectrum of Myelin Oligodendrocyte Glycoprotein (MOG) Antibody Associated Disease in Children and Adults. Front. Neurol. 11:960. doi: 10.3389/fneur.2020.00960 Received: 12 December 2019; Accepted: 24 July 2020; Published: 09 September 2020. Edited by: Reviewed by: Copyright © 2020 Parrotta and Kister. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Erica Parrotta, [email protected]
Peter Thomas Malcolm
2013 ASEE Annual Conference & Exposition Proceedings pp 23.1165.1-23.1165.15; https://doi.org/10.18260/1-2--22550

Abstract:
The Spatial Estimator: Design Feedback and the Fundamental Engineering Abilities of Mental Rotation and ApproximationThe White House has called educators to action around "science, technology, engineering andmath" ([STEM] Obama, 2010) in K-12 schools in the U.S. Meanwhile the absence of the "e" inSTEM from the traditional American curriculum is being remedied by the movement tostandardize engineering knowledge for pre-college learners (NAE, 2010). There are several keyprerequisites to engineering learning, and this paper addresses ways of teaching two thesefundamental prerequisite abilities in middle school. The abilities addressed here are: (1) mentalrotation ability, often included among a more broad set of spatial skills (Sorby, 2009) and (2) theability to make multi-step estimates. The research proposed is situated in project-basedenvironment for digital fabrication.Mental rotation ability predicts a host of engineering abilities and has been found to be mutableand teachable (Sorby, 2009). This is the ability to mentally rotate shapes and to imagine them indifferent orientations. Chemical, mechanical, and even software engineers use this ability.Numerical approximation (estimation) is a ubiquitous skill (Usiskin, 1986) used mostly in theearly stages of a project. For example, establishing feasibility depends upon using estimates.Erico Fermi (1901-1954) coined the term "back of the envelope" to refer to large estimates basedon a series of smaller estimates. These so-called Fermi problems are useful in engineeringeducation (e.g., Barak, Raviv & VanEpps, 2009; Dunn-Rankin, 2001; French & Leiffer, 2012).The Spatial Estimator is a new online tool designed to help students estimate properties of three-dimensional shapes as an early step in digital fabrication (Bell et al, 2010; Berry et al., 2010).Digital fabrication involves the design and creation of tangible objects by way of digital devices,and can include die-cutting and three-dimensional (3D) printing. As manufacturing becomessmall-scale and computer-aided-design (CAD) programs become more accessible, widespreadeducational uses of digital fabrication have great educational potential. Early research withdigital fabrication in K-12 education shows promise (Tillman, 2011).This research is based on the hypothesis that students can learn to make practical estimates aboutthe amount of time and materials needed for digital fabrication. Feedback in software willencourage students to explore aspects of their own designs to establish properties such as volumeand the necessary density of their materials. Currently, 3D printers must be used judiciously inclassrooms because of the requisite costs of time and access to fabrication materials such asplastic. These real-world constraints can be assets to a teacher since they provide authenticdesign specifications.Students will revise estimates based on interaction with three-dimensional shapes in software.This will require spatial thinking and mental rotation, in addition to the point-and-click rotationof the shapes on screen. Students' iterative estimates will elicit feedback from the computer, andstudent actions will be recorded as log files for analysis. It is anticipated that student estimatesbecome more accurate with practice and that their solutions will benefit from work with theSpatial Estimator.REFERENCES:Bell, B. L., Brown, A., Bull, G., Conly, K., Johnson, L., Mcanear, … Sprague, D., (2010). A special editorial: Digital fabrication revolution, TechTrends 54(5).Berry, R. Q., III, Bull, G., Browning, C., Thomas, C. D., Starkweather, K., & Aylor, J. H. (2010). Preliminary considerations regarding use of digital fabrication to incorporate engineering design principles in elementary mathematics education. Contemporary Issues in Technology and Teacher Education, 10(2). Retrieved from http://www.citejournal.org/vol10/iss2/editorial/article1.cfmBull, G., & Groves, J. (2009). The democratization of production. Learning and Leading with Technology, 37(3), 36-37.Dunn-rankin, D. (2001). Evaluating design alternatives – the role of simple engineering analysis and estimation. 2001 ASEE Annual Conference & Exposition.French, J. J., & Leiffer, P. R. (2012). The genesis of transformation: Preventing “failure to launch” syndrome in generation in first-year engineering students. 2012 ASEE Annual Conference & Exposition.Kolodner, J. L., Camp, P. J., Crismond, D., Fasse, B., Gray, J., Holbrook, J., Puntambekar, S., et al. (2003). Problem-based learning meets case-based reasoning in the middle-school science classroom: Putting learning by design™ into practice. Journal of the Learning Sciences, 12(4), 495–547. doi:10.1207/S15327809JLS1204_2Obama, B. (2010). Remarks by the president at the announcement of the “Change the Equation” initiative. Retrieved from the White House Web site: http://www.whitehouse.gov/the- press- office/2010/09/16/remarks - president- announcement- change- equation- initiativeRaviv, D., Barak, M., Sheva, B., Vanepps, T. J., & Raton, B. (2009). Teaching innovative thinking: Future directions. 2009 ASEE Annual Conference & Exposition.Tillman, D. (2011). Performance assessment of digital fabrication activities with embedded mathematics pedagogy. In M. Koehler & P. Mishra (Eds.), Proceedings of Society for Information Technology & Teacher Education International Conference 2011 (pp. 898– 901). Chesapeake, VA: AACE.
2014 ASEE Annual Conference & Exposition Proceedings pp 24.338.1-24.338.12; https://doi.org/10.18260/1-2--20229

Abstract:
Creativity and its Assessment in a Design and Development of Food Products and Processes CourseThe main task of a food engineer is to design and operate processes to transform raw materialsinto final products, particularly with the aim to control, prevent, or delay spoilage caused bychemical reactions, physical effects, and/or biological activity1. At ABC University foodengineering (FE) students apply their knowledge and skills required to function in the differentfields of FE in the capstone course entitled Design and Development of Food Products andProcesses, which outcomes include that students will be able to: a) Identify consumer andcommercial factors that should be considered when designing a new product, b) Describe theproduct to be developed, c) Develop and evaluate potential product formulations, d) Propose themanufacturing process for the product to be developed, e) Choose the most suitable packagingfor the product, f) Evaluate the shelf-life of the product, g) Locate and describe the lawsapplicable to the ingredients used to ensure the safety of the developed product, h) Develop anutritional label for the product, h) Identify critical control points and limits of the proposedprocess, and i) Estimate operating costs and investment required to start the production line. Creative thinking in higher education can only be expressed productively within a particulardomain. The student must have a strong foundation in the strategies and skills of the domain inorder to make connections and synthesize. While demonstrating solid knowledge of the domain'sparameters, the creative thinker, at the highest levels of performance, pushes beyond thoseboundaries in new, unique, or atypical re-combinations, uncovering or critically perceiving newsyntheses and using or recognizing creative risk-taking to achieve a solution2. With sights set onthis, the full paper will present with further detail the didactic intervention whose purpose was toenhance creative thinking, make the food product design and development processes moreefficient as well as to overall improve the creative experience in the studied capstone course3-6. Creativity assessment was grounded on the Consensual Assessment Technique7 (CAT), whichis based on the idea that the best measure of creativity regardless of what is being evaluated, isthe assessment by experts in that field. Therefore, a group of experts in the FE field were invitedto evaluate capstone course final projects and developed food products by means of the CreativeThinking VALUE Rubric, which is made up of a set of attributes that are common to creativethinking across disciplines2. Possible performance levels were entitled capstone or exemplar(value of 4), milestones (values of 3 or 2), and benchmark (value of 1). Instructor, peer-, and self-assessments were also performed throughout the course and on final project. Evaluators werefurther encouraged to assign a value of zero if work did not meet benchmark level performance.Mean values from rubric assessment of final projects were 2.35 for Acquiring Competencies(attaining strategies and skills within a particular domain), 2.42 for Taking Risks (may includepersonal risk, fear of embarrassment or rejection, or risk of failure in successfully completingassignment, i.e. going beyond original parameters of assignment, introducing new materials andforms, tackling controversial topics, advocating unpopular ideas or solutions), 2.44 for SolvingProblems, 2.44 for Embracing Contradictions, 2.40 for Innovative Thinking (novelty oruniqueness of idea, claim, question, form, etc.), and 2.24 for Connecting, Synthesizing, andTransforming. Students’ creative thinking was at an intermediate level in both the capacity tocombine or synthesize existing ideas or expertise in original ways and the experience of thinking,reacting, and working in an imaginative way.[1] XXX [For blind review purposes]. 2013. Proceedings of the 2013 ASEE Annual Conference and Exposition, Atlanta, GA, June 23 – 26.[2] AAC&U. 2013. Creative Thinking Value Rubric. Washington, DC: Association of American Colleges and Universities (AAC&U). Available online at http://www-.aacu.org/value/- rubrics/pdf/All_Rubrics.pdf[3] Baer, J. 1993. Creativity and diverge/if thinking: A task-specific approach. Hillsdale, NJ: Lawrence Erlbaum Associates.[4] Fogler, H. S. and LeBlanc S. E. 2007. Strategies for creative problem solving. 2nd Ed. Upper Saddle River, NJ: Pearson Education.[5] Guilford, J. P. 1950. Creativity. American Psychologist, 5: 444-154.[6] Sternberg, R. J. and Lubart, T. I. 1993. Creative Giftedness: A Multivariate Investment Approach. Gifted Child Quarterly, 37(1): 7-15.[7] Amabile, T. M. 1982. Social Psychology of Creativity: A Consensual Assessment Technique, Journal of Personality and Social Psychology, 43(5): 997-1013.
Page of 7
Articles per Page
by
Show export options
  Select all
Back to Top Top