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Published: 23 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18102218

Abstract:
One of pathological characteristics of Alzheimer’s disease (AD), aggregation and deposition of β amyloid (Aβ), has been accepted as a potent activator of neuronal cell death. Red ginseng is well-known for various pharmacological activities, but most studies have been focused on red ginseng water extract (RGW), which has resulted in the conception of the present study of red ginseng oil (RGO) against Aβ25–35-induced neurotoxicity. Cytotoxicity and apoptosis induction by Aβ were verified and the underlying mechanism by which RGO inhibited neuronal cell death, mitochondria dysfunction and NF-κB pathway related protein markers were evaluated. RGO attenuated Aβ25–35-induced apoptosis, not only by inhibiting calcium influx, but also by reducing mitochondrial membrane potential loss. RGO significantly decreased Bax, whereas increased Bcl-2 and inactivated of caspase-3 and -9 and PARP-1 stimulated by Aβ25–35. Anti-neuroinflammatory effect of RGO was demonstrated by downregulating c-Jun N-terminal kinase (JNK) and p38, resulting in inhibiting of the NF-κB pathway and thereby suppressing the expressions of pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), nitric oxide (NO) and tumor necrosis factor-α (TNF-α). The present study revealed that RGO is a potential natural resource of the functional foods industry as well as a promising candidate of multi-target neuronal protective agent for the prevention of AD.
Published: 23 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18102197

Abstract:
Metallothioneins (MTs) are a family of metal-binding proteins virtually expressed in all organisms including prokaryotes, lower eukaryotes, invertebrates and mammals. These proteins regulate homeostasis of zinc (Zn) and copper (Cu), mitigate heavy metal poisoning, and alleviate superoxide stress. In recent years, MTs have emerged as an important, yet largely underappreciated, component of the immune system. Innate and adaptive immune cells regulate MTs in response to stress stimuli, cytokine signals and microbial challenge. Modulation of MTs in these cells in turn regulates metal ion release, transport and distribution, cellular redox status, enzyme function and cell signaling. While it is well established that the host strictly regulates availability of metal ions during microbial pathogenesis, we are only recently beginning to unravel the interplay between metal-regulatory pathways and immunological defenses. In this perspective, investigation of mechanisms that leverage the potential of MTs to orchestrate inflammatory responses and antimicrobial defenses has gained momentum. The purpose of this review, therefore, is to illumine the role of MTs in immune regulation. We discuss the mechanisms of MT induction and signaling in immune cells and explore the therapeutic potential of the MT-Zn axis in bolstering immune defenses against pathogens.
Published: 23 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18102219

Abstract:
The original hygiene hypothesis declares “more infections in early childhood protect against later atopy”. According to the hygiene hypothesis, the increased incidence of allergic disorders in developed countries is explained by the decrease of infections. Epithelial cells and dendritic cells play key roles in bridging the innate and adaptive immune systems. Among the various pattern-recognition receptor systems of epithelial cells and dendritic cells, including toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and others, TLRs are the key systems of immune response regulation. In humans, TLRs consist of TLR1 to TLR10. They regulate cellular responses through engagement with TLR ligands, e.g., lipopolysaccharides (LPS) acts through TLR4 and dsRNA acts through TLR3, but there are certain common components between these two TLR pathways. dsRNA activates epithelial cells and dendritic cells in different directions, resulting in allergy-related Th2-skewing tendency in epithelial cells, and Th1-skewing tendency in dendritic cells. The Th2-skewing effect by stimulation of dsRNA on epithelial cells could be suppressed by the presence of LPS above some threshold. When LPS level decreases, the Th2-skewing effect increases. It may be via these interrelated networks and related factors that LPS modifies the allergic responses and provides a plausible mechanism of the hygiene hypothesis. Several hygiene hypothesis-related phenomena, seemingly conflicting, are also discussed in this review, along with their proposed mechanisms.
Published: 22 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18102213

Abstract:
Compared to other types of cells, neurons express the largest number of diverse mRNAs, including neuron-specific ones. This mRNA diversity is required for neuronal function, memory storage, maintenance and retrieval. Regulation of translation in neurons is very complicated and involves various proteins. Some proteins, implementing translational control in other cell types, are used by neurons for synaptic plasticity. In this review, we discuss the neuron-specific activity of four kinases: protein kinase R (PKR), PKR-like endoplasmic reticulum kinase (PERK), general control nonderepressible 2 kinase (GCN2), and heme-reguated eIF2α kinase (HRI), the substrate for which is α-subunit of eukaryotic initiation factor 2 (eIF2α). Phosphorylation of eIF2α is necessary for the cell during stress conditions, such as lack of amino acids, energy stress or viral infection. We propose that, during memory formation, neurons use some mechanisms similar to those involved in the cellular stress. The four eIF2α kinases regulate translation of certain mRNAs containing upstream open reading frames (uORFs). These mRNAs encode proteins involved in the processes of long-term potentiation (LTP) or long-term depression (LTD). The review examines some neuronal proteins for which translation regulation by eIF2 was suggested and checked experimentally. Of such proteins, we pay close attention to protein kinase Mζ, which is involved in memory storage and regulated at the translational level.
Published: 23 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18102216

Abstract:
In cancer, the mouse double minute 2 (MDM2) is an oncoprotein that contributes to the promotion of cell growth, survival, invasion, and therapeutic resistance. The impact of MDM2 on cell survival versus cell death is complex and dependent on levels of MDM2 isoforms, p53 status, and cellular context. Extensive investigations have demonstrated that MDM2 protein–protein interactions with p53 and other p53 family members (p63 and p73) block their ability to function as transcription factors that regulate cell growth and survival. Upon genotoxic insults, a dynamic and intricately regulated DNA damage response circuitry is activated leading to release of p53 from MDM2 and activation of cell cycle arrest. What ensues following DNA damage, depends on the extent of DNA damage and if the cell has sufficient DNA repair capacity. The well-known auto-regulatory loop between p53-MDM2 provides an additional layer of control as the cell either repairs DNA damage and survives (i.e., MDM2 re-engages with p53), or undergoes cell death (i.e., MDM2 does not re-engage p53). Furthermore, the decision to live or die is also influenced by chromatin-localized MDM2 which directly interacts with the Mre11-Rad50-Nbs1 complex and inhibits DNA damage-sensing giving rise to the potential for increased genome instability and cellular transformation.
Published: 22 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18102212

Abstract:
DNA is damaged on a daily basis, which can lead to heritable mutations and the activation of proto-oncogenes. Therefore, DNA damage and repair are critical risk factors in cancer, aging and disease, and are the underlying bases of most frontline cancer therapies. Much of our current understanding of the mechanisms that maintain DNA integrity has been obtained using antibody-based assays. The oligonucleotide equivalents of antibodies, known as aptamers, have emerged as potential molecular recognition rivals. Aptamers possess several ideal properties including chemical stability, in vitro selection and lack of batch-to-batch variability. These properties have motivated the incorporation of aptamers into a wide variety of analytical, diagnostic, research and therapeutic applications. However, their use in DNA repair studies and DNA damage therapies is surprisingly un-tapped. This review presents an overview of the progress in selecting and applying aptamers for DNA damage and repair research.
Published: 23 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18102217

Abstract:
To date, multi-drug resistant bacteria represent an increasing health threat, with a high impact on mortality, morbidity, and health costs on a global scale. The ability of bacteria to rapidly and permanently acquire new virulence factors and drug-resistance elements requires the development of new antimicrobial agents and selection of new proper targets, such as sortase A. This specific bacterial target plays an important role in the virulence of many Gram-positive pathogens, and its inhibition should produce a mild evolutionary pressure which will not favor the development of resistance. A primary screening using a fluorescence resonance energy transfer assay was used to experimentally evaluate the inhibitory activity of several compounds on sortase A. Using molecular docking and structure-activity relationship analyses, several lead inhibitors were identified, which were further tested for antimicrobial activity using the well diffusion test and minimum inhibitory concentration. The toxicity was assessed using the Daphnia magna test and used as a future screening filter. Three natural compounds were identified in this study as promising candidates for further development into therapeutically useful anti-infective agents that could be used to treat infections caused by multi-drug resistant bacterial pathogens which include sortase A in their enzymatic set.
Published: 22 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18102214

Abstract:
Oxidative stress refers to elevated reactive oxygen species (ROS) levels, and NADPH oxidases (NOXs), which are one of the most important sources of ROS. Oxidative stress plays important roles in the etiologies, pathological mechanisms, and treatment strategies of vascular diseases. Additionally, oxidative stress affects mechanisms of carcinogenesis, tumor growth, and prognosis in malignancies. Nearly all solid tumors show stimulation of neo-vascularity, termed angiogenesis, which is closely associated with malignant aggressiveness. Thus, cancers can be seen as a type of vascular disease. Oxidative stress-induced functions are regulated by complex endogenous mechanisms and exogenous factors, such as medication and diet. Although understanding these regulatory mechanisms is important for improving the prognosis of urothelial cancer, it is not sufficient, because there are controversial and conflicting opinions. Therefore, we believe that this knowledge is essential to discuss observations and treatment strategies in urothelial cancer. In this review, we describe the relationships between members of the NOX family and tumorigenesis, tumor growth, and pathological mechanisms in urological cancers including prostate cancer, renal cell carcinoma, and urothelial cancer. In addition, we introduce natural compounds and chemical agents that are associated with ROS-induced angiogenesis or apoptosis.
Published: 23 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18102220

Abstract:
Argan oil is widely used in Morocco in traditional medicine. Its ability to treat cardiovascular diseases is well-established. However, nothing is known about its effects on neurodegenerative diseases, which are often associated with increased oxidative stress leading to lipid peroxidation and the formation of 7-ketocholesterol (7KC) resulting from cholesterol auto-oxidation. As 7KC induces oxidative stress, inflammation and cell death, it is important to identify compounds able to impair its harmful effects. These compounds may be either natural or synthetic molecules or mixtures of molecules such as oils. In this context: (i) the lipid profiles of dietary argan oils from Berkane and Agadir (Morocco) in fatty acids, phytosterols, tocopherols and polyphenols were determined by different chromatographic techniques; and (ii) their anti-oxidant and cytoprotective effects in 158N murine oligodendrocytes cultured with 7KC (25–50 µM; 24 h) without and with argan oil (0.1% v/v) or α-tocopherol (400 µM, positive control) were evaluated with complementary techniques of cellular and molecular biology. Among the unsaturated fatty acids present in argan oils, oleate (C18:1 n-9) and linoleate (C18:1 n-6) were the most abundant; the highest quantities of saturated fatty acids were palmitate (C16:0) and stearate (C18:0). Several phytosterols were found, mainly schottenol and spinasterol (specific to argan oil), cycloartenol, β-amyrin and citrostadienol. α- and γ-tocopherols were also present. Tyrosol and protocatechic acid were the only polyphenols detected. Argan and extra virgin olive oils have many compounds in common, principally oleate and linoleate, and tocopherols. Kit Radicaux Libres (KRL) and ferric reducing antioxidant power (FRAP) tests showed that argan and extra virgin olive oils have anti-oxidant properties. Argan oils were able to attenuate the cytotoxic effects of 7KC on 158N cells: loss of cell adhesion, cell growth inhibition, increased plasma membrane permeability, mitochondrial, peroxisomal and lysosomal dysfunction, and the induction of oxiapoptophagy (OXIdation + APOPTOsis + autoPHAGY). Altogether, our data obtained in 158N oligodendrocytes provide evidence that argan oil is able to counteract the toxic effects of 7KC on nerve cells, thus suggesting that some of its compounds could prevent or mitigate neurodegenerative diseases to the extent that they are able to cross the blood-brain barrier.
International Journal of Molecular Sciences, Volume 14, pp 11356-11375; https://doi.org/10.3390/ijms140611356

Abstract:
Pine wilt disease (PWD) caused by pine wood nematode (PWN), Bursaphelenchus xylophilus, is the most destructive diseases of pine and poses a threat of serious economic losses worldwide. Although several of the mechanisms involved in disease progression have been discovered, the molecular response of Pinus massoniana to PWN infection has not been explored. We constructed four subtractive suppression hybridization cDNA libraries by taking time-course samples from PWN-inoculated Masson pine trees. One-hundred forty-four significantly differentially expressed sequence tags (ESTs) were identified, and 124 high-quality sequences with transcriptional features were selected for gene ontology (GO) and individual gene analyses. There were marked differences in the types of transcripts, as well as in the timing and levels of transcript expression in the pine trees following PWN inoculation. Genes involved in signal transduction, transcription and translation and secondary metabolism were highly expressed after 24 h and 72 h, while stress response genes were highly expressed only after 72 h. Certain transcripts responding to PWN infection were discriminative; pathogenesis and cell wall-related genes were more abundant, while detoxification or redox process-related genes were less abundant. This study provides new insights into the molecular mechanisms that control the biochemical and physiological responses of pine trees to PWN infection, particularly during the initial stage of infection.
International Journal of Molecular Sciences, Volume 14, pp 11347-11355; https://doi.org/10.3390/ijms140611347

Abstract:
PCA3 (prostate cancer gene 3) and multiparametric 3 tesla MRI are new promising diagnostic tools in the detection of PCa. Our aim was to study the clinical value of the Progensa PCA3-test: its predictive value for biopsy outcome, Gleason score and MRI outcome. We evaluated, retrospectively, 591 patients who underwent a Progensa PCA3-test at the Radboud University Nijmegen Medical Centre between May 2006 and December 2009. Prostate biopsies were performed in 290 patients; a multiparametric 3 tesla MRI of the prostate was performed in 163/591 patients. The PCA3-score was correlated to biopsy results and MRI outcome. The results show that PCA3 was highly predictive for biopsy outcome (p < 0.001); there was no correlation with the Gleason score upon biopsy (p = 0.194). The PCA3-score of patients with a suspicious region for PCa on MRI was significantly higher (p < 0.001) than in patients with no suspicious region on MRI (52 vs. 21). In conclusion, PCA3 is a valuable diagnostic biomarker for PCa; it did not correlate with the Gleason score. Furthermore, multiparametric MRI outcome was significantly correlated with the PCA3-score. Thus, PCA3 could be used to select patients that require MRI. However, in patients with a negative PCA3 and high clinical suspicion of PCa, a multiparametric MRI should also be done.
International Journal of Molecular Sciences, Volume 14, pp 11319-11346; https://doi.org/10.3390/ijms140611319

Abstract:
Are there common pathways underlying the broad spectrum of tissue pathologies that develop upon injuries and from subsequent tissue remodeling? Here, we explain the pathophysiological impact of a set of evolutionary conserved danger control programs for tissue pathology. These programs date back to the survival benefits of the first multicellular organisms upon traumatic injuries by launching a series of danger control responses, i.e., 1. Haemostasis, or clotting to control bleeding; 2. Host defense, to control pathogen entry and spreading; 3. Re-epithelialisation, to recover barrier functions; and 4. Mesenchymal, to repair to regain tissue stability. Taking kidney pathology as an example, we discuss how clotting, inflammation, epithelial healing, and fibrosis/sclerosis determine the spectrum of kidney pathology, especially when they are insufficiently activated or present in an overshooting and deregulated manner. Understanding the evolutionary benefits of these response programs may refine the search for novel therapeutic targets to limit organ dysfunction in acute injuries and in progressive chronic tissue remodeling
International Journal of Molecular Sciences, Volume 14, pp 11302-11318; https://doi.org/10.3390/ijms140611302

Abstract:
An amylopullulanase of the thermophilic Anoxybacillus sp. SK3-4 (ApuASK) was purified to homogeneity and characterized. Though amylopullulanases larger than 200 kDa are rare, the molecular mass of purified ApuASK appears to be approximately 225 kDa, on both SDS-PAGE analyses and native-PAGE analyses. ApuASK was stable between pH 6.0 and pH 8.0 and exhibited optimal activity at pH 7.5. The optimal temperature for ApuASK enzyme activity was 60 °C, and it retained 54% of its total activity for 240 min at 65 °C. ApuASK reacts with pullulan, starch, glycogen, and dextrin, yielding glucose, maltose, and maltotriose. Interestingly, most of the previously described amylopullulanases are unable to produce glucose and maltose from these substrates. Thus, ApuASK is a novel, high molecular-mass amylopullulanase able to produce glucose, maltose, and maltotriose from pullulan and starch. Based on whole genome sequencing data, ApuASK appeared to be the largest protein present in Anoxybacillus sp. SK3-4. The α-amylase catalytic domain present in all of the amylase superfamily members is present in ApuASK, located between the cyclodextrin (CD)-pullulan-degrading N-terminus and the α-amylase catalytic C-terminus (amyC) domains. In addition, the existence of a S-layer homology (SLH) domain indicates that ApuASK might function as a cell-anchoring enzyme and be important for carbohydrate utilization in a streaming hot spring.
International Journal of Molecular Sciences, Volume 14, pp 11277-11301; https://doi.org/10.3390/ijms140611277

Abstract:
Mass spectrometry imaging is employed for mapping proteins, lipids and metabolites in biological tissues in a morphological context. Although initially developed as a tool for biomarker discovery by imaging the distribution of protein/peptide in tissue sections, the high sensitivity and molecular specificity of this technique have enabled its application to biomolecules, other than proteins, even in cells, latent finger prints and whole organisms. Relatively simple, with no requirement for labelling, homogenization, extraction or reconstitution, the technique has found a variety of applications in molecular biology, pathology, pharmacology and toxicology. By discriminating the spatial distribution of biomolecules in serial sections of tissues, biomarkers of lesions and the biological responses to stressors or diseases can be better understood in the context of structure and function. In this review, we have discussed the advances in the different aspects of mass spectrometry imaging processes, application towards different disciplines and relevance to the field of toxicology.
International Journal of Molecular Sciences, Volume 14, pp 11259-11276; https://doi.org/10.3390/ijms140611259

Abstract:
Extending our previous observations, we have shown on HaCat cells that melatonin, at ~10−9 M concentration, transiently raises not only the expression of the neuronal nitric oxide synthase (nNOS) mRNA, but also the nNOS protein synthesis and the nitric oxide oxidation products, nitrite and nitrate. Interestingly, from the cell bioenergetic point of view, the activated NO-related chemistry induces a mild decrease of the oxidative phosphorylation (OXPHOS) efficiency, paralleled by a depression of the mitochondrial membrane potential. The OXPHOS depression is apparently balanced by glycolysis. The mitochondrial effects described have been detected only at nanomolar concentration of melatonin and within a time window of a few hours’ incubation; both findings compatible with the melatonin circadian cycle.
Published: 27 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112250

Abstract:
Konjac glucomannan (KGM) has attracted extensive attention because of its biodegradable, non-toxic, harmless, and biocompatible features. Its gelation performance is one of its most significant characteristics and enables wide applications of KGM gels in food, chemical, pharmaceutical, materials, and other fields. Herein, different preparation methods of KGM gels and their microstructures were reviewed. In addition, KGM applications have been theoretically modeled for future uses.
Published: 27 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112251

Abstract:
The antifungal activity of polygodial, a secondary metabolite extracted from Canelo, on mycelial growth of different Botrytis cinerea isolates has been evaluated. The results show that polygodial affects growth of normal and resistant isolates of B. cinerea with EC50 values ranging between 117 and 175 ppm. In addition, polygodial markedly decreases the germination of B. cinerea, i.e., after six hours of incubation the percentage of germination decreases from 92% (control) to 25% and 5% in the presence of 20 ppm and 80 ppm of polygodial, respectively. Morphological studies indicate that conidia treated with polygodial are smaller, with irregular membrane border, and a lot of cell debris, as compared to conidia in the control. The existence of polygodial-induced membrane damage was confirmed by SYTOX® Green uptake assay. Gene expression studies confirm that the effect of polygodial on B. cinerea is mainly attributed to inhibition of germination and appears at early stages of B. cinerea development. On the other hand, drimenol, a drimane with chemical structure quite similar to polygodial, inhibits the mycelial growth efficiently. Thus, both compounds inhibit mycelial growth by different mechanisms. The different antifungal activities of these compounds are discussed in terms of the electronic density on the double bond.
Published: 27 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112252

Abstract:
Cardiovascular diseases are the leading cause of mortality worldwide. Therefore, a better understanding of their pathomechanisms and the subsequent implementation of optimal prophylactic and therapeutic strategies are of utmost importance. A growing body of evidence states that low-grade inflammation is a common feature for most of the cardiovascular diseases in which the contributing factors are the activation of toll-like receptors (TLRs) and vitamin D deficiency. In this article, available data concerning the association of cardiovascular diseases with TLRs and vitamin D status are reviewed, followed by a discussion of new possible approaches to cardiovascular disease management.
Published: 27 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112256

Abstract:
Bacterial antibiotic resistance is a rapidly expanding problem in the world today. Functionalization of the outer membrane of Gram-negative bacteria provides protection from extracellular antimicrobials, and serves as an innate resistance mechanism. Lipopolysaccharides (LPS) are a major cell-surface component of Gram-negative bacteria that contribute to protecting the bacterium from extracellular threats. LPS is biosynthesized by the sequential addition of sugar moieties by a number of glycosyltransferases (GTs). Heptosyltransferases catalyze the addition of multiple heptose sugars to form the core region of LPS; there are at most four heptosyltransferases found in all Gram-negative bacteria. The most studied of the four is HepI. Cells deficient in HepI display a truncated LPS on their cell surface, causing them to be more susceptible to hydrophobic antibiotics. HepI–IV are all structurally similar members of the GT-B structural family, a class of enzymes that have been found to be highly dynamic. Understanding conformational changes of heptosyltransferases are important to efficiently inhibiting them, but also contributing to the understanding of all GT-B enzymes. Finding new and smarter methods to inhibit bacterial growth is crucial, and the Heptosyltransferases may provide an important model for how to inhibit many GT-B enzymes.
Published: 27 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112241

Abstract:
Background: Prenatal glucocorticoid administration alters the activity of the fetal hypothalamic-pituitary-adrenocortical axis (HPAA), and correspondingly the adenocorticotropic hormone (ACTH) and cortisol levels after birth. The dosages required for these effects are critically discussed. Activation of the HPAA is related to metabolic syndrome and diabetes mellitus. Hypoglycemia is the classic side effect of antidiabetic treatment. We hypothesized that a low dosage of dexamethasone in late pregnancy alters the HPAA response to hypoglycemia in pigs. Methods: 12 pregnant sows were randomly assigned to two groups which received either a low-dose intramuscular injection (99th and 100th day of gestation) of dexamethasone (0.06 μg/kg body weight) or vehicle. Three months after birth, 18 dexamethasone-treated anaesthetized offspring and 12 control offspring underwent a 75 min hypoglycemic clamp (blood glucose below 4 mmol/L) procedure. Heart rate (HR), blood pressure, ACTH and cortisol levels and body weight (at birth and after three months) were recorded. Results: Dexamethasone-treated animals exhibited significantly elevated ACTH (139.9 ± 12.7 pg/mL) and cortisol (483.1 ± 30.3 nmol/L) levels during hypoglycemia as compared to the control group (41.7 ± 6.5 pg/mL and 257.9 ± 26.7 nmol/L, respectively), as well as an elevated HR (205.5 ± 5.7 bpm) and blood pressure (systolic: 128.6 ± 1.5, diastolic: 85.7 ± 0.7 mmHg) response as compared to the control group (153.2 ± 4.5 bpm; systolic: 118.6 ± 1.6, diastolic: 79.5 ± 1.4 mmHg, respectively; p < 0.001). Conclusions: Low-dose prenatal administration of dexamethasone not only exerts effects on the HPAA (ACTH and cortisol concentration) and vital parameters (HR and diastolic blood pressure) under baseline conditions, but also on ACTH, HR and systolic blood pressure during hypoglycemia.
Published: 27 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112253

Abstract:
Megabalanus barnacle is one of the model organisms for marine biofouling research. However, further elucidation of molecular mechanisms underlying larval settlement has been hindered due to the lack of genomic information thus far. In the present study, cDNA libraries were constructed for cyprids, the key stage for larval settlement, and adults of Megabalanus volcano. After high-throughput sequencing and de novo assembly, 42,620 unigenes were obtained with a N50 value of 1532 bp. These unigenes were annotated by blasting against the NCBI non-redundant (nr), Swiss-Prot, Cluster of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Finally, 19,522, 15,691, 14,459, and 10,914 unigenes were identified correspondingly. There were 22,158 differentially expressed genes (DEGs) identified between two stages. Compared with the cyprid stage, 8241 unigenes were down-regulated and 13,917 unigenes were up-regulated at the adult stage. The neuroactive ligand-receptor interaction pathway (ko04080) was significantly enriched by KEGG enrichment analysis of the DEGs, suggesting that it possibly involved in larval settlement. Potential functions of three conserved allatostatin neuropeptide-receptor pairs and two light-sensitive opsin proteins were further characterized, indicating that they might regulate attachment and metamorphosis at cyprid stage. These results provided a deeper insight into the molecular mechanisms underlying larval settlement of barnacles.
Published: 27 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112259

Abstract:
The treatment of patients with acute myeloid leukemia (AML) with targeted immunotherapy is challenged by the heterogeneity of the disease and a lack of tumor-exclusive antigens. Conventional immunotherapy targets for AML such as CD33 and CD123 have been proposed as targets for chimeric antigen receptor (CAR)-engineered T-cells (CAR-T-cells), a therapy that has been highly successful in the treatment of B-cell leukemia and lymphoma. However, CD33 and CD123 are present on hematopoietic stem cells, and targeting with CAR-T-cells has the potential to elicit long-term myelosuppression. C-type lectin-like molecule-1 (CLL1 or CLEC12A) is a myeloid lineage antigen that is expressed by malignant cells in more than 90% of AML patients. CLL1 is not expressed by healthy Hematopoietic Stem Cells (HSCs), and is therefore a promising target for CAR-T-cell therapy. Here, we describe the development and optimization of an anti-CLL1 CAR-T-cell with potent activity on both AML cell lines and primary patient-derived AML blasts in vitro while sparing healthy HSCs. Furthermore, in a disseminated mouse xenograft model using the CLL1-positive HL60 cell line, these CAR-T-cells completely eradicated tumor, thus supporting CLL1 as a promising target for CAR-T-cells to treat AML while limiting myelosuppressive toxicity.
Published: 27 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112258

Abstract:
The conversion of peptidylarginine into peptidylcitrulline by calcium-dependent peptidylarginine deiminases (PADs) has been implicated in the pathogenesis of a number of diseases, identifying PADs as therapeutic targets for various diseases. The PAD inhibitor Cl-amidine ameliorates the disease course, severity, and clinical manifestation in multiple disease models, and it also modulates dendritic cell (DC) functions such as cytokine production, antigen presentation, and T cell proliferation. The beneficial effects of Cl-amidine make it an attractive compound for PAD-targeting therapeutic strategies in inflammatory diseases. Here, we found that Cl-amidine inhibited nitric oxide (NO) generation in a time- and dose-dependent manner in maturing DCs activated by lipopolysaccharide (LPS). This suppression of NO generation was independent of changes in NO synthase (NOS) enzyme activity levels but was instead dependent on changes in inducible NO synthase (iNOS) transcription and expression levels. Several upstream signaling pathways for iNOS expression, including the mitogen-activated protein kinase, nuclear factor-κB p65 (NF-κB p65), and hypoxia-inducible factor 1 pathways, were not affected by Cl-amidine. By contrast, the LPS-induced signal transducer and the activator of transcription (STAT) phosphorylation and activator protein-1 (AP-1) transcriptional activities (c-Fos, JunD, and phosphorylated c-Jun) were decreased in Cl-amidine-treated DCs. Inhibition of Janus kinase/STAT signaling dramatically suppressed iNOS expression and NO production, whereas AP-1 inhibition had no effect. These results indicate that Cl-amidine-inhibited STAT activation may suppress iNOS expression. Additionally, we found mildly reduced cyclooxygenase-2 expression and prostaglandin E2 production in Cl-amidine-treated DCs. Our findings indicate that Cl-amidine acts as a novel suppressor of iNOS expression, suggesting that Cl-amidine has the potential to ameliorate the effects of excessive iNOS/NO-linked immune responses.
Published: 27 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112255

Abstract:
Aquaporins are tetrameric membrane-bound channels that facilitate transport of water and other small solutes across cell membranes. In eukaryotes, they are frequently regulated by gating or trafficking, allowing for the cell to control membrane permeability in a specific manner. Protein–protein interactions play crucial roles in both regulatory processes and also mediate alternative functions such as cell adhesion. In this review, we summarize recent knowledge about aquaporin protein–protein interactions; dividing the interactions into three types: (1) interactions between aquaporin tetramers; (2) interactions between aquaporin monomers within a tetramer (hetero-tetramerization); and (3) transient interactions with regulatory proteins. We particularly focus on the structural aspects of the interactions, discussing the small differences within a conserved overall fold that allow for aquaporins to be differentially regulated in an organism-, tissue- and trigger-specific manner. A deep knowledge about these differences is needed to fully understand aquaporin function and regulation in many physiological processes, and may enable design of compounds targeting specific aquaporins for treatment of human disease.
Published: 27 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112254

Abstract:
Endothelial cells (ECs) play a pivotal role in the crosstalk between blood coagulation and inflammation. Endothelial cellular dysfunction underlies the development of vascular inflammatory diseases. Recent studies have revealed that aberrant gap junctions (GJs) and connexin (Cx) hemichannels participate in the progression of cardiovascular diseases such as cardiac infarction, hypertension and atherosclerosis. ECs can communicate with adjacent ECs, vascular smooth muscle cells, leukocytes and platelets via GJs and Cx channels. ECs dynamically regulate the expression of numerous Cxs, as well as GJ functionality, in the context of inflammation. Alterations to either result in various side effects across a wide range of vascular functions. Here, we review the roles of endothelial GJs and Cx channels in vascular inflammation, blood coagulation and leukocyte adhesion. In addition, we discuss the relevant molecular mechanisms that endothelial GJs and Cx channels regulate, both the endothelial functions and mechanical properties of ECs. A better understanding of these processes promises the possibility of pharmacological treatments for vascular pathogenesis.
Published: 27 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112260

Abstract:
Olfaction is often deregulated in Alzheimer’s disease (AD) patients, and is also impaired in transgenic Tg2576 AD mice, which overexpress the Swedish mutated form of human amyloid precursor protein (APP). However, little is known about the molecular mechanisms that accompany the neurodegeneration of olfactory structures in aged Tg2576 mice. For that, we have applied proteome- and transcriptome-wide approaches to probe molecular disturbances in the olfactory bulb (OB) dissected from aged Tg2576 mice (18 months of age) as compared to those of age matched wild-type (WT) littermates. Some over-represented biological functions were directly relevant to neuronal homeostasis and processes of learning, cognition, and behavior. In addition to the modulation of CAMP responsive element binding protein 1 (CREB1) and APP interactomes, an imbalance in the functionality of the IκBα-NFκB p65 complex was observed during the aging process in the OB of Tg2576 mice. At two months of age, the phosphorylated isoforms of olfactory IκBα and NFκB p65 were inversely regulated in transgenic mice. However, both phosphorylated proteins were increased at 6 months of age, while a specific drop in IκBα levels was detected in 18-month-old Tg2576 mice, suggesting a transient activation of NFκB in the OB of Tg2576 mice. Taken together, our data provide a metabolic map of olfactory alterations in aged Tg2576 mice, reflecting the progressive effect of APP overproduction and β-amyloid (Aβ) accumulation on the OB homeostasis in aged stages.
Published: 27 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112261

Abstract:
Monocytes and macrophages express a repertoire of cell surface P2 receptors for adenosine 5′-triphosphate (ATP) a damage-associated molecular pattern molecule (DAMP), which are capable of raising cytoplasmic calcium when activated. This is achieved either through direct permeation (ionotropic P2X receptors) or by mobilizing intracellular calcium stores (metabotropic P2Y receptors). Here, a side-by-side comparison to investigate the contribution of P2X4 receptor activation in ATP-evoked calcium responses in model human monocytes and macrophages was performed. The expression of P2X1, P2X4, P2X5 and P2X7 was confirmed by qRT-PCR and immunocytochemistry in both model monocyte and macrophage. ATP evoked a concentration-dependent increase in intracellular calcium in both THP-1 monocyte and macrophages. The sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thasigargin (Tg) responses to the maximal ATP concentration (100 μM) in THP-1 monocytes, and responses in macrophage were significantly attenuated. Tg-resistant ATP-evoked calcium responses in the model macrophage were dependent on extracellular calcium, suggesting a requirement for calcium influx. Ivermectin (IVM) potentiated the magnitude of Tg-resistant component and slowed the decay of response in the model macrophage. The Tg-resistant component was attenuated by P2X4 antagonists 5-BDBD and PSB-12062 but not by the P2X1 antagonist Ro0437626 or the P2X7 antagonist A438079. shRNA-mediated P2X4 knockdown resulted in a significant reduction in Tg-resistant ATP-evoked calcium response as well as reduced sensitivities towards P2X4-specific pharmacological tools, IVM and PSB-12062. Inhibition of endocytosis with dynasore significantly reduced the magnitude of Tg-resistant component but substantially slowed decay response. Inhibition of calcium-dependent exocytosis with vacuolin-1 had no effect on the Tg-resistant component. These pharmacological data suggest that P2X4 receptor activation contributed significantly towards the ionotropic calcium response evoked by ATP of the model human macrophage.
Published: 27 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112257

Abstract:
The periosteum is a thin membrane that surrounds the outer surface of bones and participates in fracture healing. However, the molecular signals that trigger/initiate the periosteal reaction are not well established. We fractured the rat femoral bone at the diaphysis and fixed it with an intramedullary inserted wire, and the expression of regenerating gene (Reg) I, which encodes a tissue regeneration/growth factor, was analyzed. Neither bone/marrow nor muscle showed Reg I gene expression before or after the fracture. By contrast, the periosteum showed an elevated expression after the fracture, thereby confirming the localization of Reg I expression exclusively in the periosteum around the fractured areas. Expression of the Reg family increased after the fracture, followed by a decrease to basal levels by six weeks, when the fracture had almost healed. In vitro cultures of periosteal cells showed no Reg I expression, but the addition of IL-6 significantly induced Reg I gene expression. The addition of IL-6 also increased the cell number and reduced pro-apoptotic gene expression of Bim. The increased cell proliferation and reduction in Bim gene expression were abolished by transfection with Reg I siRNA, indicating that these IL-6-dependent effects require the Reg I gene expression. These results indicate the involvement of the IL-6/Reg pathway in the osteogenic response of the periosteum, which leads to fracture repair.
Published: 25 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112239

Abstract:
Acute pancreatitis refers to the sudden inflammation of the pancreas. It is associated with premature activation and release of digestive enzymes into the pancreatic interstitium and systemic circulation, resulting in pancreatic tissue autodigestion and multiple organ dysfunction, as well as with increased cytokine production, ultimately leading to deleterious local and systemic effects. Although mechanisms involved in pathogenesis of acute pancreatitis have not been completely elucidated, oxidative stress is regarded as a major risk factor. In human acute pancreatitis, lipid peroxide levels in pancreatic tissues increase. Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid (C22:6n-3), exerts anti-inflammatory and antioxidant effects on various cells. Previous studies have shown that DHA activates peroxisome proliferator-activated receptor-γ and induces catalase, which inhibits oxidative stress-mediated inflammatory signaling required for cytokine expression in experimental acute pancreatitis using cerulein. Cerulein, a cholecystokinin analog, induces intra-acinar activation of trypsinogen in the pancreas, which results in human acute pancreatitis-like symptoms. Therefore, DHA supplementation may be beneficial for preventing or inhibiting acute pancreatitis development. Since DHA reduces serum triglyceride levels, addition of DHA to lipid-lowering drugs like statins has been investigated to reduce hypertriglyceridemic acute pancreatitis. However, high DHA concentrations increase cytosolic Ca2+, which activates protein kinase C and may induce hyperlipidemic acute pancreatitis. In this review, effect of DHA on cerulein-induced and hypertriglyceridemic acute pancreatitis has been discussed. The relation of high concentration of DHA to hyperlipidemic acute pancreatitis has been included.
Published: 25 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112230

Abstract:
Neurological disorders such as stroke, Alzheimer’s and Parkinson’s diseases are associated with high morbidity and mortality, and few or no effective options are available for their treatment. These disorders share common pathological characteristics like the induction of oxidative stress, abnormal protein aggregation, perturbed Ca2+ homeostasis, excitotoxicity, inflammation and apoptosis. A large body of evidence supports the beneficial effects of the Mediterranean diet in preventing neurodegeneration. As the Mediterranean diet is characterized by a high consumption of extra-virgin olive oil it has been hypothesized that olive oil, and in particular its phenols, could be responsible for the beneficial effect of the Mediterranean diet. This review provides an updated vision of the beneficial properties of olive oil and olive oil phenols in preventing/counteracting both acute and chronic neurodegenerative diseases.
Published: 25 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112233

Abstract:
The radiation-induced damage to mitochondrial oxidative respiratory chain could lead to generating of superoxide anions (O2−) and secondary reactive oxygen species (ROS), which are the major resources of continuous ROS production after radiation. Scavenging radiation-induced ROS effectively can help mitochondria to maintain their physiological function and relief cells from oxidative stress. Dihydropyridines (DHPs) are biomimetic hydrogen sources that could protect cells against radiation damage. In this study, we designed and synthetized three novel mitochondrial-targeted dihydropyridines (Mito-DHPs) that utilize the mitochondrial membrane potential to enter the organelle and scavenge ROS. MitoTracker confirmed Mito-DHPs accumulation in mitochondria, and the DCFH-DA assay demonstrated effective ROS scavenging activity. In addition, the γ-H2AX and comet assay demonstrated the ability of Mito-DHPs to protect against both radiation and ROS-induced DNA strand breaks. Furthermore, Mito-DHP1 proved to be non-toxic and displayed significant radioprotection activity (p < 0.05) in vitro. Mito-DHPs are therefore promising antioxidants that could penetrate the membrane of mitochondria, scavenge excessive ROS, and protect cells against radiation-induced oxidative damage.
Published: 25 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112240

Abstract:
In this study, the tetrad stage of microspore development in a new cotton (Gossypium barbadense L.) cytoplasmic male sterility (CMS) line, H276A, was identified using paraffin sections at the abortion stage. To explore the molecular mechanism underlying CMS in cotton, a comparative transcriptome analysis between the CMS line H276A and its maintainer line H276B at the tetrad stage was conducted using an Illumina HiSeq 4000 platform. The comparison of H276A with H276B revealed a total of 64,675 genes, which consisted of 59,255 known and 5420 novel genes. An analysis of the two libraries with a given threshold yielded a total of 3603 differentially expressed genes (DEGs), which included 1363 up- and 2240 down-regulated genes. Gene Ontology (GO) annotation showed that 2171 DEGs were distributed into 38 categories, and a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 2683 DEGs were classified into 127 groups. Thirteen DEGs were randomly selected and detected by quantitative reverse-transcribed PCR (qRT-PCR), and the results indicated that the transcriptome sequencing results were reliable. The bioinformatic analysis results in conjunction with previously reported data revealed key DEGs that might be associated with the male sterility features of H276A. Our results provide a comprehensive foundation for understanding anther development and will accelerate the study of the molecular mechanisms of CMS in cotton.
Published: 25 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112231

Abstract:
Sulfated quercetin derivatives are important authentic standards for metabolic studies. Quercetin-3′-O-sulfate, quercetin-4′-O-sulfate, and quercetin-3-O-sulfate as well as quercetin-di-O-sulfate mixture (quercetin-7,3′-di-O-sulfate, quercetin-7,4′-di-O-sulfate, and quercetin-3′,4′-di-O-sulfate) were synthetized by arylsulfotransferase from Desulfitobacterium hafniense. Purified monosulfates and disulfates were fully characterized using MS and NMR and tested for their 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) and N,N-dimethyl-p-phenylenediamine (DMPD) radical scavenging, Folin-Ciocalteau reduction (FCR), ferric reducing antioxidant power (FRAP), and anti-lipoperoxidant activities in rat liver microsomes damaged by tert-butylhydroperoxide. Although, as expected, the sulfated metabolites were usually less active than quercetin, they remained still effective antiradical and reducing agents. Quercetin-3′-O-sulfate was more efficient than quercetin-4′-O-sulfate in DPPH and FCR assays. In contrast, quercetin-4′-O-sulfate was the best ferric reductant and lipoperoxidation inhibitor. The capacity to scavenge ABTS+• and DMPD was comparable for all substances, except for disulfates, which were the most efficient. Quantum calculations and molecular dynamics simulations on membrane models supported rationalization of free radical scavenging and lipid peroxidation inhibition. These results clearly showed that individual metabolites of food bioactives can markedly differ in their biological activity. Therefore, a systematic and thorough investigation of all bioavailable metabolites with respect to native compounds is needed when evaluating food health benefits.
Published: 25 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112232

Abstract:
The E3 ubiquitin ligase, von Hippel–Lindau (VHL), regulates protein expression by polyubiquitination. Although the protein VHL (pVHL) was reported to be involved in the heart function, the underlying mechanism is unclear. Here, we show that pVHL was upregulated in hearts from two types of genetically dilated cardiomyopathy (DCM) mice models. In comparison with the wild-type mouse, both DCM mice models showed a significant reduction in the expression of phospholamban (PLN), a potent inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPase, and enhanced interaction between pVHL and PLN. To clarify whether pVHL is involved in PLN degradation in failing hearts, we used carbonylcyanide m-chlorophenylhydrazone (CCCP), a mitochondrial membrane potential (MMP)-lowering reagent, to mimic the heart failure condition in PLN-expressing HEK293 cells and found that CCCP treatment resulted in PLN degradation and increased interaction between PLN and pVHL. However, these effects were reversed with the addition of N-acetyl-l-cysteine. Furthermore, the co-transfection of VHL and PLN in HEK293 cells decreased PLN expression under oxidative stress, whereas knockdown of VHL increased PLN expression both under normal and oxidative stress conditions. Together, we propose that oxidative stress upregulates pVHL expression to induce PLN degradation in failing hearts.
Published: 25 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112237

Abstract:
The abalone Haliotis discus hannai is an important aquaculture species that is grown for human consumption. However, little is known of the genetic mechanisms governing muscle growth in this species, particularly with respect to proteomics. The isobaric tag for relative and absolute quantitation (iTRAQ) method allows for sensitive and accurate protein quantification. Our study was the first to use iTRAQ-based quantitative proteomics to investigate muscle growth regulation in H. discus hannai. Among the 1904 proteins identified from six samples, 125 proteins were differentially expressed in large specimens of H. discus hannai as compared to small specimens. In the large specimens, 47 proteins were upregulated and 78 were downregulated. Many of the significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including these differentially expressed proteins, were closely related to muscle growth, including apoptosis, thyroid hormone signaling, regulation of the actin cytoskeleton, and viral myocarditis (p < 0.05). Our quantitative real-time polymerase chain reaction (qRT-PCR) analyses suggested that the alterations in expression levels observed in the differentially expressed proteins were consistent with the alterations observed in the encoding mRNAs, indicating the repeatability of our proteomic approach. Our findings contribute to the knowledge of the molecular mechanisms of muscle growth in H. discus hannai.
Published: 25 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112235

Abstract:
Lung cancer (LC) screening will be more efficient if it is applied to a well-defined high-risk population. Characteristics including metabolic byproducts may be taken into account to access LC risk more precisely. Breath examination provides a non-invasive method to monitor metabolic byproducts. However, the association between volatile organic compounds (VOCs) in exhaled breath and LC risk or LC risk factors is not studied. Exhaled breath samples from 122 healthy persons, who were given routine annual exam from December 2015 to December 2016, were analyzed using thermal desorption coupled with gas chromatography mass spectrometry (TD-GC-MS). Smoking characteristics, air quality, and other risk factors for lung cancer were collected. Univariate and multivariate analyses were used to evaluate the relationship between VOCs and LC risk factors. 7, 7, 11, and 27 VOCs were correlated with smoking status, smoking intensity, years of smoking, and depth of inhalation, respectively. Exhaled VOCs are related to smoking and might have a potential to evaluate LC risk more precisely. Both an assessment of temporal stability and testing in a prospective study are needed to establish the performance of VOCs such as 2,5-dimethylfuranm and 4-methyloctane as lung cancer risk biomarkers.
Published: 25 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112234

Abstract:
Background: Diabetes mellitus (DM) is a multifactorial disease orphan of a cure. Regenerative medicine has been proposed as novel strategy for DM therapy. Human fibroblast growth factor (FGF)-2b controls β-cell clusters via autocrine action, and human placental lactogen (hPL)-A increases functional β-cells. We hypothesized whether FGF-2b/hPL-A treatment induces β-cell differentiation from ductal/non-endocrine precursor(s) by modulating specific genes expression. Methods: Human pancreatic ductal-cells (PANC-1) and non-endocrine pancreatic cells were treated with FGF-2b plus hPL-A at 500 ng/mL. Cytofluorimetry and Immunofluorescence have been performed to detect expression of endocrine, ductal and acinar markers. Bromodeoxyuridine incorporation and annexin-V quantified cells proliferation and apoptosis. Insulin secretion was assessed by RIA kit, and electron microscopy analyzed islet-like clusters. Results: Increase in PANC-1 duct cells de-differentiation into islet-like aggregates was observed after FGF-2b/hPL-A treatment showing ultrastructure typical of islets-aggregates. These clusters, after stimulation with FGF-2b/hPL-A, had significant (p < 0.05) increase in insulin, C-peptide, pancreatic and duodenal homeobox 1 (PDX-1), Nkx2.2, Nkx6.1, somatostatin, glucagon, and glucose transporter 2 (Glut-2), compared with control cells. Markers of PANC-1 (Cytokeratin-19, MUC-1, CA19-9) were decreased (p < 0.05). These aggregates after treatment with FGF-2b/hPL-A significantly reduced levels of apoptosis. Conclusions: FGF-2b and hPL-A are promising candidates for regenerative therapy in DM by inducing de-differentiation of stem cells modulating pivotal endocrine genes.
Published: 25 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112215

Abstract:
The development of low-cost catalytic systems that mimic the activity of tyrosinase enzymes (Catechol oxidase) is of great promise for future biochemistry technologic demands. Herein, we report the synthesis of new biomolecules systems based on hydrazone derivatives containing a pyrazole moiety (L1–L6) with superior catecholase activity. Crystal structures of L1 and L2 biomolecules were determined by X-ray single crystal diffraction (XRD). Optimized geometrical parameters were calculated by density functional theory (DFT) at B3LYP/6–31G (d, p) level and were found to be in good agreement with single crystal XRD data. Copper (II) complexes of the compounds (L1–L6), generated in-situ, were investigated for their catalytic activities towards the oxidation reaction of catechol to ortho-quinone with the atmospheric dioxygen, in an attempt to model the activity of the copper containing enzyme tyrosinase. The studies showed that the activities depend on four parameters: the nature of the ligand, the nature of counter anion, the nature of solvent and the concentration of ligand. The Cu(II)-ligands, given here, present the highest catalytic activity (72.920 μmol·L−1·min−1) among the catalysts recently reported in the existing literature.
Published: 25 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112236

Abstract:
Bacterial cellulose (BC) is an excellent biomaterial with many medical applications. In this study, resorbable BC membranes were prepared for guided bone regeneration (GBR) using an irradiation technique for applications in the dental field. Electron beam irradiation (EI) increases biodegradation by severing the glucose bonds of BC. BC membranes irradiated at 100 kGy or 300 kGy were used to determine optimal electron beam doses. Electron beam irradiated BC membranes (EI-BCMs) were evaluated by scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, thermal gravimetric analysis (TGA), and using wet tensile strength measurements. In addition, in vitro cell studies were conducted in order to confirm the cytocompatibility of EI-BCMs. Cell viabilities of NIH3T3 cells on 100k and 300k EI-BCMs (100 kGy and 300 kGy irradiated BC membranes) were significantly greater than on NI-BCMs after 3 and 7 days (p < 0.05). Bone regeneration by EI-BCMs and their biodegradabilities were also evaluated using in vivo rat calvarial defect models for 4 and 8 weeks. Histometric results showed 100k EI-BCMs exhibited significantly larger new bone area (NBA; %) than 300k EI-BCMs at 8 weeks after implantation (p < 0.05). Mechanical, chemical, and biological analyses showed EI-BCMs effectively interacted with cells and promoted bone regeneration.
Published: 25 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112238

Abstract:
In the pathogenesis of psoriasis, systemic inflammation and oxidative stress play mutual roles interrelated with metabolic syndrome (MetS). This study aims to map the selected markers of inflammation (C-reactive protein (CRP)), oxidative damage to nucleic acids (DNA/RNA damage; 8-hydroxy-2′-deoxyguanosine, 8-hydroxyguanosine, and 8-hydroxyguanine), and the parameters of MetS (waist circumference, fasting glucose, triglycerides, high-density lipoprotein (HDL) cholesterol, diastolic and systolic blood pressure) in a group of 37 patients with psoriasis (62% of MetS) and in 43 healthy controls (42% of MetS). Levels of CRP, DNA/RNA damage, fasting glucose, and triglycerides were significantly elevated in patients. MetS in conjunction with psoriasis was associated with high levels of CRP, significantly higher than in control subjects without MetS. Patients with MetS exhibited further DNA/RNA damage, which was significantly higher in comparison with the control group. Our study supports the independent role of psoriasis and MetS in the increase of CRP and DNA/RNA damage. The psoriasis contributes to an increase in the levels of both effects more significantly than MetS. The psoriasis also diminished the relationship between CRP and oxidative damage to nucleic acids existent in controls.
Published: 26 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112243

Abstract:
Chronic high caloric intake (HCI) is a risk factor for multiple major human disorders, from diabetes to neurodegeneration. Mounting evidence suggests a significant contribution of circadian misalignment and sleep alterations to this phenomenon. An inverse temporal relationship between sleep, activity, food intake, and clock mechanisms in nocturnal and diurnal animals suggests that a search for effective therapeutic approaches can benefit from the use of diurnal animal models. Here, we show that, similar to normal aging, HCI leads to the reduction in daily amplitude of expression for core clock genes, a decline in sleep duration, an increase in scoliosis, and anxiety-like behavior. A remarkable decline in adult neurogenesis in 1-year old HCI animals, amounting to only 21% of that in age-matched Control, exceeds age-dependent decline observed in normal 3-year old zebrafish. This is associated with misalignment or reduced amplitude of daily patterns for principal cell cycle regulators, cyclins A and B, and p20, in brain tissue. Together, these data establish HCI in zebrafish as a model for metabolically induced premature aging of sleep, circadian functions, and adult neurogenesis, allowing for a high throughput approach to mechanistic studies and drug trials in a diurnal vertebrate.
Published: 26 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112244

Abstract:
Glucose-6-phosphate dehydrogenase (G6PD) is a key regulatory enzyme that plays a crucial role in the regulation of cellular energy and redox balance. Mutations in the gene encoding G6PD cause the most common enzymopathy that drives hereditary nonspherocytic hemolytic anemia. To gain insights into the effects of mutations in G6PD enzyme efficiency, we have investigated the biochemical, kinetic, and structural changes of three clinical G6PD variants, the single mutations G6PD A+ (Asn126AspD) and G6PD Nefza (Leu323Pro), and the double mutant G6PD A− (Asn126Asp + Leu323Pro). The mutants showed lower residual activity (≤50% of WT G6PD) and displayed important kinetic changes. Although all Class III mutants were located in different regions of the three-dimensional structure of the enzyme and were not close to the active site, these mutants had a deleterious effect over catalytic activity and structural stability. The results indicated that the G6PD Nefza mutation was mainly responsible for the functional and structural alterations observed in the double mutant G6PD A−. Moreover, our study suggests that the G6PD Nefza and G6PD A− mutations affect enzyme functions in a similar fashion to those reported for Class I mutations.
Published: 26 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112249

Abstract:
Ras proteins, as small GTPases, mediate cell proliferation, survival and differentiation. Ras mutations have been associated with a broad spectrum of human cancers and thus targeting Ras represents a potential way forward for cancer therapy. A recently reported monobody NS1 allosterically disrupts the Ras-mediated signaling pathway, but its efficacy is reduced by R135K mutation in H-Ras. However, the detailed mechanism is unresolved. Here, using molecular dynamics (MD) simulations and dynamic network analysis, we explored the molecular mechanism for the unbinding of NS1 to H-Ras and shed light on the underlying allosteric network in H-Ras. MD simulations revealed that the overall structures of the two complexes did not change significantly, but the H-Ras–NS1 interface underwent significant conformational alteration in the mutant Binding free energy analysis showed that NS1 binding was unfavored after R135K mutation, which resulted in the unfavorable binding of NS1. Furthermore, the critical residues on H-Ras responsible for the loss of binding of NS1 were identified. Importantly, the allosteric networks for these important residues were revealed, which yielded a novel insight into the allosteric regulatory mechanism of H-Ras.
Published: 26 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112245

Abstract:
The rapid development of genome modification technology has provided many great benefits in diverse areas of research and industry. Genome modification technologies have also been actively used in a variety of research areas and fields of industry in avian species. Transgenic technologies such as lentiviral systems and piggyBac transposition have been used to produce transgenic birds for diverse purposes. In recent years, newly developed programmable genome editing tools such as transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) have also been successfully adopted in avian systems with primordial germ cell (PGC)-mediated genome modification. These genome modification technologies are expected to be applied to practical uses beyond system development itself. The technologies could be used to enhance economic traits in poultry such as acquiring a disease resistance or producing functional proteins in eggs. Furthermore, novel avian models of human diseases or embryonic development could also be established for research purposes. In this review, we discuss diverse genome modification technologies used in avian species, and future applications of avian biotechnology.
Published: 26 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112246

Abstract:
We performed targeted re-sequencing to identify the genetic etiology of early-onset postlingual deafness and encountered a frequent TMPRSS3 allele harboring two variants in a cis configuration. We aimed to evaluate the pathogenicity of the allele. Among 88 cochlear implantees with autosomal recessive non-syndromic hearing loss, subjects with GJB2 and SLC26A4 mutations were excluded. Thirty-one probands manifesting early-onset postlingual deafness were sorted. Through targeted re-sequencing, we detected two families with a TMPRSS3 mutant allele containing p.V116M and p.V291L in a cis configuration, p.[p.V116M; p.V291L]. A minor allele frequency was calculated and proteolytic activity was measured. A p.[p.V116M; p.V291L] allele demonstrated a significantly higher frequency compared to normal controls and merited attention due to its high frequency (4.84%, 3/62). The first family showed a novel deleterious splice site variant—c.783-1G>A—in a trans allele, while the other showed homozygosity. The progression to deafness was noted within the first decade, suggesting DFNB10. The proteolytic activity was significantly reduced, confirming the severe pathogenicity. This frequent mutant allele significantly contributes to early-onset postlingual deafness in Koreans. For clinical implication and proper auditory rehabilitation, it is important to pay attention to this allele with a severe pathogenic potential.
Published: 26 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18112247

Abstract:
Severe trauma constitutes a major cause of death and disability, especially in younger patients. The cerebral autoregulatory capacity only protects the brain to a certain extent in states of hypovolemia; thereafter, neurological deficits and apoptosis occurs. We therefore set out to investigate neuroprotective strategies during haemorrhagic shock. This review was performed in accordance to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Before the start of the search, a review protocol was entered into the PROSPERO database. A systematic literature search of Pubmed, Web of Science and CENTRAL was performed in August 2017. Results were screened and evaluated by two researchers based on a previously prepared inclusion protocol. Risk of bias was determined by use of SYRCLE’s risk of bias tool. The retrieved results were qualitatively analysed. Of 9093 results, 119 were assessed in full-text form, 16 of them ultimately adhered to the inclusion criteria and were qualitatively analyzed. We identified three subsets of results: (1) hypothermia; (2) fluid therapy and/or vasopressors; and (3) other neuroprotective strategies (piracetam, NHE1-inhibition, aprotinin, human mesenchymal stem cells, remote ischemic preconditioning and sevoflurane). Overall, risk of bias according to SYRCLE’s tool was medium; generally, animal experimental models require more rigorous adherence to the reporting of bias-free study design (randomization, etc.). While the individual study results are promising, the retrieved neuroprotective strategies have to be evaluated within the current scientific context—by doing so, it becomes clear that specific promising neuroprotective strategies during states of haemorrhagic shock remain sparse. This important topic therefore requires more in-depth research.
Published: 21 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18102210

Abstract:
Maintenance of mesenchymal stem cells (MSCs) requires a tissue-specific microenvironment (i.e., niche), which is poorly represented by the typical plastic substrate used for two-dimensional growth of MSCs in a tissue culture flask. The objective of this study was to address the potential use of collagen-based medical devices (HEMOCOLLAGENE®, Saint-Maur-des-Fossés, France) as mimetic niche for MSCs with the ability to preserve human MSC stemness in vitro. With a chemical composition similar to type I collagen, HEMOCOLLAGENE® foam presented a porous and interconnected structure (>90%) and a relative low elastic modulus of around 60 kPa. Biological studies revealed an apparently inert microenvironment of HEMOCOLLAGENE® foam, where 80% of cultured human MSCs remained viable, adopted a flattened morphology, and maintained their undifferentiated state with basal secretory activity. Thus, three-dimensional HEMOCOLLAGENE® foams present an in vitro model that mimics the MSC niche with the capacity to support viable and quiescent MSCs within a low stiffness collagen I scaffold simulating Wharton’s jelly. These results suggest that haemostatic foam may be a useful and versatile carrier for MSC transplantation for regenerative medicine applications.
Published: 21 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18102206

Abstract:
Venous leg ulcer (VLU) is a huge healthcare problem with poorly understood pathophysiology. Transforming growth factor-β (TGF-β) and endoglin (Eng), are inflammatory and wound healing mediators. Eng, co-receptor for TGF-β type-II receptors, may be cleaved forming soluble Eng (sEng), antagonizing TGF-β signaling, a crucial process in vascular pathologies. We evaluated the accumulation in wound fluid (WF) of TGF-β isoforms and sEng in healing stages, showing the effects of sulodexide treatments, a glycosaminoglycan with clinical efficacy in VLU healing. Patients with inflammatory (Infl) and granulating (Gran) VLU were recruited. WFs and THP-1 monocytes exposed to Infl and Gran WF (treated/untreated with sulodexide) were analyzed for TGF-β isoforms and sEng by multiplex immunoassay. In both Infl and Gran WF, TGF-β1 and β2 were similar; TGF-β3 was significantly increased in Infl compared to Gran WFs (p = 0.033). sEng was significantly elevated in Gran compared to Infl WFs (p = 0.002). In THP-1 monocytes there was a significant increase in sEng after co-treatment of WF and sulodexide. The increase in TGF-β3 found in Infl WF highlights its negative effect on wound healing, while the increased levels of sEng in Gran WF affects the leukocyte adhesion/transmigration through the endothelium, reducing the inflammatory response and favoring the wound healing. Glycosaminoglycan sulodexide potentiates the effects of sEng release from monocyte, representing an important therapeutic option for wound healing.
Published: 21 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18102198

Abstract:
Matricellular proteins differ from other classical extracellular matrix proteins; for instance, they are transiently expressed as soluble proteins rather than being constitutively expressed in pathological conditions, such as acute viral infections. Accumulating studies have revealed that matricellular proteins, including osteopontin and tenascin-C, both of which interact with integrin heterodimers, are involved in inflammatory diseases, autoimmune disorders, and cancers. The concentrations of these matricellular proteins are elevated in the plasma of patients with certain types of cancers, indicating that they play important roles in oncogenesis. Chronic viral infections are associated with certain cancers, which are distinct from non-viral cancers. Viral oncogenes play critical roles in the development and progression of such cancers. It is vital to investigate the mechanisms of tumorigenesis and, particularly, the mechanism by which viral proteins induce tumor progression. Viral proteins have been shown to influence not only the viral-infected cancer cells, but also the stromal cells and matricellular proteins that constitute the extracellular matrix that surrounds tumor tissues. In this review, we summarize the recent progress on the involvement of matricellular proteins in oncogenic virus-induced cancers to elucidate the mechanism of oncogenesis and consider the possible role of matricellular proteins as therapeutic targets in virus-induced cancers.
Published: 21 October 2017
by MDPI
International Journal of Molecular Sciences, Volume 18; https://doi.org/10.3390/ijms18102208

Abstract:
A recent genetic analysis of infertile globozoospermic patients identified causative mutations in three genes: a protein interacting with C kinase 1 (PICK1), dpy 19-like 2 (DPY19L2), and spermatogenesis associated 16 (SPATA16). Although mouse models have clarified the physiological functions of Pick1 and Dpy19l2 during spermatogenesis, Spata16 remains to be determined. Globozoospermic patients carried a homozygous point mutation in SPATA16 at 848G→A/R283Q. We generated CRISPR/Cas9-mediated mutant mice with the same amino acid substitution in the fourth exon of Spata16 to analyze the mutation site at R284Q, which corresponded with R283Q of mutated human SPATA16. We found that the point mutation in Spata16 was not essential for male fertility; however, deletion of the fourth exon of Spata16 resulted in infertile male mice due to spermiogenic arrest but not globozoospermia. This study demonstrates that Spata16 is indispensable for male fertility in mice, as well as in humans, as revealed by CRISPR/Cas9-mediated mouse models.
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