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, , , Jana Kenney, Eduard Kelemen,
Published: 9 October 2017
Abstract:
We used the psychotomimetic phencyclidine (PCP) to investigate the relationships between cognitive behavior, coordinated neural network function and information processing within the hippocampus place cell system. We report in rats that PCP (5mg/kg i.p.) impairs a well-learned hippocampus-dependent place avoidance behavior in rats that requires cognitive control, even when PCP is injected directly into dorsal hippocampus. PCP increases 60-100 Hz medium gamma oscillations in hippocampus CA1 and these increases correlate with the cognitive impairment caused by systemic PCP administration. PCP discoordinates theta-modulated medium and slow gamma oscillations in CA1 local field potentials (LFP) such that medium gamma oscillations become more theta-organized than slow gamma oscillations. CA1 place cell firing fields are preserved under PCP but the drug discoordinates the sub-second temporal organization of discharge amongst place cells. This discoordination causes place cell ensemble representations of a familiar space to cease resembling pre-PCP representations, despite preserved place fields. These findings point to the cognitive impairments caused by PCP arising from neural discoordination. PCP disrupts the timing of discharge with respect to the sub-second timescales of theta and gamma oscillations in the LFP. Because these oscillations arise from local inhibitory synaptic activity, these findings point to excitation-inhibition discoordination as the root of PCP-induced cognitive impairment.SIGNIFICANCE STATEMENT: Hippocampal neural discharge is temporally coordinated on timescales of theta and gamma oscillations in the local field potential, and the discharge of a subset of pyramidal neurons called “place cells” is spatially organized such that discharge is restricted to locations called a cell’s “place field.” Because this temporal coordination and spatial discharge organization is thought to represent spatial knowledge, we used the psychotomimetic phencyclidine (PCP) to disrupt cognitive behavior and assess the importance of neural coordination and place fields for spatial cognition. PCP impaired the judicious use of spatial information and discoordinated hippocampal discharge, without disrupting firing fields. These findings dissociate place fields from spatial cognitive behavior and suggest that hippocampus discharge coordination is crucial to spatial cognition.
Published: 9 October 2017
Abstract:
A major challenge in cancer research is to determine the biological and clinical significance of somatic mutations in non-coding regions. This has been studied in terms of recurrence, functional impact, and association to individual regulatory sites, but the combinatorial contribution of mutations to common RNA regulatory motifs has not been explored. We developed a new method, MIRA, to perform the first comprehensive study of significantly mutated regions (SMRs) affecting binding sites for RNA-binding proteins (RBPs) in cancer. Extracting signals related to RNA-related selection processes and using RNA sequencing data from the same samples we identified alterations in RNA expression and splicing linked to mutations on RBP binding sites. We found SRSF10 and MBNL1 motifs in introns, HNRPLL motifs at 5’ UTRs, as well as 5’ and 3’ splice-site motifs, among others, with specific mutational patterns that disrupt the motif and impact RNA processing. MIRA facilitates the integrative analysis of multiple genome sites that operate collectively through common RBPs and can aid in the interpretation of non-coding variants in cancer. MIRA is available athttps://github.com/comprna/mira.
, Kazuya Ohara, Kentaro Ito, Nobuo Kokubun, Takuma Kitanishi, Daisuke Takaichi, , , , , et al.
Published: 9 October 2017
Abstract:
Animal behavior is the final and integrated output of the brain activity. Thus, recording and analyzing behavior is critical to understand the underlying brain function. While recording animal behavior has become easier than ever with the development of compact and inexpensive devices, detailed behavioral data analysis requires sufficient previous knowledge and/or high content data such as video images of animal postures, which makes it difficult for most of the animal behavioral data to be efficiently analyzed to understand brain function. Here, we report a versatile method using a hybrid supervised/unsupervised machine learning approach to efficiently estimate behavioral states and to extract important behavioral features only from low-content animal trajectory data. As proof of principle experiments, we analyzed trajectory data of worms, fruit flies, rats, and bats in the laboratories, and penguins and flying seabirds in the wild, which were recorded with various methods and span a wide range of spatiotemporal scales—from mm to 1000 km in space and from sub-seconds to days in time. We estimated several states during behavior and comprehensively extracted characteristic features from a behavioral state and/or a specific experimental condition. Physiological and genetic experiments in worms revealed that the extracted behavioral features reflected specific neural or gene activities. Thus, our method provides a versatile and unbiased way to extract behavioral features from simple trajectory data to understand brain function.
Published: 9 October 2017
Abstract:
The effectiveness of most cancer targeted therapies is short lived since tumors evolve and develop resistance. Combinations of drugs offer the potential to overcome resistance, however the number of possible combinations is vast necessitating data-driven approaches to find optimal treatments tailored to a patient’s tumor. AstraZeneca carried out 11,576 experiments on 910 drug combinations across 85 cancer cell lines, recapitulating in vivo response profiles. These data, the largest openly available screen, were hosted by DREAM alongside deep molecular characterization from the Sanger Institute for a Challenge to computationally predict synergistic drug pairs and associated biomarkers. 160 teams participated to provide the most comprehensive methodological development and subsequent benchmarking to date. Winning methods incorporated prior knowledge of putative drug target interactions. For >60% of drug combinations synergy was reproducibly predicted with an accuracy matching biological replicate experiments, however 20% of drug combinations were poorly predicted by all methods. Genomic rationale for synergy predictions were identified, including antagonism unique to combined PIK3CB/D inhibition with the ADAM17 inhibitor where synergy is seen with other PI3K pathway inhibitors. All data, methods and code are freely available as a resource to the community.
Jaspreet S. Khurana, Derek M. Clay, , ,
Published: 27 October 2017
RNA, Volume 24, pp 18-29; https://doi.org/10.1261/rna.061333.117

Abstract:
Dicer-dependent small noncoding RNAs play important roles in gene regulation in a wide variety of organisms. Endogenous small interfering RNAs (siRNAs) are part of an ancient pathway of transposon control in plants and animals. The ciliate, Oxytricha trifallax, has approximately 16,000 gene-sized chromosomes in its somatic nucleus. Long noncoding RNAs establish high ploidy levels at the onset of sexual development, but the factors that regulate chromosome copy numbers during cell division and growth have been a mystery. We report a novel function of a class of Dicer (Dcl-1)- and RNA-dependent RNA polymerase (RdRP)-dependent endogenous small RNAs in regulating chromosome copy number and gene dosage in O. trifallax. Asexually growing populations express an abundant class of 21-nt sRNAs that map to both coding and noncoding regions of most chromosomes. These sRNAs are bound to chromatin and their levels surprisingly do not correlate with mRNA levels. Instead, the levels of these small RNAs correlate with genomic DNA copy number. Reduced sRNA levels in dcl-1 or rdrp mutants lead to concomitant reduction in chromosome copy number. Furthermore, these cells show no signs of transposon activation, but instead display irregular nuclear architecture and signs of replication stress. In conclusion, Oxytricha Dcl-1 and RdRP-dependent small RNAs that derive from the somatic nucleus contribute to the maintenance of gene dosage, possibly via a role in DNA replication, offering a novel role for these small RNAs in eukaryotes.
Louis Philip Benoit Bouvrette, Neal A.L. Cody, Julie Bergalet, Fabio Alexis Lefebvre, Cédric Diot, Xiaofeng Wang, Mathieu Blanchette,
Published: 27 October 2017
RNA, Volume 24, pp 98-113; https://doi.org/10.1261/rna.063172.117

Abstract:
Cells are highly asymmetrical, a feature that relies on the sorting of molecular constituents, including proteins, lipids, and nucleic acids, to distinct subcellular locales. The localization of RNA molecules is an important layer of gene regulation required to modulate localized cellular activities, although its global prevalence remains unclear. We combine biochemical cell fractionation with RNA-sequencing (CeFra-seq) analysis to assess the prevalence and conservation of RNA asymmetric distribution on a transcriptome-wide scale in Drosophila and human cells. This approach reveals that the majority (∼80%) of cellular RNA species are asymmetrically distributed, whether considering coding or noncoding transcript populations, in patterns that are broadly conserved evolutionarily. Notably, a large number of Drosophila and human long noncoding RNAs and circular RNAs display enriched levels within specific cytoplasmic compartments, suggesting that these RNAs fulfill extra-nuclear functions. Moreover, fraction-specific mRNA populations exhibit distinctive sequence characteristics. Comparative analysis of mRNA fractionation profiles with that of their encoded proteins reveals a general lack of correlation in subcellular distribution, marked by strong cases of asymmetry. However, coincident distribution profiles are observed for mRNA/protein pairs related to a variety of functional protein modules, suggesting complex regulatory inputs of RNA localization to cellular organization.
Published: 27 October 2017
Genome Research, Volume 27, pp 1988-2000; https://doi.org/10.1101/gr.219956.116

Abstract:
Mutations provide the raw material of evolution, and thus our ability to study evolution depends fundamentally on having precise measurements of mutational rates and patterns. We generate a data set for this purpose using (1) de novo mutations from mutation accumulation experiments and (2) extremely rare polymorphisms from natural populations. The first, mutation accumulation (MA) lines are the product of maintaining flies in tiny populations for many generations, therefore rendering natural selection ineffective and allowing new mutations to accrue in the genome. The second, rare genetic variation from natural populations allows the study of mutation because extremely rare polymorphisms are relatively unaffected by the filter of natural selection. We use both methods in Drosophila melanogaster, first generating our own novel data set of sequenced MA lines and performing a meta-analysis of all published MA mutations (∼2000 events) and then identifying a high quality set of ∼70,000 extremely rare (≤0.1%) polymorphisms that are fully validated with resequencing. We use these data sets to precisely measure mutational rates and patterns. Highlights of our results include: a high rate of multinucleotide mutation events at both short (∼5 bp) and long (∼1 kb) genomic distances, showing that mutation drives GC content lower in already GC-poor regions, and using our precise context-dependent mutation rates to predict long-term evolutionary patterns at synonymous sites. We also show that de novo mutations from independent MA experiments display similar patterns of single nucleotide mutation and well match the patterns of mutation found in natural populations.
R.S. Clymo
Published: 25 March 2014
Abstract:
Most bioscientists need to report mean values, yet many have little idea of how many digits are significant, and at what point further digits are mere random junk. Thus a recent report that the mean of 17 values was 3.863 with a standard error of the mean (SEM) of 2.162 revealed only that none of the seven authors understood the limitations of their work. The simple rule derived here by experiment for restricting a mean value to its significant digits (sig-digs) is this: the last sig‑dig in the mean value is at the same decimal decade as the first sig-dig (the first non-zero) in the SEM. An extended rule for the mean, and a different rule for the SEM itself are also derived. For the example above the reported values should be a mean of 4 with SEM 2.2. Routine application of these simple rules will often show that a result is not as compelling as one had hoped.
Published: 6 May 2014
Abstract:
In developing embryos, gene regulatory networks canalize cells towards discrete terminal fates. We studied the behavior of the anterior-posterior segmentation network in Drosophila melanogaster embryos depleted of a key maternal input, bicoid (bcd), by building a cellular- resolution gene expression atlas containing measurements of 12 core patterning genes over 6 time points in early development. With this atlas, we determine the precise perturbation each cell experiences, relative to wild type, and observe how these cells assume cell fates in the perturbed embryo. The first zygotic layer of the network, consisting of the gap and terminal genes, is highly robust to perturbation: all combinations of transcription factor expression found in bcd depleted embryos were also found in wild type embryos, suggesting that no new cell fates were created even at this very early stage. All of the gap gene expression patterns in the trunk expand by different amounts, a feature that we were unable to explain using two simple models of the effect of bcd depletion. In the second layer of the network, depletion of bcd led to an excess of cells expressing both even skipped and fushi tarazu early in the blastoderm stage, but by gastrulation this overlap resolved into mutually exclusive stripes. Thus, following depletion of bcd, individual cells rapidly canalize towards normal cell fates in both layers of this gene regulatory network. Our gene expression atlas provides a high resolution picture of a classic perturbation and will enable further modeling of canalization in this transcriptional network.
Sergio Arredondo-Alonso, , Rob J. Willems,
Published: 14 November 2016
Abstract:
Plasmids are autonomous extra-chromosomal elements in bacterial cells that can carry genes that are important for bacterial survival. To benchmark algorithms for automated plasmid sequence reconstruction from short read sequencing data, we selected 42 publicly available complete bacterial genome sequences which were assembled by a combination of long- and short-read data. The selected bacterial genome sequence projects span 12 genera, containing 148 plasmids. We predicted plasmids from short-read data with four different programs (PlasmidSPAdes, Recycler, cBar and PlasmidFinder) and compared the outcome to the reference sequences.PlasmidSPAdes reconstructs plasmids based on coverage differences in the assembly graph. It reconstructed most of the reference plasmids (recall = 0.82) but approximately a quarter of the predicted plasmid contigs were false positives (precision = 0.76). PlasmidSPAdes merged 83 % of the predictions from genomes with multiple plasmids in a single bin. Recycler searches the assembly graph for sub-graphs corresponding to circular sequences and correctly predicted small plasmids but failed with long plasmids (recall = 0.12, precision = 0.30). cBar, which applies pentamer frequency composition analysis to detect plasmid-derived contigs, showed an overall recall and precision of 0.78 and 0.64. However, cBar only categorizes contigs as plasmid-derived and does not bin the different plasmids correctly within a bacterial isolate. PlasmidFinder, which searches for matches in a replicon database, had the highest precision (1.0) but was restricted by the contents of its database and the contig length obtained from de novo assembly (recall = 0.36).Surprisingly, PlasmidSPAdes and Recycler detected single isolated components corresponding to putative novel small plasmids (50 kbp) containing repeated sequences remains challenging and limits the high-throughput analysis of WGS data.Author Summary: Short read sequencing of the DNA of bacteria is often used to understand characteristics such as antibiotic resistance. However the assembly of short read sequencing data with the goal of reconstructing a complete genome is often fragmented and leaves gaps. Therefore independently replicating DNA fragments called plasmids cannot easily be identified from an assembly. Lately a number of programs have been developed to enable the automated prediction of the sequences of plasmids. Here we tested these programs by comparing their outcomes with complete genome sequences. None of the tested programs were able to fully and unambiguously predict distinct plasmid sequences. All programs performed best with the prediction of plasmids smaller than 50 kbp. Larger plasmids were only correctly predicted if they were present as a single contig in the assembly. While predictions by PlasmidSPAdes and cBar contained most of the plasmids, they were merged with or indistinguishable from other plasmids and sometimes chromosome sequences. PlasmidFinder missed most plasmids but all its predictions were correct. Without manual steps or long-read sequencing information, plasmid reconstruction from short read sequencing data remains challenging.
, , Sebastian Steinhauser, Philipp Rentzsch, Stephen Krämer, Carolin Andresen, , , ,
Published: 8 October 2017
Abstract:
Metazoans are crucially dependent on multiple layers of gene regulatory mechanisms which allow them to control gene expression across developmental stages, tissues and cell types. Multiple recent research consortia have aimed to generate comprehensive datasets to profile the activity of these cell type- and condition-specific regulatory landscapes across many different cell lines and primary cells. However, extraction of genes or regulatory elements specific to certain entities from these datasets remains challenging. We here propose a novel method based on non-negative matrix factorization for disentangling and associating huge multi-assay datasets including chromatin accessibility and gene expression data. Taking advantage of implementations of NMF algorithms in the GPU CUDA environment full datasets composed of tens of thousands of genes as well as hundreds of samples can be processed without the need for prior feature selection to reduce the input size. Applying this framework to multiple layers of genomic data derived from human blood cells we unravel mechanisms of regulation of cell type-specific expression in T-cells and monocytes.
Published: 7 October 2017
Abstract:
Evolution of cis-properties (such as enhancers) often plays an important role in the production of diverse morphology. However, a mechanistic understanding is often limited by the absence of methods to study enhancers in species outside of established model systems. Here, we sought to establish methods to identify and test enhancer activity in the red flour beetle, Tribolium castaneum. To identify possible enhancer regions, we first obtained genome-wide chromatin profiles from various tissues and stages of Tribolium via FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements)-sequencing. Comparison of these profiles revealed a distinct set of open chromatin regions in each tissue and stage. Second, we established the first reporter assay system that works in both Drosophila and Tribolium, using nubbin in the wing and hunchback in the embryo as case studies. Together, these advances will be useful to study the evolution of cis-language and morphological diversity in Tribolium and other insects.
William M. Fricke,
Genes & Development, Volume 17, pp 1768-1778; https://doi.org/10.1101/gad.1105203

Abstract:
The RecQ DNA helicases human BLM and yeast Sgs1 interact with DNA topoisomerase III and are thought to act on stalled replication forks to maintain genome stability. To gain insight into this mechanism, we previously identifiedSLX1andSLX4as genes that are required for viability and for completion of rDNA replication in the absence ofSGS1–TOP3. Here we show thatSLX1andSLX4encode a heteromeric structure-specific endonuclease. The Slx1–Slx4 nuclease is active on branched DNA substrates, particularly simple-Y, 5′-flap, or replication forkstructures. It cleaves the strand bearing the 5′ nonhomologous arm at the branch junction and generates ligatable nicked products from 5′-flap or replication fork substrates. Slx1 is the founding member of a family of proteins with a predicted URI nuclease domain and PHD-type zinc finger. This subunit displays weakstructure-specific endonuclease activity on its own, is stimulated 500-fold by Slx4, and requires the PHD finger for activity in vitro and in vivo. Both subunits are required in vivo for resistance to DNA damage by methylmethane sulfonate (MMS). We propose that Sgs1–Top3 acts at the termination of rDNA replication to decatenate stalled forks, and, in its absence, Slx1–Slx4 cleaves these stalled forks.
Published: 6 October 2017
Abstract:
Dimorphic fungal pathogens cause a significant human disease burden and unlike most fungal pathogens affect immunocompetent hosts. To examine the origin of virulence of these fungal pathogens, we compared genomes of classic systemic, opportunistic, and non-pathogenic species, includingEmmonsiaand two basal branching, non-pathogenic species in the Ajellomycetaceae,Helicocarpus griseusandPolytolypa hystricis. We found that gene families related to plant degradation, secondary metabolites synthesis, and amino acid and lipid metabolism are retained inH. griseusandP. hystricis. While genes involved in the virulence of dimorphic pathogenic fungi are conserved in saprophytes, changes in the copy number of proteases, kinases and transcription factors in systemic dimorphic relative to non-dimorphic species may have aided the evolution of specialized gene regulatory programs to rapidly adapt to higher temperatures and new nutritional environments. Notably, both of the basal branching, non-pathogenic species appear homothallic, with both mating type locus idiomorphs fused at a single locus, whereas all related pathogenic species are heterothallic. These differences revealed that independent changes in nutrient acquisition capacity have occurred in the Onygenaceae and Ajellomycetaceae, and underlie how the dimorphic pathogens have adapted to the human host and decreased their capacity for growth in environmental niches.
Published: 6 October 2017
Abstract:
Single cell RNA sequencing (scRNA-seq) is a powerful technique to analyze the transcriptomic heterogeneities in single cell level. It is an important step for studying cell sub-populations and lineages based on scRNA-seq data by finding an effective low-dimensional representation and visualization of the original data. The scRNA-seq data are much noiser than traditional bulk RNA-Seq: in the single cell level, the transcriptional fluctuations are much larger than the average of a cell population and the low amount of RNA transcripts will increase the rate of technical dropout events. In this study, we proposed VASC (deep Variational Autoencoder for scRNA-seq data), a deep multi-layer generative model, for the unsupervised dimension reduction and visualization of scRNA-seq data. It can explicitly model the dropout events and find the nonlinear hierarchical feature representations of the original data. Tested on twenty datasets, VASC shows superior performances in most cases and broader dataset compatibility compared with four state-of-the-art dimension reduction methods. Then, for a case study of pre-implantation embryos, VASC successfully re-establishes the cell dynamics and identifies several candidate marker genes associated with the early embryo development.
Published: 6 October 2017
Abstract:
The oncogeneMYBL2(encoding B-Myb) is a poor prognostic biomarker in many cancers. B-Myb interacts with the MuvB core of five proteins (LIN9, LIN37, LIN52, LIN53/RBBP4, and LIN54) to form the MMB (Myb-MuvB) complex and promotes expression of late cell cycle genes necessary for progression through mitosis. BothMYBL2amplification and over-expression are associated with deregulation of the cell cycle and increased cell proliferation. Alternatively, by interacting with E2F4-DP1 and p130 or p107, the MuvB core becomes part of the DREAM complex (DP, RB-like, E2F, and MuvB). The DREAM complex opposes MMB by globally repressing cell cycle genes in G0/G1, maintaining the cell in a quiescent state. However, the specific mechanism by which B-Myb alters the cell cycle is not well understood. Our analysis of The Cancer Genome Atlas data revealed significant upregulation of DREAM and MMB target genes in breast and ovarian cancer withMYBL2gain. Given that most of the DREAM target genes are not directly regulated by B-Myb, we investigated the effects of B-Myb on DREAM formation. We found that depletion of B-Myb results in increased DREAM formation in human cancer cells, while its overexpression inhibits DREAM formation in the non-transformed cells. Since the MuvB core subunit LIN52 is essential for assembly of both the DREAM and MMB complexes, we tested whether B-Myb disrupts DREAM by sequestering LIN52. Overexpression of LIN52 did not increase either DREAM or MMB formation, but instead increased the turnover rate of the endogenous LIN52 protein. Interestingly, co-expression of B-Myb increased the expression of both endogenous and overexpressed LIN52 while knockdown of B-Myb had an opposite effect. We found that regulation of LIN52 occurs at the protein level, and that activity of DYRK1A kinase, the enzyme that triggers DREAM complex formation by phosphorylating LIN52, is required for this regulation. These findings are the first to implicate B-Myb in the disassembly of the DREAM complex and offer insight into the underlying mechanisms of poor prognostic value ofMYBL2amplification in cancer. We conclude that B-Myb mediates its oncogenic effects not only by increasing mitotic gene expression by the MMB complex, but also by broad disruption of cell cycle gene regulatory programs through compromised DREAM formation.
Published: 5 October 2017
Abstract:
Although it is widely recognised as involving two stages (poor placentation followed by oxidative stress/inflammation), the precise originating causes of pre-eclampsia (PE) remain elusive. We have previously brought together some of the considerable evidence that a (dormant) microbial component is commonly a significant part of its aetiology. However, apart from recognising, consistent with this view, that the many inflammatory markers of PE are also increased in infection, we had little to say about immunity, whether innate or adaptive. In addition, we focussed on the gut, oral and female urinary tract microbiomes as the main sources of the infection. We here marshall further evidence for an infectious component in PE, focussing on the immunological tolerance characteristic of pregnancy, and the well-established fact that increased exposure to the father’s semen assists this immunological tolerance. As well as these benefits, however, semen is not sterile, microbial tolerance mechanisms may exist, and we also review the evidence that semen may be responsible for inoculating the developing conceptus with microbes, not all of which are benign. It is suggested that when they are not, this may be a significant cause of preeclampsia. A variety of epidemiological and other evidence is entirely consistent with this, not least correlations between semen infection, infertility and PE. Our view also leads to a series of other, testable predictions. Overall, we argue for a significant paternal role in the development of PE through microbial infection of the mother via insemination.“In one of the last articles which he wrote, the late Professor F J Browne (1958) expressed the opinion that all the essential facts about pregnancy toxaemia are now available and that all that is required to solve the problem is to fit them together in the right order, like the pieces of a jigsaw puzzle” [1] “It appears astonishing how little attention has been given in reproductive medicine to the maternal immune system over the last few decades.” [2]
Published: 5 October 2017
Abstract:
Mentally imagining another’s perspective is a high-level social process, reliant on manipulating internal representations of the self in an embodied manner. Recently Wang et al., (1) showed that theta-band (3-7Hz) brain oscillations within the right temporo-parietal junction (rTPJ) and brain regions coding for motor/body schema contribute to the process of perspective-taking. Using a task requiring participants to engage in embodied perspective-taking, we set out to unravel the extended functional brain network and its connections in detail. We found that increasing the angle of disparity between self and other perspective was accompanied by longer reaction times and increases in theta power within rTPJ, right lateral pre-frontal cortex (PFC) and right anterior cingulate cortex (ACC). Using nonparametric Granger-causality, we showed that during later stages of perspective-taking, the lateral PFC and ACC exert top-down influences over rTPJ, indicative of executive control processes required for managing conflicts between self and other perspectives. Finally, we quantified patterns of whole-brain phase coupling (imaginary coherence) in relation to rTPJ during high-level perspective taking. Results suggest that rTPJ increases its theta-band phase synchrony with brain regions involved in mentalizing and regions coding for motor/body schema; whilst decreasing its synchrony to visual regions. Implications for neurocognitive models are discussed, and it is proposed that rTPJ acts as a ‘hub’ to route bottom-up visual information to internal representations of the self during perspective-taking, co-ordinated by theta-band oscillations. The self is then projected onto the other’s perspective via embodied motor/body schema transformations, regulated by top-down cingulo-frontal activity.Significance Statement: High-level social processing, such as the ability to imagine another’s visuospatial experience of the world (perspective taking), is a core part of what makes us human. Building on a substantial body of converging previous evidence, our study reveals how concerted activity across the cortex in low frequencies (theta: 3-7 Hz) implements this crucial human process. We found that oscillatory power and connectivity (imaginary coherence, nonparametric Granger causality) at theta frequency linked functional sub-networks of executive control, mentalizing, and sensorimotor/body schema via a main hub located in the right temporo-parietal junction (rTPJ). Our findings inform neurocognitive models of social cognition by describing the co-ordinated changes in brain network connectivity, mediated by theta oscillations, during perspective-taking.
Edith Jones, , , Neda Nourabadi, Françoise Van Den Bergh, Hyo Sub Choi, Ali Haidar, ,
Published: 5 October 2017
Abstract:
Calcium ion concentration modulates the function of pyruvate dehydrogenase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase. Previous studies have shown that despite its ability to affect the function of these dehydrogenases, [Ca2+] does not substantially alter mitochondrial ATP synthesis in vitro under physiological sub-strate conditions. We hypothesize that, rather than contributing to respiratory control, [Ca2+] governs fuel selection. Specifically, cardiac mitochondria are able to use different primary carbon substrates to synthesize ATP aerobically. To determine if and how [Ca2+] affects the relative use of carbohydrates versus fatty acids we measured oxygen consumption and tricarboxylic acid cycle intermediate concentrations in suspensions of cardiac mitochondria with different combinations of pyruvate and palmitoyl-L-carnitine in the media at various [Ca2+] and ADP infusion rates. Results reveal that when both fatty acid and carbohydrate substrates are available, fuel selection is sensitive to both calcium and ATP synthesis rate. When no Ca2+ is added under low ATP-demand conditions, β-oxidation provides roughly half of acetyl-CoA for the citrate synthase reaction with the rest coming from the pyruvate dehydrogenase reaction. Under low demand conditions with increasing [Ca2+], the fuel utilization ratio shifts to increased fractional consumption of pyruvate, with 83±10% of acetyl-CoA derived from pyruvate at the highest [Ca2+] evaluated. With high ATP demand, the majority of acetyl-CoA is derived from pyruvate, regardless of the Ca2+ level. Our results suggest that changes in work rate alone are enough to effect a switch to carbohydrate use while in vivo the rate at which this switch happens may depend on mitochondrial calcium.Key Points: Despite its effects on activity of mitochondrial dehydrogenases, Ca2+ does not substantially alter mitochondrial ATP synthesis in vitro under physiological substrate conditions. Nor does is appear to play an important role in respiratory control in vivo in the myocardium. We hypothesize that Ca2+ plays a role mediating the switch in fuel selection to increasing carbohydrate oxidation and decreasing fatty acid oxidation with increasing work rate. To determine if and how Ca2+ affects the relative use of carbohydrates versus fatty acids in vitro we measured oxygen consumption and TCA cycle intermediate concentrations in suspensions of purified rat ventricular mitochondria with carbohydrate, fatty acid, and mixed substrates at various [Ca2+] and ATP demand rates. Our results suggest that changes in work rate alone are enough to effect a switch to carbohydrate use in vitro while in vivo the rate at which this switch happens may depend on mitochondrial calcium.
Published: 5 October 2017
Abstract:
In studying the cellular interactions within complex tissues, it is extremely valuable to be able to reproducibly and flexibly target transgene expression to restricted subsets of cells. This approach is particularly valuable in studying the nervous system, with its bewildering diversity of neuronal cell types. We report here the generation of over 18,000 driver lines (the VT collection) that exploit the GAL4, LexA, and split-GAL4 systems to express transgenes in distinct and highly specific cell types in Drosophila. We document the expression patterns of over 14,000 of these lines in the adult male brain.
, Joey Kuok Hoong Yam, Ziyan Hong, May Margarette Santillan Salido, Bau Yi Woo, Sam Fong Yau Li, Liang Yang, Michael Givskov,
Published: 5 October 2017
Abstract:
Pseudomonas aeruginosa is widely attributed as the leading cause of hospital-acquired infections. Due to intrinsic antibiotic resistance mechanisms and the ability to form biofilms, P. aeruginosa infections are challenging to treat. P. aeruginosa employs multiple virulence mechanisms to establish infections, many of which are controlled by the global virulence regulator Vfr. An attractive strategy to combat P. aeruginosa infections is thus the use of anti-virulence compounds. Here, we report the discovery that FDA-approved drug auranofin attenuates virulence in P. aeruginosa. We demonstrate that auranofin acts by targeting Vfr, which in turn leads to inhibition of quorum sensing (QS) and Type IV pili (TFP). Consistent with inhibition of QS and TFP expression, we show that auranofin attenuates biofilm maturation, and when used in combination with colistin, displays strong synergy in eradicating P. aeruginosa biofilms. Auranofin may have immediate applications as an anti-virulence drug against P. aeruginosa infections.
Published: 5 October 2017
Abstract:
Primary cilia are cylindrical organelles extending from the surface of most animal cells that have been implicated in a host of signaling and sensory functions. Genetic defects in their component molecules, known as “ciliopathies” give rise to devastating symptoms, ranging from defective development, to kidney disease, to progressive blindness. The detailed structures of these organelles and the true functions of proteins encoded by ciliopathy genes are poorly understood because of the small size of cilia and the limitations of conventional microscopic techniques. We describe the combination of cryo-electron tomography, enhanced by sub-tomogram averaging, with super-resolution stochastic reconstruction microscopy (STORM) to define substructures and subdomains within the light-sensing rod sensory cilium of the mammalian retina. Longitudinal and radial domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of these compartments, including Ca2+-binding protein centrin-2 in the lumen of the axoneme, acetylated tubulin forming the axoneme, the glycocalyx extending outward from the surface of the plasma membrane, and molecular residents of the space between axoneme and ciliary membrane, including Arl13B, intraflagellar transport proteins, BBS5, and syntaxin-3. Within this framework we document that deficiencies in the ciliopathy proteins BBS2, BBS7 and BBS9 lead to inappropriate accumulation of proteins in rod outer segments while largely preserving their sub-domain localization within the connecting cilium region, but alter the distribution of syntaxin-3 clusters.
Published: 5 October 2017
Abstract:
Withdrawal of the growth factor interleukin 3 from IL3-dependent myeloid cells causes them to undergo Bax/Bak1-dependent apoptosis, whereas factor-deprivedBax-/-Bak1-/-cells remain viable, but arrest and shrink. It was reported that withdrawal of IL3 fromBax-/-Bak1-/-cells caused decreased expression of the glucose transporter Glut1, leading to reduced glucose uptake, so that arrested cells required Atg5-dependent autophagy for long-term survival. In other cell types, a decrease in Glut1 is mediated by the thioredoxin-interacting protein Txnip, which is induced in IL3-dependent myeloid cells when growth factor is removed. We mutatedAtg5andTxnipby CRISPR/Cas9 and found that Atg5-dependent autophagy was not necessary for the long-term viability of cycling or arrestedBax-/-Bak1-/-cells, and that Txnip was not required for the decrease in Glut1 expression in response to IL3 withdrawal. Surprisingly, Atg5-deficientBax/Bak1double mutant cells survived for several weeks in medium supplemented with 10% fetal bovine serum (FBS), without high concentrations of added glucose or glutamine. When serum was withdrawn, the provision of an equivalent amount of glucose present in 10% FBS (~0.5 mM) was sufficient to support cell survival for more than a week, in the presence or absence of IL3. Thus,Bax-/-Bak1-/-myeloid cells deprived of growth factor consume extracellular glucose to maintain long-term viability, without a requirement for Atg5-dependent autophagy.
Genes & Development, Volume 18, pp 1926-1945; https://doi.org/10.1101/gad.1212704

Abstract:
The evolutionarily conserved checkpoint protein kinase, TOR (target of rapamycin), has emerged as a major effector of cell growth and proliferation via the regulation of protein synthesis. Work in the last decade clearly demonstrates that TOR controls protein synthesis through a stunning number of downstream targets. Some of the targets are phosphorylated directly by TOR, but many are phosphorylated indirectly. In this review, we summarize some recent developments in this fast-evolving field. We describe both the upstream components of the signaling pathway(s) that activates mammalian TOR (mTOR) and the downstream targets that affect protein synthesis. We also summarize the roles of mTOR in the control of cell growth and proliferation, as well as its relevance to cancer and synaptic plasticity.
, Feng Wang, Jan Wisniewski, Alexey K. Shaytan, Rodolfo Ghirlando, Peter C. FitzGerald, Yingzi Huang, Debbie Wei, Shipeng Li, David Landsman, et al.
Genes & Development, Volume 31, pp 1958-1972; https://doi.org/10.1101/gad.304782.117

Abstract:
Histone CENP-A-containing nucleosomes play an important role in nucleating kinetochores at centromeres for chromosome segregation. However, the molecular mechanisms by which CENP-A nucleosomes engage with kinetochore proteins are not well understood. Here, we report the finding of a new function for the budding yeast Cse4/CENP-A histone-fold domain interacting with inner kinetochore protein Mif2/CENP-C. Strikingly, we also discovered that AT-rich centromere DNA has an important role for Mif2 recruitment. Mif2 contacts one side of the nucleosome dyad, engaging with both Cse4 residues and AT-rich nucleosomal DNA. Both interactions are directed by a contiguous DNA- and histone-binding domain (DHBD) harboring the conserved CENP-C motif, an AT hook, and RK clusters (clusters enriched for arginine–lysine residues). Human CENP-C has two related DHBDs that bind preferentially to DNA sequences of higher AT content. Our findings suggest that a DNA composition-based mechanism together with residues characteristic for the CENP-A histone variant contribute to the specification of centromere identity.
Published: 26 October 2017
Genome Research, Volume 27, pp 2108-2119; https://doi.org/10.1101/gr.223735.117

Abstract:
Deep sequencing of size-selected DNase I–treated chromatin (DNase-seq) allows high-resolution measurement of chromatin accessibility to DNase I cleavage, permitting identification of de novo active cis-regulatory modules (CRMs) and individual transcription factor (TF) binding sites. We adapted DNase-seq to nuclei isolated from C. elegans embryos and L1 arrest larvae to generate high-resolution maps of TF binding. Over half of embryonic DNase I hypersensitive sites (DHSs) were annotated as noncoding, with 24% in intergenic, 12% in promoters, and 28% in introns, with similar statistics observed in L1 arrest larvae. Noncoding DHSs are highly conserved and enriched in marks of enhancer activity and transcription. We validated noncoding DHSs against known enhancers from myo-2, myo-3, hlh-1, elt-2, and lin-26/lir-1 and recapitulated 15 of 17 known enhancers. We then mined DNase-seq data to identify putative active CRMs and TF footprints. Using DNase-seq data improved predictions of tissue-specific expression compared with motifs alone. In a pilot functional test, 10 of 15 DHSs from pha-4, icl-1, and ceh-13 drove reporter gene expression in transgenic C. elegans. Overall, we provide experimental annotation of 26,644 putative CRMs in the embryo containing 55,890 TF footprints, as well as 15,841 putative CRMs in the L1 arrest larvae containing 32,685 TF footprints.
, Armando Reimer, , Xiao-Yong Li, Michael Stadler, Hernan Garcia, ,
Published: 1 September 2017
Genes & Development, Volume 31, pp 1784-1794; https://doi.org/10.1101/gad.305078.117

Abstract:
Morphogen gradients direct the spatial patterning of developing embryos; however, the mechanisms by which these gradients are interpreted remain elusive. Here we used lattice light-sheet microscopy to perform in vivo single-molecule imaging in early Drosophila melanogaster embryos of the transcription factor Bicoid that forms a gradient and initiates patterning along the anteroposterior axis. In contrast to canonical models, we observed that Bicoid binds to DNA with a rapid off rate throughout the embryo such that its average occupancy at target loci is on-rate-dependent. We further observed Bicoid forming transient “hubs” of locally high density that facilitate binding as factor levels drop, including in the posterior, where we observed Bicoid binding despite vanishingly low protein levels. We propose that localized modulation of transcription factor on rates via clustering provides a general mechanism to facilitate binding to low-affinity targets and that this may be a prevalent feature of other developmental transcription factors.
, Sharon E. Torigoe, , Daniel H. Mai, Li Li, Fred P. Davis, Peng Dong, Herve Marie-Nelly, Jonathan Grimm, Luke Lavis, et al.
Published: 1 September 2017
Genes & Development, Volume 31, pp 1795-1808; https://doi.org/10.1101/gad.303321.117

Abstract:
Transcription factor (TF)-directed enhanceosome assembly constitutes a fundamental regulatory mechanism driving spatiotemporal gene expression programs during animal development. Despite decades of study, we know little about the dynamics or order of events animating TF assembly at cis-regulatory elements in living cells and the long-range molecular “dialog” between enhancers and promoters. Here, combining genetic, genomic, and imaging approaches, we characterize a complex long-range enhancer cluster governing Krüppel-like factor 4 (Klf4) expression in naïve pluripotency. Genome editing by CRISPR/Cas9 revealed that OCT4 and SOX2 safeguard an accessible chromatin neighborhood to assist the binding of other TFs/cofactors to the enhancer. Single-molecule live-cell imaging uncovered that two naïve pluripotency TFs, STAT3 and ESRRB, interrogate chromatin in a highly dynamic manner, in which SOX2 promotes ESRRB target search and chromatin-binding dynamics through a direct protein-tethering mechanism. Together, our results support a highly dynamic yet intrinsically ordered enhanceosome assembly to maintain the finely balanced transcription program underlying naïve pluripotency.
Michael G. Kearse,
Published: 1 September 2017
Genes & Development, Volume 31, pp 1717-1731; https://doi.org/10.1101/gad.305250.117

Abstract:
Although it was long thought that eukaryotic translation almost always initiates at an AUG start codon, recent advancements in ribosome footprint mapping have revealed that non-AUG start codons are used at an astonishing frequency. These non-AUG initiation events are not simply errors but instead are used to generate or regulate proteins with key cellular functions; for example, during development or stress. Misregulation of non-AUG initiation events contributes to multiple human diseases, including cancer and neurodegeneration, and modulation of non-AUG usage may represent a novel therapeutic strategy. It is thus becoming increasingly clear that start codon selection is regulated by many trans-acting initiation factors as well as sequence/structural elements within messenger RNAs and that non-AUG translation has a profound impact on cellular states.
Comment
Published: 1 September 2017
Genes & Development, Volume 31, pp 1715-1716; https://doi.org/10.1101/gad.306837.117

Abstract:
Medulloblastomas are among the most common malignant brain cancers in the pediatric population and consist of at least four distinct subgroups with unique molecular and genetic features and clinical outcomes. In this issue of Genes & Development, Niklison-Chirou and colleagues (pp. 1738–1753) identify the p53 family member and p73 isoform TAp73 as a crucial factor causing glutamine addiction in aggressive medulloblastomas. Their findings pave the way for the use of glutamine restriction as an adjuvant treatment for TAp73-expressing medulloblastomas.
, Beth Hayes, Matthew C. Radey, Xia Lee, Tanya Josek, , David Neitzel, Susan Paskewitz, ,
Published: 4 October 2017
Abstract:
Hard ticks of the order Ixodidae serve as vectors for numerous human pathogens, including the causative agent of Lyme DiseaseBorrelia burgdorferi. Tick-associated microbes can influence pathogen colonization, offering the potential to inhibit disease transmission through engineering of the tick microbiota. Here, we investigate whetherB. burgdorferiencounters abundant bacteria within the midgut of wild adultIxodes scapularis, its primary vector. Through the use of controlled sequencing methods and confocal microscopy, we find that the majority of field-collected adultI. scapularisharbor limited internal microbial communities that are dominated by endosymbionts. A minority ofI. scapularisticks harbor abundant midgut bacteria and lackB. burgdorferi. We find that the lack of a stable resident midgut microbiota is not restricted toI. scapularissince extension of our studies toI. pacificus, Amblyomma maculatum, andDermacentorspp showed similar patterns. Finally, bioinformatic examination of theB. burgdorferigenome revealed the absence of genes encoding known interbacterial interaction pathways, a feature unique to theBorreliagenus within the phylumSpirochaetes. Our results suggest that reduced selective pressure from limited microbial populations within ticks may have facilitated the evolutionary loss of genes encoding interbacterial competition pathways fromBorrelia.
Published: 4 October 2017
Abstract:
Fluorescence microscopy is a powerful approach for studying sub-cellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel LSFM method: Lateral Interference Tilted Excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically-limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution.
Published: 4 October 2017
Abstract:
Cellular redox status affects diverse cellular functions, including proliferation, protein homeostasis, and aging. Thus, individual differences in redox status can give rise to distinct sub-populations even among cells with identical genetic backgrounds. Here, we have created a novel methodology to track redox status at single cell resolution using the redox-sensitive probe roGFP. Our method allows identification and sorting of sub-populations with different oxidation levels in either the cytosol, mitochondria or peroxisomes. Using this approach we defined redox-dependent heterogeneity of yeast cells, and characterized growth, as well as proteomic and transcriptomic profiles of subpopulations of cells that differ in their redox status, but are similar in age. We report that, starting in late logarithmic growth, cells of the same age have a bi-modal distribution of oxidation status. A comparative proteomic analysis between these populations identified three key proteins, Hsp30, Dhh1, and Pnc1, which affect basal oxidation levels and may serve as first line of defense proteins in redox homeostasis.
Published: 3 October 2017
Abstract:
Polycyclic tetramate macrolactam (PTM) natural products are produced by actinomycetes and other bacteria. PTMs are often bioactive, and the simplicity of their biosynthetic clusters make them attractive for bioengineering. Clifednamide-type PTMs fromStreptomycessp. JV178 contain a distinctive ketone group, suggesting the existence of a novel PTM oxidizing enzyme. Here, we report the new cytochrome P450 enzyme (CftA) is required for clifednamide production. Genome mining was used to identify several new clifednamide producers, some having improved clifednamide yields. Using a parallel synthetic biology approach, CftA isozymes were used to engineer the ikarugamycin pathway ofStreptomycessp. NRRL F-2890 to yield clifednamides. Further, we observed that strong CftA expression leads to the production of a new PTM, clifednamide C. We demonstrate the utility of both genome mining and synthetic biology to rapidly increase clifednamide production and identify a PTM tailoring enzyme for rational molecule design.
Published: 3 October 2017
Abstract:
Visual analysis of histopathology slides of lung cell tissues is one of the main methods used by pathologists to assess the stage, types and sub-types of lung cancers. Adenocarcinoma and squamous cell carcinoma are two most prevalent sub-types of lung cancer, but their distinction can be challenging and time-consuming even for the expert eye. In this study, we trained a deep learning convolutional neural network (CNN) model (inception v3) on histopathology images obtained from The Cancer Genome Atlas (TCGA) to accurately classify whole-slide pathology images into adenocarcinoma, squamous cell carcinoma or normal lung tissue. Our method slightly outperforms a human pathologist, achieving better sensitivity and specificity, with ∼0.97 average Area Under the Curve (AUC) on a held-out population of whole-slide scans. Furthermore, we trained the neural network to predict the ten most commonly mutated genes in lung adenocarcinoma. We found that six of these genes – STK11, EGFR, FAT1, SETBP1, KRAS and TP53 – can be predicted from pathology images with an accuracy ranging from 0.733 to 0.856, as measured by the AUC on the held-out population. These findings suggest that deep learning models can offer both specialists and patients a fast, accurate and inexpensive detection of cancer types or gene mutations, and thus have a significant impact on cancer treatment.
Published: 3 October 2017
Abstract:
It is widely conjectured that the mutually antagonizing pair of transcription factors GATA1 and PU.1, deter-mines the choice between the erythroid and myeloid lineages in hematopoiesis. In theoretical approaches, this appears natural with a bistable switch driving the decision. Recent extensive binding and gene expression experiments with some focus on the triad GATA1, GATA2 and PU.1 indicate that GATA2 may be more involved in this lineage decision than previously anticipated. Here, we analyze these experimental data by modeling regulatory sub-networks with deterministic rate equations. Using network dynamical parameters determined by the data, we deduce from increasing the self-interaction bindings in silico among the triad genes that GATA2 and PU.1 exhibit non-linear behavior with one unstable and one stable state. This is in contrast to GATA1, which shows smoother behavior. We extend the network to include the downstream regulators FOG1 and CEBPA, and extract the nature of the corresponding regulatory interactions, excitatory or suppressing, between this pair and the triad by fitting to experimental gene expression time series. Based on this extended network, we simulate and explore different knockout scenarios, providing insight into the role of these regulators in the process of lineage specification, as well as predictions for future experimental validation. We address the mechanism of GATA switching as a mechanism of lineage differentiation by investigating the dynamics of FOG1 regulation by GATA2 and GATA1. Overall, this analysis strongly suggests that within this network, GATA2 is the key driver of erythroid lineage specification through its repression of PU.1, whereas GATA1 appears to be more relevant for the downstream differentiation events.
, , Guila Ganem, Charlotte Faurie
Published: 3 October 2017
Abstract:
Theoretical and empirical studies in humans suggest that cooperative behaviors may act as signals during mate choice. However, cooperation is not always observable by potential partners before mate choice. To address whether cooperative phenotypes are preferred based on cues different from cooperative behaviors per se, we designed an experimental paradigm using wild-born mound-building mice (Mus spicilegus), a species with biparental care. In this species, females cannot observe male cooperative behaviors: mate choice occurs in the spring, whereas mounds are cooperatively built in the fall. We first assessed the variation in mound building investment and identified males exhibiting high and low amounts of cooperation. Second, we presented these males to females during two-way choice tests. As offspring survival relies on mound protection, we hypothesized that mound building could be a form of paternal care and assessed whether cooperative males were more involved in offspring attendance using pup-retrieval experiments. Our results indicate that females were more attracted to highly cooperative males over less cooperative, even when they did not observe them build. This finding suggests that female mate choice is influenced either by cues of cooperativeness different than cooperative behaviors or by preferences for traits associated with cooperativeness. Moreover, male offspring attendance was negatively correlated with cooperativeness, suggesting the potential existence of two alternative paternal strategies in offspring care (mound building versus offspring attendance). Overall, our findings support the existence of preference for cooperative phenotypes in a non-human species and suggest that sexual selection might be involved in the evolution of cooperation.
Published: 3 October 2017
Abstract:
Mannheimia haemolytica serotype A2 is a common commensal species present in the nasopharynx of healthy cattle. However, prior to the onset of bovine pneumonic pasteurellosis, there is sudden increase in M. haemolytica serotype A1 within the upper respiratory tract. The events during this selective proliferation of serotype A1 strains are poorly characterised. In this investigation, a differentiated bovine airway epithelial cell culture was used to study the interactions of A1 and A2 bovine isolates with the respiratory epithelium. This model reproduced the key defences of the airway epithelium, including tight junctions and mucociliary clearance. Although initial adherence of the serotype A1 strains was low, by 12 hours post-infection the bacteria was able to traverse the tight junctions to form foci of infection below the apical surface. The size, density and number of these foci increased with time, as did the cytopathic effects observed in the bovine bronchial epithelial cells. Penetration of M. haemolytica A1 into the sub-apical epithelium was shown to be through transcytosis but not paracytosis. Commensal A2 bovine isolates however were not capable of colonising the model to a high degree, and did not penetrate the epithelium following initial adherence at the apical surface. This difference in their ability to colonise the respiratory epithelium may account for the sudden proliferation of serotype A1 in the onset of pneumonia pasteurellosis. The pathogenesis observed was replicated by virulent A2 ovine isolates; however colonisation was 10-fold lower in comparison to bovine A1 strains. This investigation provides new insight into the interactions of M. haemolytica with bovine airway epithelial cells which are occurring in vivo during pneumonia pasteurellosis.
, Jia Ling, Jesse Kurland, Xiaotong Ren, Zhe Xu, Gozde Yucel, Jackie Moore, Leila Shokri, Isabel Baker, Timothy Bishop, et al.
Published: 3 October 2017
Abstract:
The K50 homeodomain (K50HD) protein Orthodenticle (Otd) is critical for anterior patterning and brain and eye development in most metazoans. In Drosophila melanogaster, another K50HD protein, Bicoid (Bcd), has evolved to replace Otd’s ancestral function in embryo patterning. Bcd is distributed as a long-range maternal gradient and activates transcription of a large number of target genes including otd. Otd and Bcd bind similar DNA sequences in vitro, but how their transcriptional activities are integrated to pattern anterior regions of the embryo is unknown. Here we define three major classes of enhancers that are differentially sensitive to binding and transcriptional activation by Bcd and Otd. Class 1 enhancers are initially activated by Bcd, and activation is transferred to Otd via a feed-forward relay (FFR) that involves sequential binding of the two proteins to the same DNA motif. Class 2 enhancers are activated by Bcd, and maintained by an Otd-independent mechanism. Class 3 enhancers are never bound by Bcd, but Otd binds and activates them in a second wave of zygotic transcription. The specific activities of enhancers in each class are mediated by DNA motif variants preferentially bound by Bcd or Otd, and the presence or absence of sites for cofactors that interact with these proteins. Our results define specific patterning roles for Bcd and Otd, and provide mechanisms for coordinating the precise timing of gene expression patterns during embryonic development.
, , Andrey S. Krasilnikov
Published: 29 September 2017
RNA, Volume 24, pp 1-5; https://doi.org/10.1261/rna.063107.117

Abstract:
RNase P catalyzes 5′-maturation of tRNAs in all three domains of life. This primary function is accomplished by either a ribozyme-centered ribonucleoprotein (RNP) or a protein-only variant (with one to three polypeptides). The large, multicomponent archaeal and eukaryotic RNase P RNPs appear disproportionate to the simplicity of their role in tRNA 5′-maturation, prompting the question of why the seemingly gratuitously complex RNP forms of RNase P were not replaced with simpler protein counterparts. Here, motivated by growing evidence, we consider the hypothesis that the large RNase P RNP was retained as a direct consequence of multiple roles played by its components in processes that are not related to the canonical RNase P function.
Kristin R. Gleitsman, Raghuvir N. Sengupta, Daniel Herschlag
Published: 28 September 2017
RNA, Volume 23, pp 1745-1753; https://doi.org/10.1261/rna.062026.117

Abstract:
Molecular recognition is central to biological processes, function, and specificity. Proteins associate with ligands with a wide range of association rate constants, with maximal values matching the theoretical limit set by the rate of diffusional collision. As less is known about RNA association, we compiled association rate constants for all RNA/ligand complexes that we could find in the literature. Like proteins, RNAs exhibit a wide range of association rate constants. However, the fastest RNA association rates are considerably slower than those of the fastest protein associations and fall well below the diffusional limit. The apparently general observation of slow association with RNAs has implications for evolution and for modern-day biology. Our compilation highlights a quantitative molecular property that can contribute to biological understanding and underscores our need to develop a deeper physical understanding of molecular recognition events.
, David Wilson, Michael Weigert, Stefan Wyder,
Published: 2 October 2017
Abstract:
Background and objectives: Treatments that inhibit the expression or functioning of bacterial virulence factors hold great promise to be both effective and exert weaker selection for resistance than conventional antibiotics. However, the evolutionary robustness argument, based on the idea that anti-virulence treatments disarm rather than kill pathogens, is controversial. Here we probe the evolutionary robustness of two repurposed drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine of the opportunistic human pathogenPseudomonas aeruginosa.Methodology: We subjected replicated cultures of bacteria to two concentrations of each drug for 20 consecutive days in human serum as an ex-vivo infection model. We screened evolved populations and clones for resistance phenotypes, including the restoration of growth and pyoverdine production, and the evolution of iron uptake by-passing mechanisms. We whole-genome sequenced evolved clones to identify the genetic basis of resistance.Results: We found that mutants resistant against anti-virulence treatments readily arose, but their selective spreading varied between treatments. Flucytosine resistance quickly spread in all populations due to disruptive mutations inupp, a gene encoding an enzyme required for flucytosine activation. Conversely, resistance against gallium arose only sporadically, and was based on mutations in transcriptional regulators, upregulating pyocyanin production, a redox-active molecule promoting siderophore-independent iron acquisition. The spread of gallium resistance could be hampered because pyocyanin-mediated iron delivery benefits resistant and susceptible cells alike.Conclusions and implications: Our work highlights that anti-virulence treatments are not evolutionarily robustper se. Instead, evolutionary robustness is a relative measure, with specific treatments occupying different positions on a continuous scale.
Published: 2 October 2017
Abstract:
Despite major strides in the treatment of cancer, the development of drug resistance remains a major hurdle. One strategy which has been proposed to address this is the sequential application of drug therapies where resistance to one drug induces sensitivity to another drug, a concept called collateral sensitivity. The optimal timing of drug switching in these situations, however, remains unknown.To study this, we developed a dynamical model of sequential therapy on heterogeneous tumors comprised of resistant and sensitive cells. A pair of drugs (DrugA, DrugB) are utilized and are periodically switched during therapy. Assuming resistant cells to one drug are collaterally sensitive to the opposing drug, we classified cancer cells into two groups,ARandBR, each of which is a subpopulation of cells resistant to the indicated drug and concurrently sensitive to the other, and we subsequently explored the resulting population dynamics.Specifically, based on a system of ordinary differential equations forARandBR, we determined that the optimal treatment strategy consists of two stages: an initial stage in which a chosen effective drug is utilized until a specific time point,T, and a second stage in which drugs are switched repeatedly, during which each drug is used for a relative duration (i.e.fΔt-long forDrugAand (1 –f) Δt-long forDrugBwith 0 ≤f≤ 1 and Δt≥ 0). We prove that the optimal duration of the initial stage, in which the first drug is administered,T, is shorter than the period in which it remains effective in decreasing the total population, contrary to current clinical intuition.We further analyzed the relationship between population makeup, , and the effect of each drug. We determine a critical ratio, which we term , at which the two drugs are equally effective. As the first stage of the optimal strategy is applied, changes monotonically to and then, during the second stage, remains at thereafter.Beyond our analytic results, we explored an individual based stochastic model and presented the distribution of extinction times for the classes of solutions found. Taken together, our results suggest opportunities to improve therapy scheduling in clinical oncology.
Hyuckjoon Kang, Youngsook L. Jung, Kyle A. McElroy, , Heather A. Wallace, Jessica L. Woolnough, Peter J. Park,
Genes & Development, Volume 31, pp 1988-2002; https://doi.org/10.1101/gad.305987.117

Abstract:
Regulatory decisions in Drosophila require Polycomb group (PcG) proteins to maintain the silent state and Trithorax group (TrxG) proteins to oppose silencing. Since PcG and TrxG are ubiquitous and lack apparent sequence specificity, a long-standing model is that targeting occurs via protein interactions; for instance, between repressors and PcG proteins. Instead, we found that Pc-repressive complex 1 (PRC1) purifies with coactivators Fs(1)h [female sterile (1) homeotic] and Enok/Br140 during embryogenesis. Fs(1)h is a TrxG member and the ortholog of BRD4, a bromodomain protein that binds to acetylated histones and is a key transcriptional coactivator in mammals. Enok and Br140, another bromodomain protein, are orthologous to subunits of a mammalian MOZ/MORF acetyltransferase complex. Here we confirm PRC1–Br140 and PRC1–Fs(1)h interactions and identify their genomic binding sites. PRC1–Br140 bind developmental genes in fly embryos, with analogous co-occupancy of PRC1 and a Br140 ortholog, BRD1, at bivalent loci in human embryonic stem (ES) cells. We propose that identification of PRC1–Br140 “bivalent complexes” in fly embryos supports and extends the bivalency model posited in mammalian cells, in which the coexistence of H3K4me3 and H3K27me3 at developmental promoters represents a poised transcriptional state. We further speculate that local competition between acetylation and deacetylation may play a critical role in the resolution of bivalent protein complexes during development.
Sarah B. Reiff,
Published: 25 July 2017
Abstract:
Background: Stentor coeruleus is a large ciliated protist, about 1mm in length, with the extraordinary ability to fully regenerate each fragment after being cut into pieces, perfectly restoring cell polarity and morphology. Single-cell regeneration in Stentor remains one of the greatest long-standing mysteries of biology, but the recently published Stentor genome now enables studies on this organism at the molecular and genetic levels. Here we characterize the complete complement of kinases, or kinome, of Stentor, in order to begin to understand the signaling capacities that underlie Stentor’s unique biology.Results: The genome of S. coeruleus contains over 2000 kinases, representing 6% of the predicted proteome. Classification of the kinase genes reveals large expansions in several kinase families, particularly in the CDPKs, the DYRKs, and in several mitotic kinase families including the PLKs, NEKs, and Auroras. The large expansion of the CDPK and DYRK kinase families is an unusual feature of the Stentor kinome compared to other ciliates with sequenced genomes. The DYRK family in Stentor, notably, contains only a single pseudokinase which may suggest an important role in Stentor growth and survival, while the smaller PEK family contains a novel pseudokinase subfamily. The Stentor kinome also has examples of new domain architectures that have not been previously observed in other organisms.Conclusion: Our analysis provides the first gene-level view into the signaling capabilities of Stentor and will lay the foundation for unraveling how this organism can coordinate processes as complex as regeneration throughout a giant cell.
Published: 30 December 2015
Abstract:
Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of cfDNA in blood, can be readily adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparation, however, is insensitive to ultrashort, degraded and single-stranded cfDNA. Drawing inspiration from recent technical advances in ancient genome analyses, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in the plasma of lung transplant recipients (40 samples, six patients). We found that the ssDNA library preparation yields a greater portion of sub-100 bp DNA, as well as an increased relative abundance of human mitochondrial cfDNA (10.7x) and microbial cfDNA (71.3x). We report the fragmentation pattern of mitochondrial, nuclear genomic and microbial cfDNA over a broad fragment length range. We furthermore report the first observation of donor-specific mitochondrial cfDNA in the circulation of lung transplant recipients. We found that donor-specific mitochondrial cfDNA molecules are significantly shorter than those specific to the recipient. The higher yield of viral, microbial and fungal sequences that result from the single-stranded ligation approach reduces the cost and increase the sensitivity of cfDNA-based monitoring for infectious complications after transplantation. An ssDNA library preparation method provides a more informative window into understudied forms of cfDNA, including mitochondrial and microbial derived cfDNA and short fragment nuclear genomic cfDNA, while retaining information provided by standard dsDNA library preparation methods.
Published: 1 May 2014
Abstract:
Short Tandem Repeats are among the most polymorphic loci in the human genome. These loci play a role in the etiology of a range of genetic diseases and have been frequently utilized in forensics, population genetics, and genetic genealogy. Despite this plethora of applications, little is known about the variation of most STRs in the human population. Here, we report the largest-scale analysis of human STR variation to date. We collected information for nearly 700,000 STR loci across over 1,000 individuals in phase 1 of the 1000 Genomes Project. This process nearly saturated common STR variations. After employing a series of quality controls, we utilize this call set to analyze determinants of STR variation, assess the human reference genome?s representation of STR alleles, find STR loci with common loss-of-function alleles, and obtain initial estimates of the linkage disequilibrium between STRs and common SNPs. Overall, these analyses further elucidate the scale of genetic variation beyond classical point mutations. The resource is publicly available at http://strcat.teamerlich.org/ both in raw format and via a graphical interface.
Published: 3 April 2014
Abstract:
In tropical forests, one of the most common relationships between parasites and insects is that between the fungus Ophiocordyceps (Ophiocordycipitaceae, Hypocreales, Ascomycota) and ants, especially within the tribe Camponotini. Here, we describe three new and host-specific species of the genus Ophiocordyceps on Camponotus ants from the central Amazonian region of Brazil, which can readily be separated using morphological traits, in particular, ascospore form and function. In addition, we use sequence data to infer phylogenetic relationships between these taxa and closely related species within the Ophiocordyceps unilateralis complex, as well as with other members of the family Ophiocordycipitaceae.
, Cassandra Falckenhayn, Anne Schrimpf, Katharina Schmid, Katharina Hanna, Jörn Panteleit, , ,
Published: 23 August 2015
Abstract:
The parthenogenetic all-female marbled crayfish is a novel research model and potent invader of freshwater ecosystems. It is a triploid descendant of the sexually reproducing slough crayfish,Procambarus fallax, but its taxonomic status has remained unsettled. By cross-breeding experiments and parentage analysis we show here that marbled crayfish andP. fallaxare reproductively separated. Both crayfish copulate readily, suggesting that the reproductive barrier is set at the cytogenetic rather than the behavioural level. Analysis of complete mitochondrial genomes of marbled crayfish from laboratory lineages and wild populations demonstrates genetic identity and indicates a single origin. Flow cytometric comparison of DNA contents of haemocytes and analysis of nuclear microsatellite loci confirm triploidy and suggest autopolyploidization as its cause. Global DNA methylation is significantly reduced in marbled crayfish implying the involvement of molecular epigenetic mechanisms in its origination. Morphologically, both crayfish are very similar but growth and fecundity are considerably larger in marbled crayfish, making it a different animal with superior fitness. These data and the high probability of a divergent future evolution of the marbled crayfish andP. fallaxclusters suggest that marbled crayfish should be considered as an independent asexual species. Our findings also establish theP. fallax-marbled crayfish pair as a novel paradigm for rare chromosomal speciation by autopolyploidy and parthenogenesis in animals and for saltational evolution in general.
Published: 17 August 2015
Abstract:
Meiotic recombination breaks down linkage disequilibrium and forms new haplotypes, meaning thatit is an important driver of diversity in eukaryotic genomes. Understanding the causes of variation in recombination rate is important in interpreting and predicting evolutionary phenomena and forunderstanding the potential of a population to respond to selection. However, despite attention inmodel systems, there remains little data on how recombination rate varies at the individual level in natural populations. Here, we used extensive pedigree and high-density SNP information in a wild population of Soay sheep (Ovis aries) to investigate the genetic architecture of individual autosomal recombination rate. Individual rates were high relative to other mammal systems, and were higher in males than in females (autosomal map lengths of 3748 cM and 2860 cM, respectively). The heritability of autosomal recombination rate was low but significant in both sexes(h2= 0.16 & 0.12 in females and males, respectively). In females, 46.7% of the heritable variation was explained by a sub-telomeric region on chromosome 6; a genome-wide association study showed the strongest associations at the locusRNF212, with further associations observed at a nearby ~374kb region of complete linkage disequilibrium containing three additional candidate loci,CPLX1,GAKandPCGF3. A second region on chromosome 7 containingREC8andRNF212Bexplained 26.2% of the heritable variation in recombination rate in both sexes. Comparative analyses with 40 other sheep breeds showed that haplotypes associated with recombination rates are both old and globally distributed. Both regions have been implicated in rate variation in mice, cattle and humans, suggesting a common genetic architecture of recombination rate variation in mammals.AUTHOR SUMMARY: Recombination offers an escape from genetic linkage by forming new combinations of alleles, increasing the potential for populations to respond to selection. Understanding the causes and consequences of individual recombination rates are important in studies of evolution and genetic improvement, yet little is known on how rates vary in natural systems. Using data from a wild population of Soay sheep, we show that individual recombination rate is heritable and differs between the sexes, with the majority of genetic variation in females explained by a genomic region containing thegenesRNF212andCPLX1.
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