title: Toxoplasma-proximal and distal control by GBPs in human macrophages

abstract: Human guanylate-binding proteins (GBPs) are key players of interferon-gamma (IFN{gamma})-induced cell intrinsic defense mechanisms targeting intracellular pathogens. In this study we combine the well-established Toxoplasma gondii infection model with three in vitro macrophage culture systems to delineate the contribution of individual GBP family members to control this apicomplexan parasite. Use of high-throughput imaging assays and genome engineering allowed us to define a role for GBP1, 2 and 5 in parasite infection control. While GBP1 performs a pathogen-proximal, parasiticidal and growth-restricting function through accumulation at the parasitophorous vacuole of intracellular Toxoplasma, GBP2 and 5 perform a pathogen-distal, growth-restricting role. We further find that mutants of the GTPase or isoprenylation site of GBP1/2/5 affect their normal function in Toxoplasma control by leading to mis-localization of the proteins.


title: Genomic screening of antimicrobial resistance markers in UK and US Campylobacter isolates highlights stability of resistance over an 18 year period

abstract: Campylobacter jejuni and Campylobacter coli are important bacterial causes of human foodborne illness. Despite several years of reduced antibiotics usage in livestock production in the UK and US, high prevalence of antimicrobial resistance (AMR) persists in Campylobacter. Both countries have instigated genome sequencing-based surveillance programs for Campylobacter, and here we have identified AMR genes in 32,256 C. jejuni and 8,776 C. coli publicly available genome sequences to compare the prevalence and trends of AMR in Campylobacter isolated in the UK and US between 2001-2018. AMR markers were detected in 68% of C. coli and 53% of C. jejuni, with 15% of C. coli being multi-drug resistant (MDR) compared to only 2% of C. jejuni. The prevalence of aminoglycoside, macrolide, quinolone and tetracycline resistance remained fairly stable from 2001-2018 in both C. jejuni and C. coli, but statistically significant differences were observed between the UK and US. There was a statistically significant higher prevalence of aminoglycoside and tetracycline resistance for US C. coli and C. jejuni, and macrolide resistance for US C. coli. In contrast, UK C. coli and C. jejuni showed a significantly higher prevalence of quinolone resistance. Specific MLST clonal complexes (e.g. ST-353/464) showed >95% quinolone resistance. This large-scale comparison of AMR prevalence has shown that the prevalence of AMR remains stable for Campylobacter in the UK and the US. This suggests that antimicrobial stewardship and restricted antibiotic usage may help contain further expansion of AMR prevalence in Campylobacter, but are unlikely to reduce it in the short term.

category: microbiology


title: Defining dysfunction due to loss of MECP2 in Rett Patient Brain

abstract: Rett Syndrome is characterized by a postnatal loss of neurophysiological function and regression of childhood development. Because the syndrome is X-linked and males with MECP2 mutations generally do not survive birth, the study of this syndrome has been complicated by the fact that in female brain, a portion of neurons express wild type MECP2, and another portion express a non-functional allele of MECP2. Therefore, bulk-RNA-sequencing of Rett brain is confounded by the presence of chimerism of neurons for functional MECP2 in neurons. We developed an approach that allows for single-nuclei transcriptional profiling of individual neurons and a direct comparison between neurons that express functional MECP2 with those that express the disease-causing allele. We found that mutant neurons from Rett brain show patterns of aberrant expression of synaptic and metabolic genes, both of which can be detected in in vitro models of Rett Syndrome. We used these resources to identify a role for POU2F1/OCT1 transcription factor in mediating the response to stress due to loss of MECP2, highlighting a potential key molecular regulator of stress in Rett neurons. Together, our new sorting approach enables us to highlight defective molecular and metabolic pathways in Rett brain neurons and suggests that in vitro models could serve as valuable tools to further study this syndrome and potentially for development of novel therapeutics.

category: molecular biology


title: Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export

abstract: Nuclear clearance of the DNA/RNA-binding protein TDP-43 is a pathologic hallmark of amyotrophic lateral sclerosis and frontotemporal dementia that remains unexplained. Moreover, our current understanding of TDP-43 nucleocytoplasmic shuttling does not fully explain the predominantly nuclear localization of TDP-43 in healthy cells. Here, we used permeabilized and live-cell models to investigate TDP-43 nuclear export and the role of RNA in TDP-43 localization. We show that TDP-43 nuclear efflux occurs in low-ATP conditions and independent of active mRNA export, consistent with export by passive diffusion through nuclear pore channels. TDP-43 nuclear residence requires binding to GU-rich nuclear intronic pre-mRNAs, based on the induction of TDP-43 nuclear efflux by RNase and GU-rich oligomers and TDP-43 nuclear retention conferred by pre-mRNA splicing inhibitors. Mutation of TDP-43 RNA recognition motifs disrupts TDP-43 nuclear accumulation and abolishes transcriptional blockade-induced TDP-43 nuclear efflux, demonstrating strict dependence of TDP-43 nuclear localization on RNA binding. Thus, the nuclear abundance of GU-rich intronic pre-mRNAs, as dictated by the balance of transcription and pre-mRNA processing, regulates TDP-43 nuclear sequestration and availability for passive nuclear export.

category: cell biology


title: Inferring and perturbing cell fate regulomes in human cerebral organoids

abstract: Self-organizing cerebral organoids grown from pluripotent stem cells combined with single-cell genomic technologies provide opportunities to explore gene regulatory networks (GRNs) underlying human brain development. Here we acquire single-cell transcriptome and accessible chromatin profiling data over a dense time course covering multiple phases of organoid development including neuroepithelial formation, patterning, brain regionalization, and neurogenesis. We identify temporally dynamic and brain region-specific regulatory regions, and cell interaction analysis reveals emergent patterning centers associated with regionalization. We develop Pando, a flexible linear model-based framework that incorporates multi-omic data and transcription binding site predictions to infer a global GRN describing organoid development. We use pooled genetic perturbation with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state regulation in organoid. We find that certain factors regulate the abundance of cell fates, whereas other factors impact neuronal cell states after differentiation. We show that the zinc finger protein GLI3 is required for cortical fate establishment in humans, recapitulating previous work performed in mammalian model systems. We measure transcriptome and chromatin accessibility in normal or GLI3-perturbed cells and identify a regulome central to the dorsoventral telencephalic fate decision. This regulome suggests that Notch effectors HES4/5 are direct GLI3 targets, which together coordinate cortex and ganglionic eminence diversification. Altogether, we provide a framework for how multi-brain region model systems and single-cell technologies can be leveraged to reconstruct human brain developmental biology.

category: developmental biology


title: Long-read and chromosome-scale assembly of the hexaploid wheat genome achieves higher resolution for research and breeding

abstract: The sequencing of the wheat (Triticum aestivum) genome has been a methodological challenge for many years due to its large size (15.5 Gb), repeat content, and hexaploidy. Many initiatives aiming at obtaining a reference genome of cultivar Chinese Spring have been launched in the past years and it was achieved in 2018 as the result of a huge effort to combine short-read sequencing with many other resources. Reference-quality genome assemblies were then produced for other accessions but the rapid evolution of sequencing technologies offers opportunities to reach high-quality standards at lower cost. Here, we report on an optimized procedure based on long-reads produced on the ONT (Oxford Nanopore Technology) PromethION device to assemble the genome of the French bread wheat cultivar Renan. We provide the most contiguous and complete chromosome-scale assembly of a bread wheat genome to date. Coupled with an annotation based on RNA-Seq data, this resource will be valuable for the crop community and will facilitate the rapid selection of agronomically important traits. We also provide a framework to generate high-quality assemblies of complex genomes using ONT.

category: genomics


title: Long-read and chromosome-scale assembly of the hexaploid wheat genome achieves high resolution for research and breeding


title: Carbon Nanotube Electrodes for Electrochemical Detection of Dopamine

abstract:
Carbon nanotubes (CNTs) are suited for neurochemistry because of their biological inertness, ability to withstand biofouling, and superior electron transport kinetics. Dopamine, the canonical monoaminergic neuromodulator, contributes to reward, cognition and attention, however, its detection in real-time is challenging due to its low basal concentration in the brain (100nM L-1). In our present work, we fabricate pyrolytic carbon electrodes and perform a CNT coating to improve the electrochemical kinetics of dopamine. Upon CNTs coating, dopamine shows a sensitivity of 9{+/-}18nA/M for a cylindrical electrode having a mean surface diameter of 8{+/-}4m. Increasing the scan frequency from 10-100 Hz shows that dopamine electron transfer kinetics improves; wherein dopamine is oxidized at 0.35{+/-}0.09V and reduced to -0.10{+/-}0.05V for 10 Hz. Increasing the frequency results in a shift of oxidation peak towards the anodic region, wherein dopamine oxidizes at 0.08{+/-}3V and reduces at -0.1{+/-}0.05V for 100 Hz, thus showing that dopamine redox is reversible which can be attributed to the superior electron transport kinetics of CNTs. The sensor was able to distinguish dopamine signals against other neurochemicals like serotonin and foulant 3,4-Dihydroxyphenylacetic acid (DOPAC). The minimum chemical detection that can be performed using these nanopipettes is 50{+/-}18nM L-1, which is well below the physiological concentrations of dopamine in the brain.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/457511v1_ufig1.gif" ALT="Figure 1">
View larger version (34K):
org.highwire.dtl.DTLVardef@bf710aorg.highwire.dtl.DTLVardef@966bcforg.highwire.dtl.DTLVardef@1800530org.highwire.dtl.DTLVardef@1d4a37e_HPS_FORMAT_FIGEXP  M_FIG A: Pictorial view of background-subtracted voltammetry. The waveform used was -0.4V to 1.3 V and cycled back to -0.4V at 10 Hz. B: The voltammogram was converted as a 2-D representation, into current, voltage, and repetition to understand the dopamine oxidation. C: Background subtracted voltammetry for dopamine using 100 Hz waveform. D: The 2-D representation of current, voltage, and repetition.

C_FIG

category: biochemistry


title: Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding

abstract: Transmembrane {beta}-barrel proteins are folded into the outer membrane (OM) of Gram-negative bacteria by the {beta}-barrel assembly machine (BAM) via an unexplained process that occurs without known external energy sources. Here we used single-particle cryo-EM to visualize the folding dynamics of a model {beta}-barrel protein (EspP) by BAM. We found that BAM binds the highly conserved "{beta}-signal" motif of EspP to correctly orient {beta}-strands in the OM during folding. We also found that the folding of EspP proceeds via remarkable "hybrid-barrel" intermediates in which membrane integrated {beta}-sheets are attached to the essential BAM subunit, BamA. The structures show an unprecedented deflection of the membrane surrounding the EspP intermediates and suggest that {beta}-sheets progressively fold towards BamA to form a {beta}-barrel. Along with in vivo experiments that tracked {beta}-barrel folding while the OM tension was modified, our results support a model in which BAM harnesses OM elasticity to accelerate {beta}-barrel folding.


title: Principles of Dopamine Binding to Carbon Surfaces

abstract:
Fast Scan Cyclic Voltammetry (FSCV) combined with carbon electrodes is considered as the gold standard method for real-time detection of oxidizable neurotransmitters. The bioinert nature, rapid electron transfer kinetics and long-term stability make carbon an attractive material for probing brain electrochemistry. Herein, we first demonstrate a rapid fabrication process of carbonized nanopipettes and subsequently perform experimental measurements and theoretical simulations to study mechanisms of dopamine binding on carbonized surfaces. To explain the kinetics of dopamine oxidation on carbonized electrodes we adapted the electron-proton transfer model originally developed by Compton and found that the electron-proton transfer model best explains the experimental observations. We further investigated the electron-proton transfer theory by constructing a Density Function Theory (DFT) for visualization of dopamine binding to graphite-like surfaces consisting of heteroatoms. For graphite surfaces that are capped with hydrogen alone, we found that dopamine is oxidized, whereas, on graphite surfaces doped with heteroatoms such as nitrogen and oxygen, we found deprotonation of dopamine along with oxidation thus validating our experimental and theoretical data. These observations provide mechanistic insights into multistep electron transfer during dopamine oxidation on graphite surfaces.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/457508v1_ufig1.gif" ALT="Figure 1">
View larger version (29K):
org.highwire.dtl.DTLVardef@8bee9corg.highwire.dtl.DTLVardef@de66ecorg.highwire.dtl.DTLVardef@1373032org.highwire.dtl.DTLVardef@3d61d0_HPS_FORMAT_FIGEXP  M_FIG A: Pictorial view of the experimental setup of carbonized electrodes. The application of waveform causes the oxidation of dopamine. B. Background subtracted voltammogram of dopamine, wherein the waveform applied is -0.4V to 1.3V and cycled back at -0.4V at 200 V s-1 at 10 Hz. C: A hotspot showing the oxidation and reduction of dopamine, wherein two distinct redox spots can be seen. The first redox spot can be seen at 0.0V and the second one at 0.5V. Thus showing a multistep electron transfer for dopamine. D: A DFT model for dopamines interaction with graphite surfaces doped with nitrogen atoms. Oxidation of oxygen (red) can be seen with loss of protons.

C_FIG


title: Spontaneous emergence of non-convertible cell states with CD24-High phenotype results in phenotypic heterogeneity that associates with poor prognosis in oral cancer

abstract:
PurposeCellular dynamics between phenotypically heterogeneous subpopulations of cancer cells within individual tumor is shown to be responsible for drug tolerance and overall poor prognosis; however, evidences were largely missing in oral cancer. Therefore, this study was undertaken to describe the dynamic phenotypic states among oral cancer cells, its influence on transcriptomic heterogeneity as well as its clinical significance.

Experimental DesignWe multiplexed phenotypic markers of putative oral-stem-like cancer cells (SLCCs) and characterized diversity among CD44-positive oral cancer cell subpopulations with respect to distinct expression of CD24 and aldehyde dehydrogenase (ALDH)-activity in multiple cell lines. Population trajectories were characterized by Markov model and cell states were defined based on the population specific RNA sequencing (RNAseq). ssGSEA based gene expression signatures were explore for prognostic significance.

ResultsOral cancer cells followed two distinct patterns of spontaneous repopulation dynamics with stochastic inter-conversions on  ALDH-axis, however a strict non-interconvertible transition on  CD24-axis. Interestingly, plastic  ALDH-axis was harnessed to enrich ALDHHigh subpopulations in response to Cisplatin treatment, to adapt a drug tolerant state. Phenotype-specific RNAseq results suggested the organization of subpopulations into hierarchical structure with possible maintenance of intermediate states of stemness within the differentiating oral cancer cells. Further, survival analysis with each subpopulation-specific gene signature strongly suggested that the cell-state dynamics may act as possible mechanism to drive ITH, resulting in poor prognosis in patient.

ConclusionsOur results emphasized the prognostic power of the population dynamics in oral cancer. Importantly, we have described the phenotypic-composition of heterogeneous subpopulations critical for global tumor behaviour in oral cancer; which is a prerequisite knowledge important for precision treatment, however largely lacking for most solid tumors.

Graphical AbstractWe have characterized diversity among CD44-positive oral cancer cells lines with respect to distinct expression of CD24 and ALDH-activity. Subpopulations showed stochastic inter-conversions on ALDH-axis but a strict non-interconvertible transition of CD24Low to CD24High phenotype, even in response to chemotherapy-induced stress. RNAseq study suggested the organization of subpopulations into hierarchical structure with possible maintenance of intermediate alternate states of stemness within the differentiating oral cancer cells. The described population dynamics demonstrtaed influence tumor behaviour possibly by increasing intratumoral heterogeneity in aggressive oral tumors.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/457509v3_ufig1.gif" ALT="Figure 1">
View larger version (20K):
org.highwire.dtl.DTLVardef@1dc6a7org.highwire.dtl.DTLVardef@dd0948org.highwire.dtl.DTLVardef@18c9603org.highwire.dtl.DTLVardef@cca8fb_HPS_FORMAT_FIGEXP  M_FIG C_FIG Translational relevanceIntratumoral heterogeneity (ITH) has been the clinically important factor, impacting aggressive cancer behaviour, drug tolerance and overall poor prognosis. Recent high-throughput studies have provided better cellular and molecular resolution of ITH; however, the prerequisite knowledge which defines the composition of subpopulations critical for global tumor behaviour is majorly lacking for most of the solid tumors. By combining phenotypic markers, we have defined four subpopulations of oral cancer cells. These subpopulations showed stochastic inter-conversions as well as a strict non-interconvertible transition among them to acheive heterogeneity. Importantly, transcriptional states of each subpopulations indicated a clinically relevant signatures for patient prognosis. Also, we observed interconversions of these subpopulations in response to Cisplatin to accumulate drug-tolerant cell state, as rapid and reversible strategy to respond to chemotherapy induced stress. Thus, the characteristics of described phenotypic subgroups may be translated to the clinic for estimating the extent of intratumoral heterogeneity in oral cancer patients.

category: cancer biology


title: Spontaneous differentiation leads to emergence of hybrid cell states relate to poor prognosis in oral cancer


title: PU.1 and MYC transcriptional network defines synergistic drug responses to KIT and LSD1 inhibition in acute myeloid leukemia

abstract:
Activating mutations in the KIT tyrosine receptor kinase confer an adverse prognosis for patients with acute myeloid leukemia (AML). Outside of bone marrow transplantation, treatment options are limited. Here we demonstrate combined KIT and LSD1 inhibition produces synergistic cell death against KIT mutant AML cells. This combination suppresses MYC expression to drive cell cycle exit and apoptosis. This decreased MYC expression results from a loss of PU.1 binding at downstream MYC enhancers. The drug combination also inactivates PI3K/AKT/GSK3a/b signaling to decrease MYC protein abundance. KIT-mutant AML cells rapidly adapt to KIT inhibitor monotherapy by restoring PI3K/AKT activity, but cannot when treated with combined KIT and LSD1 inhibitor. In addition, we validate MYC suppression as a mechanism of synergy in KIT-mutant AML patient samples. Collectively, this work provides rational for a clinical trial to assess the efficacy of KIT and LSD1 inhibition in patients with KIT-mutant AML.

Statement of significanceEffective treatment options for AML are limited. We describe the synergistic response to combined KIT and LSD1 inhibition in KIT-mutant AML and identify key biomarkers of drug response. The specificity and efficacy of this combination in cell lines and patient samples provides rationale for investigation in early phase clinical trials.


title: Defective ORF8 dimerization in delta variant of SARS CoV2 leads to abrogation of ORF8 MHC-I interaction and overcome suppression of adaptive immune response

abstract:
In India, the breakthrough infections during second wave of COVID-19 pandemic was due to SARS-COV-2 delta variant (B.1.617.2). It was reported that majority of the infections were caused by the delta variant and only 9.8% percent cases required hospitalization whereas, only 0.4% fatality was observed. Sudden dropdown in COVID-19 infections was observed within a short timeframe, suggesting better host adaptation with evolved delta variant. Down regulation of host immune response against SARS-CoV-2 by ORF8 induced MHC-I degradation has been reported earlier. The Delta variant carried mutations (deletion) at Asp119 and Phe120 amino acids which are critical for ORF8 dimerization. The deletions of amino acids Asp119 and Phe120 in ORF8 of delta variant results in structural instability of ORF8 dimer caused by disruption of hydrogen bonding and salt bridges as revealed by structural analysis and MD simulation studies of ORF8 dimer. Further, flexible docking of wild type and mutant ORF8 dimer revealed reduced interaction of mutant ORF8 dimer with MHC-I as compared to wild type ORF8 dimer with MHC-1, thus implicating its possible role in MHC-I expression and host immune response against SARS-CoV-2. We thus propose that mutant ORF8 may not hindering the MHC-I expression thereby resulting in better immune response against SARS-CoV-2 delta variant, which partly explains the sudden drop of SARS-CoV-2 infection rate in the second wave of SARS-CoV-2 predominated by delta variant in India

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/457457v1_ufig1.gif" ALT="Figure 1">
View larger version (40K):
org.highwire.dtl.DTLVardef@751eeaorg.highwire.dtl.DTLVardef@140b5b5org.highwire.dtl.DTLVardef@159a3a5org.highwire.dtl.DTLVardef@6c206_HPS_FORMAT_FIGEXP  M_FIG C_FIG

category: biophysics


title: Generating surrogates for significance estimation ofspatio-temporal spike patterns

abstract:
The generation of surrogate data, i.e., the modification of original data to destroy a certain feature, is used for the implementation of a null-hypothesis whenever an analytical approach is not feasible. Thus, surrogate data generation has been extensively used to assess the significance of spike correlations in parallel spike trains. In this context, one of the main challenges is to properly construct the desired null-hypothesis distribution and to avoid a bias in the null-hypothesis by altering the spike train statistics.

A classical surrogate technique is uniform dithering (UD), which displaces spikes locally and uniformly. In this study, we compare UD against five surrogate techniques (two newly introduced) in the context of the detection of significant spatio-temporal spike patterns. We evaluate the surrogates for their performance, first on spike trains based on point process models with constant firing rate, and second on modeled non-stationary artificial data serving as ground truth to assess the pattern detection in a more complex and realistic setting. We determine which statistical features of the original spike trains are modified and to which extent. Moreover, we find that UD fails as an appropriate surrogate because it leads to a loss of spikes in the context of binning and clipping, and thus to a large number of false-positive patterns. The other surrogates achieve a better performance in detecting precisely timed higher-order correlations. Based on these insights, we analyze experimental data from pre-/motor cortex of macaque monkeys during a reaching-and-grasping task for spatio-temporal spike patterns.

Significance statementTemporal jittering or dithering of single spikes or subsections of spike trains is a common method of generating surrogate data for the statistical analysis of temporal spike correlations. We discovered a serious problem with the classical and widely used method of uniform dithering that can lead to an overestimation of significance, i.e., to false positives in the statistical evaluation of spatio-temporal spike patterns. Therefore we consider 5 other dithering methods, compare and evaluate their statistical properties. Finally, we apply a much better method (trial shifting) to the analysis of experimental multiple-unit recordings and find several highly significant patterns that also reflect different experimental situations.

category: neuroscience


title: Comparing surrogates to evaluate precisely timed higher-order spike correlations


title: A yeast model of the ALS protein Matrin3 uncovers Hsp90 and its co-chaperone Sti1 as modifiers of misfolding and toxicity

abstract:
The MATR3 gene encoding the protein Matrin3 is implicated in the pathogenesis of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Matrin3 forms neuronal cytoplasmic and nuclear inclusions in ALS-affected neurons. Additionally, 13 heterozygous missense mutations in MATR3 are identified in ALS patients. To further explore Matrin3 misfolding and toxicity, we established and characterized a yeast model. We demonstrate that wild type Matrin3 and the ALS-associated variant F115C are toxic and form inclusions in yeast. Our further characterization uncovers substantial modification of Matrin3 toxicity and inclusion formation by Hsp90 and its co-chaperones, specifically Sti1. Thus, our study demonstrates how specific branches of cellular protein quality control regulate the misfolding and toxicity of Matrin3.

Summary StatementWe established and characterized a yeast model expressing human Matrin3, a protein implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Using this yeast model and mammalian neuronal cells, we showed that Matrin3 mislocalizes and forms inclusions, is cytotoxic, and increases sensitivity to cellular stress. We also uncovered that Hsp90 and particularly its co-chaperone Sti1 alter Matrin3 toxicity.


title: Landscaping the behavioural ecology of primate stone tool use

abstract:
Ecology is fundamental to the development, transmission, and perpetuity of primate technology. Previous studies on tool site selection have addressed the relevance of targeted resources and raw materials for tools, but few have considered the broader foraging landscape. In this first landscape-scale study of the ecological contexts of wild chimpanzee (Pan troglodytes verus) tool-use, we investigate the conditions required for nut-cracking to occur and persist over time at discrete locations in Bossou (Guinea). We examine this at three levels: selection, frequency of use, and inactivity. We find that, further to the presence of a nut tree and availability of raw materials, abundance of food-providing trees as well as proximity to nest sites were significant predictors of nut-cracking occurrence. This suggests that the spatial distribution of nut-cracking sites is mediated by the broader behavioural landscape and is influenced by non-extractive foraging of predictable resources, as well as non-foraging activities. Additionally, the number of functional tools was greater at sites with higher frequency of nut-cracking and was negatively correlated with site inactivity. Our findings indicate that the technological landscape of the Bossou chimpanzees shares affinities with the  favoured places model of hominin site formation and provides new insights for reconstructing ancient patterns of landscape use.

ResumeLecologie est fondamentale pour le developpement, la transmission et la perennite de la technologie des primates. Des etudes anterieures ont identifie la disponibilite des ressources cibles ainsi que les matieres premieres pour les outils comme des facteurs influents dans la selection des emplacements pour les activites technologiques. Cependant, il y a peu detudes qui abordent cette recherche a lechelle du paysage et du comportement fourrager. Dans cette premiere etude paysagere sur lutilisation doutils par le chimpanze sauvage (Pan troglodytes verus), nous recherchons les conditions ecologiques qui influencent la selection, lutilisation et linactivite des emplacements utilises pour le cassage des noix en Bossou, Guinee. Nos resultats montrent quen plus de la presence dun noyer et de la disponibilite des matieres premieres, labondance darbres nourriciers ainsi que la proximite des sites de nidification etaient des predicteurs significatifs de loccurrence du cassage des noix. Cela suggere que la distribution spatiale des sites de cassage de noix est influencee par le paysage comportemental et est influencee par le fourrage non-extractive de ressources previsibles, ainsi que par des activites non-fourragers. Nos resultats indiquent que le paysage technologique des chimpanzes de Bossou partage des affinites avec le modele des << lieux favoris >> de la formation des sites hominides et fournit de nouvelles perspectives pour reconstruire les modes dutilisation du paysage anciens.

category: ecology


title: The Evolution of Nest Site Specialization and its Macroevolutionary Consequences in Passerine Birds

abstract: A long-standing hypothesis in evolutionary biology is that the evolution of resource specialization can lead to an evolutionary dead end, where specialists have low diversification rates and limited ability to evolve into generalists. However, in recent years, advances in comparative methods investigating trait-based differences associated with diversification have enabled more robust tests of this idea and have found mixed support. Here we test the evolutionary dead end hypothesis by estimating net diversification rate differences associated with nest site specialization among 3,224 species of passerine birds. In particular, we test whether the adoption of hole-nesting, a nest site specialization that decreases predation, results in reduced diversification rates relative to nesting outside of holes. Further, we examine whether evolutionary transitions to the specialist hole-nesting state have been more frequent than transitions out of hole-nesting. Using diversification models that accounted for background rate heterogeneity and different extinction rate scenarios, we found that hole-nesting specialization was not associated with diversification rate differences. Furthermore, contrary to the assumption that specialists rarely evolve into generalists, we found that transitions out of hole-nesting occur more frequently than transitions into hole-nesting. These results suggest that interspecific competition may limit adoption of hole-nesting, but that such competition does not result in limited diversification of hole-nesters. In conjunction with other recent studies using robust comparative methods, our results add to growing evidence that evolutionary dead ends are not a typical outcome of resource specialization.

category: evolutionary biology


title: VTA-projecting cerebellar neurons mediate stress-dependent depression-like behavior

abstract: Although cerebellar alterations have been implicated in mental depression, the exact contribution of the cerebellum to depressive symptoms remains to be elucidated. Here, we demonstrated the crucial role of cerebellar neurons projecting to the ventral tegmental area (VTA) in the chronic stress-induced development of depression-like behavior. The combination of adeno-associated virus-based circuit mapping and electrophysiological recording identified network connections from crus I to the VTA via the dentate nucleus (DN) of the deep cerebellar nuclei. Chronic chemogenetic activation of inhibitory Purkinje cells in crus I suppressed c-Fos expression in the DN and depression-like behavior, which were triggered by chronic stress application. Furthermore, specific inhibition of neurons in the DN that project to the VTA prevented stressed mice from showing depression-like behavior, whereas specific activation of these neurons alone triggered depression-like behavior that was comparable with the one triggered by chronic stress application. Our results indicate that the VTA-projecting cerebellar neurons proactively regulate depression-like behavior, raising the possibility that cerebellum may be an effective target for the prevention of depressive disorders.


title: Electrochemical Detection of Adrenaline and Hydrogen Peroxide on Carbon Nanotube Electrodes

abstract: Adrenaline and hydrogen peroxide have neuromodulatory functions in the brain. Considerable interest exists in developing electrochemical sensors that can detect their levels in vivo due to their important biochemical roles. Challenges associated with electrochemical detection of hydrogen peroxide and adrenaline are that the oxidation of these molecules usually requires highly oxidising potentials (beyond 1.4 V vs Ag/AgCl) where electrode damage and biofouling are likely and the signals of adrenaline, hydrogen peroide and adenosine overlap. To address these issues we fabricated pyrolysed carbon electrodes coated with oxidised carbon nanotubes (CNTs). Using these electrodes for fast-scan cyclic voltammetric (FSCV) measurements showed that the electrode offers reduced overpotentials compared with graphite and improved resistance to biofouling. The Adrenaline peak is reached at 0.75({+/-}0.1) V and reduced back at -0.2({+/-}0.1) V while hydrogen peroxide is detected at 0.85({+/-}0.1) V on this electrode. The electrodes are highly sensitive with a sensitivity of 16nA {micro}M-1 for Adrenaline and 11nA {micro}M-1 for hydrogen peroxide on an 80 {micro}m2 electrode. They are also suitable to distinguish between adrenaline, hydrogen peroxide and adenosine thus these probes can be used for multimodal detection of analytes.


title: Production and composition of group B streptococcal membrane vesicles varies across diverse lineages

abstract: Although the neonatal and fetal pathogen Group B Streptococcus (GBS) asymptomatically colonizes the vaginal tract of [~]30% of pregnant women, only a fraction of their offspring develops invasive disease. We and others have postulated that these dimorphic clinical phenotypes are driven by strain variability; however, the bacterial factors that promote these divergent clinical phenotypes remain unclear. It was previously shown that GBS produces membrane vesicles (MVs) that contain active virulence factors capable of inducing adverse pregnancy outcomes. Because the relationship between strain variation and vesicle composition or production is unknown, we sought to quantify MV production and examine the protein composition, using label-free proteomics on MVs produced by diverse clinical GBS strains representing three phylogenetically distinct lineages. We found that MV production varied across strains, with certain strains displaying nearly two-fold increases in production relative to others. Hierarchical clustering and principal component analysis of the proteomes revealed that MV composition is lineage-dependent but independent of clinical phenotype. Multiple proteins that contribute to virulence or immunomodulation, including hyaluronidase, C5a peptidase, and sialidases, were differentially abundant in MVs, and were partially responsible for this divergence. Together, these data indicate that production and composition of GBS MVs vary in a strain-dependent manner, suggesting that MVs have lineage-specific functions relating to virulence. Such differences may contribute to variation in clinical phenotypes observed among individuals infected with GBS strains representing distinct lineages.


title: bletl - A Python package for integrating microbioreactors in the design-build-test-learn cycle

abstract:
Microbioreactor (MBR) devices have emerged as powerful cultivation tools for tasks of microbial phenotyping and bioprocess characterization and provide a wealth of online process data in a highly parallelized manner. Such datasets are difficult to interpret in short time by manual workflows. In this study, we present the Python package bletl and show how it enables robust data analyses and the application of machine learning techniques without tedious data parsing and preprocessing. bletl reads raw result files from BioLector I, II and Pro devices to make all the contained information available to Python-based data analysis workflows. Together with standard tooling from the Python scientific computing ecosystem, interactive visualizations and spline-based derivative calculations can be performed. Additionally, we present a new method for unbiased quantification of time-variable specific growth rate [Formula] based on a novel method of unsupervised switchpoint detection with Student-t distributed random walks. With an adequate calibration model, this method enables practitioners to quantify time-variable growth rate with Bayesian uncertainty quantification and automatically detect switch-points that indicate relevant metabolic changes. Finally, we show how time series feature extraction enables the application of machine learning methods to MBR data, resulting in unsupervised phenotype characterization. As an example, t-distributed Stochastic Neighbor Embedding (t-SNE) is performed to visualize datasets comprising a variety of growth/DO/pH phenotypes.

Practical ApplicationThe bletl package can be used to analyze microbioreactor datasets in both data analysis and autonomous experimentation workflows. Using the example of BioLector datasets, we show that loading such datasets into commonly used data structures with one line of Python code is a significant improvement over spreadsheet or hand-crafted scripting approaches. On top of established standard data structures, practitioners may continue with their favorite data analysis routines, or make use of the additional analysis functions that we specifically tailored to the analysis of microbioreactor time series.

Particularly our function to fit cross-validated smoothing splines can be used for on-line signals from any microbioreactor system and has the potential to improve robustness and objectivity of many data analyses. Likewise, our random walk based [Formula] method for inferring growth rates under uncertainty, but also the time-series feature extraction may be applied to on-line data from other cultivation systems as well.

category: bioengineering


title: Mining folded proteomes in the era of accurate structure prediction

abstract: Protein structure fundamentally underpins the function and processes of numerous biological systems. Fold recognition algorithms offer a sensitive and robust tool to detect structural, and thereby functional, similarities between distantly related homologs. In the era of accurate structure prediction owing to advances in machine learning techniques, previously curated sequence databases have become a rich source of biological information. Here, we use bioinformatic fold recognition algorithms to scan the entire AlphaFold structure database to identify novel protein family members, infer function and group predicted protein structures. As an example of the utility of this approach, we identify novel, previously unknown members of various pore-forming protein families, including MACPFs, GSDMs and aerolysin-like proteins. Further, we explore the use of structure-based mining for functional inference.

category: bioinformatics


title: Systemic regulation of nitrogen acquisition and use in Oryza longistaminata ramets under nitrogen heterogeneity

abstract:
Oryza longistaminata, a wild rice, vegetatively reproduces and forms a networked clonal colony consisting of ramets connected by rhizomes. Although water, nutrients, and other molecules can be transferred between ramets via the rhizomes, inter-ramet communication in response to spatially heterogeneous nitrogen availability is not well understood. We studied the response of ramet pairs to heterogeneous nitrogen availability by using a split hydroponic system that allowed each ramet root to be exposed to different conditions. Ammonium uptake was compensatively enhanced in the sufficient-side root when roots of the ramet pairs were exposed to ammonium-sufficient and deficient conditions. Comparative transcriptome analysis revealed that a gene regulatory network for effective ammonium assimilation and amino acid biosynthesis was activated in the sufficient-side roots. Allocation of absorbed nitrogen from the nitrogen-sufficient to the deficient ramets was rather limited. Nitrogen was preferentially used for newly growing axillary buds on the sufficient-side ramets. Biosynthesis of trans-zeatin, a cytokinin, was up-regulated in response to the nitrogen supply, but trans-zeatin appears not to target the compensatory regulation. Our results also implied that the O. longistaminata ortholog of OsCEP1 plays a role as a nitrogen-deficient signal in inter-ramet communication, providing compensatory up-regulation of nitrogen assimilatory genes. These results provide insights into the molecular basis for efficient growth strategies of asexually proliferating plants growing in areas where the distribution of ammonium ions is spatially heterogeneous.

One sentence summaryOryza longistaminata, a rhizomatous wild rice, systemically regulates ammonium acquisition and use in response to spatially heterogeneous nitrogen availability via inter-ramet communication.

category: plant biology


title: Regulation of ammonium acquisition and use in Oryza longistaminata ramets under nitrogen source heterogeneity


title: 24-nt phasiRNAs move from tapetal to meiotic cells in maize anthers

abstract:
O_LIIn maize, 24-nt phased, secondary small interfering RNAs (phasiRNAs) are abundant in meiotic stage anthers, but their distribution and functions are not precisely known.
C_LIO_LIUsing laser capture microdissection, we analyzed tapetal cells, meiocytes, and other somatic cells at several stages of anther development to establish the timing of 24-PHAS precursor transcripts and the 24-nt phasiRNA products.
C_LIO_LIBy integrating RNA and small RNA profiling plus single-molecule and small RNA FISH (smFISH or sRNA-FISH) spatial detection, we demonstrate that the tapetum is the primary site of 24-PHAS precursor and Dcl5 transcripts and the resulting 24-nt phasiRNAs. Interestingly, 24-nt phasiRNAs accumulate in all cell types, with the highest levels in meiocytes, followed by tapetum.
C_LIO_LIOur data support the conclusion that 24-nt phasiRNAs are mobile from tapetum to meiocytes and to other somatic cells. We discuss possible roles for 24-nt phasiRNAs in anther cell types.
C_LI


title: ICOS expression is required for maintenance but not the formation of germinal centers in the spleen in response to P. yoelii infection.

abstract: Inducible T cell co-stimulator (ICOS) plays a key role in the differentiation and maintenance of follicular helper T (Tfh) cells and thus germinal center (GC) formation. Previously, our lab showed in a Plasmodium chabaudi infection model that Icos-/- mice did not form GCs despite a persistent infection and thus a continued antigen (Ag) load. Here, we show that resolution of a primary infection with P. yoelii, was delayed in Icos-/- mice. This phenotype was associated with a reduction in the accumulation of Tfh-like and GC Tfh cells and an early deficiency in Ag-specific antibody (Ab) production. However, Icos-/- mice maintained their ability to form GCs, though they were less frequent in number than in wild-type (WT) mice. Furthermore, while Ab production in Icos-/- mice matched that of WT mice after the infection cleared, the Abs lacked signs of affinity maturation, suggesting functional defects associated with these GCs. Eventually, these GC structures dissipated more rapidly in Icos-/- mice than in WT mice. Moreover, the ability of Icos-/- mice to form these GC structures is not reliant on the high Ag load associated with P. yoelii infections, as GC formation was preserved in Icos-/- mice treated with early with atovaquone. Finally, mice were unable to form secondary GCs in the absence of ICOS after re-challenge. Overall, these data demonstrate the necessity of ICOS in the maintenance of Tfh cells, the formation and maintenance of sufficient numbers of functioning GCs, and the ability to generate new GC structures after re-infection with P. yoelii.

category: immunology


title: The cyclin Cln1 controls polyploid titan cell formation following a stress-induced G2 arrest in Cryptococcus

abstract:
The pathogenic yeast Cryptococcus neoformans produces polyploid titan cells in response to the host lung environment that are critical for host adaptation and subsequent disease. We analyzed the in vivo and in vitro cell cycles to identify key aspects of the C. neoformans cell cycle that are important for the formation of titan cells. We identified unbudded 2C cells, referred to as a G2 arrest, produced both in vivo and in vitro in response to various stresses. Deletion of the non-essential cyclin Cln1 resulted in over-production of titan cells in vivo, and transient morphology defects upon release from stationary phase in vivo. Using a copper-repressible promoter PCTR4-CLN1 strain and a two-step in vitro titan cell formation assay, our in vitro studies revealed Cln1 functions after the G2 arrest. These studies highlight unique cell cycle alterations in C. neoformans that ultimately promote genomic diversity and virulence in this important fungal pathogen.

ImportanceDysregulation of the cell cycle underlies many human genetic diseases and cancers. Yet numerous organisms, including microbes, also manipulate the cell cycle to generate both morphologic and genetic diversity as a natural mechanism to enhance their chances for survival. The eukaryotic pathogen Cryptococcus neoformans generates morphologically distinct polyploid titan cells critical for host adaptation and subsequent disease. We analyzed the C. neoformans in vivo and in vitro cell cycles to identify changes required to generate the polyploid titan cells. C. neoformans paused cell cycle progression in response to various environmental stresses after DNA replication and before morphological changes associated with cell division, referred to as a G2 arrest. Release from this G2 arrest was coordinated by the cyclin Cln1. Reduced CLN1 expression after the G2 arrest was associated with polyploid titan cell production. These results demonstrate a mechanism to generate genomic diversity in eukaryotic cells through manipulation of the cell cycle that has broad disease implications.


title: Neutrophil-associated responses to Vibrio cholerae infection in a natural host model

abstract: Vibrio cholerae, the cause of human cholera, is an aquatic bacterium found in association with a variety of animals in the environment, including many teleost fish species. V. cholerae infection induces a pro-inflammatory response followed by a non-inflammatory convalescent phase. Neutrophils are integral to this early immune response. However, the relationship between the neutrophil-associated protein calprotectin and V. cholerae has not been investigated, nor have the effects of limiting transition metals on V. cholerae growth. Zebrafish are useful as a natural V. cholerae model as the entire infectious cycle can be recapitulated in the presence of an intact intestinal microbiome and mature immune responses. Here, we demonstrate that zebrafish produce a significant neutrophil, IL-8, and calprotectin response following V. cholerae infection. Bacterial growth was completely inhibited by purified calprotectin protein or the chemical chelator TPEN, but growth was recovered by addition of transition metals zinc and manganese. Expression of downstream calprotectin targets also significantly increased in the zebrafish. These findings are the first to illuminate the role of calprotectin and nutritional immunity in combating V. cholerae infection. Inhibition of V. cholerae growth through metal limitation may provide new approaches in the development of anti-V. cholerae therapeutics. This study also establishes a major role for calprotectin in combating infectious diseases in zebrafish.


title: Warming decreases host survival with multiple parasitoids, but parasitoid performance also decreases

abstract: Current global changes are reshaping ecological communities and modifying environmental conditions. We need to recognize the combined impact of these biotic and abiotic factors on species interactions, community dynamics, and ecosystem functioning. Specifically, the strength of predator-prey interactions often depends on the presence of other natural enemies: it weakens with competition and interference or strengthens with facilitation. Such effects of multiple predators on prey are likely to be affected by changes in the abiotic environment, altering top-down control, a key structuring force in natural and agricultural ecosystems. Here, we investigated how warming alters the effects of multiple predators on prey suppression using a dynamic model coupled with empirical laboratory experiments with Drosophila-parasitoid communities. While multiple parasitoids enhanced top-down control under warming, parasitoid performance generally declined when another parasitoid was present due to competitive interactions. This could reduce top-down control over multiple generations. Our study highlights the importance of accounting for interactive effects between abiotic and biotic factors to better predict community dynamics in a rapidly changing world and thus better preserve ecosystem functioning and services such as biological control.


title: CDCP: a visualization and analyzing platform for single-cell datasets

abstract: Advances in single-cell sequencing technology provide a unique approach to characterize the heterogeneity and distinctive functional states at single-cell resolution, leading to rapid accumulation of large-scale single-cell datasets. A big challenge undertaken by research community especially bench scientists is how to simplify the way of retrieving, processing and analyzing the huge number of datasets. Towards this end, we developed Cell-omics Data Coordinate Platform (CDCP), a platform that aims to share and integrate comprehensive single-cell datasets, and to provide a network analysis toolkit for personalized analysis. CDCP contains single-cell RNA-seq and ATAC-seq datasets of 474 572 cells from 6 459 samples in species covering humans, non-human primate models and other animals. It allows querying and visualization of interested datasets and the expression profile of distinct genes in different cell clusters and cell types. Besides, this platform provides an analysis pipeline for non-bioinformatician experimental scientists to address questions not focused by the submitters of the datasets. In summary, CDCP provides a user-friendly interface for researchers to explore, visualize, analyze, download and submit published single-cell datasets and it will be a valuable resource for investigators to explore the global transcriptome profiling at single-cell level.


title: Structural mechanism underpinning Thermus oshimai Pif1-mediated G-quadruplex unfolding

abstract: G-quadruplexes (G4s) are unusual DNA structures and can stall DNA replication, causing genomic instability for the cell. Although the solved crystal structure of the DHX36 helicase demonstrated that G4 was specifically targeted by a DHX36-specific motif (DSM), lack of complete structural details for general G4-resolving helicases without specific target motifs remains a barrier to the complete understanding of the molecular basis underlying the recognition and unfolding of G4s. Herein, we present the first X-ray crystal structure of the Thermus oshimai Pif1 (ToPif) complexed with a G4, thereby mimicking the physiological G4 formed during DNA replication. Strictly different from the previous determined G4-helicase structure of DHX36, our structure revealed that ToPif1 recognizes the entire native G4 via a cluster of amino acids at domains 1B/2B constituting a G4-Recognizing Surface (GRS). The overall topology of the G4 structure solved in this work maintains its three-layered propeller-type G4 topology, with no significant reorganization of G-tetrads upon protein binding. The three G-tetrads in G4 were differentially recognized by GRS residues mainly through electrostatic, ionic interactions and hydrogen bonds formed between the GRS residues and the ribose-phosphate backbone. Our structure explains how helicases from distinct superfamilies adopt different strategies for recognizing and unfolding G4s.


title: Phospholipase Dα1 acts as a negative regulator of high Mg2+-induced leaf senescence in Arabidopsis

abstract: Magnesium is a macronutrient involved in essential cellular processes. Its deficiency or excess is a stress factor for plants, seriously affecting their growth and development and therefore, its accurate regulation is essential. Recently, we discovered that phospholipase D1 (PLD1) activity is vital in the stress response to high-magnesium conditions in Arabidopsis roots. This study shows that PLD1 acts as a negative regulator of high-Mg2+-induced leaf senescence in Arabidopsis. The level of phosphatidic acid produced by PLD1 and the amount of PLD1 in the leaves increase in plants treated with high Mg2+. A knockout mutant of PLD1 (plda1-1), exhibits premature leaf senescence under high-Mg2+ conditions. In pld1-1 plants, higher accumulation of abscisic and jasmonic acid and impaired magnesium, potassium and phosphate homeostasis were observed under high-Mg2+ conditions. High Mg2+ also led to an increase of starch and proline content in Arabidopsis plants. While the starch content was higher in plda1-1 plants, proline content was significantly lower in plda1-1 compared with WT. Our results show that PLD1 is essential for Arabidopsis plants to cope with the pleiotropic effects of high-Mg2+ stress and delay the leaf senescence.


title: Genome-wide characterization of the R2R3-MYB transcription factors in pepper (Capsicum spp.) unveils the role of CaMYB101 as repressor in anthocyanin biosynthesis

abstract: Fruit colour is one of the most important commercial traits of pepper (Capsicum spp.), a major horticultural crop worldwide. Some pepper accessions temporarily accumulate anthocyanins during fruit development and gradually lose them upon fruit ripening. Meanwhile, anthocyanin biosynthesis gradually stops. However, how this process is exactly regulated is still largely unknown. R2R3-MYB is one of the largest plant transcription factor families, and it is considered the most important regulator for the biosynthesis of anthocyanins and other flavonoids. Although R2R3-MYBs are widely studied in many plants, research in pepper has been limited. In this study, we performed a genome-wide analysis of R2R3-MYBs across three cultivated pepper species (C. annuum, C. baccatum, and C. chinense ) involving identification, chromosome localization, gene structure analysis, phylogenetic analysis and collinearity analysis. Candidate R2R3-MYB repressors were further identified based on repression motifs. An R2R3-MYB gene, CaMYB101, was selected based on its high homology with anthocyanin biosynthesis repressors in tomato and petunia as well as its high expression level in fruit when purple pigmentation started to discolour. By using virus-induced gene silencing, CaMYB101 was characterized as an anthocyanin biosynthesis repressor. To our knowledge, CaMYB101 is the first transcriptional repressor associated with anthocyanin biosynthesis identified in pepper.


title: Mean-field theory accurately captures the variation of copy number distributions across the mRNA's life cycle

abstract: We consider a stochastic model where a gene switches between two states, an mRNA transcript is released in the active state and subsequently it undergoes an arbitrary number of sequential unimolecular steps before being degraded. The reactions effectively describe various stages of the mRNA life cycle such as initiation, elongation, termination, splicing, export and degradation. We construct a novel mean-field approach that leads to closed-form steady-state distributions for the number of transcript molecules at each stage of the mRNA life cycle. By comparison with stochastic simulations, we show that the approximation is highly accurate over all of parameter space, independent of the type of expression (constitutive or bursty) and of the shape of the distribution (unimodal, bimodal and nearly bimodal). The theory predicts that in a population of identical cells, any bimodality is gradually washed away as the mRNA progresses through its life cycle.

category: systems biology


title: CD72-semaphorin3A axis: a possible new player in immune regulation

abstract:
Semaphorin3A (sema3A) inhibits the activity of B and T cells in autoimmune diseases such as Systemic Lupus Erythematosus (SLE). We have now found that CD72 functions as a novel sema3A binding and signal-transducing receptor. These functions of CD72 are independent of the known sema3A receptor neuropilin-1 (NRP-1). We find that sema3A induces the phosphorylation of CD72 on tyrosine residues and the association of CD72 with SHP-1 and SHP-2. In contrast, sema4D/CD100 inhibits these functions. sema3A signals mediated by CD72, inhibit the phosphorylation of STAT-4 and HDAC-1 and induce the phosphorylation of p38-MAPK and PKC-theta in B-cells derived B-lymphoblastoid (BLCL) cells lacking NRP-1 expression, and in primary B-cells isolated from either healthy donors or SLE (Systemic Lupus Erythematosus) patients. We have also generated a modified truncated sema3A (T-sema3A) which cannot signal via NRP-1 yet still activates inhibitory CD72 signaling. We propose that T-sema3A may have potential as a possible therapeutic for autoimmune diseases such as SLE.

One Sentence SummaryCD72 found as a novel sema3A receptor transduces inhibitory signals in Bcells. A modified sema3A can be used to treat autoimmunity.


title: Plant Polymerase IV sensitizes chromatin through histone modifications to preclude spread of silencing into protein-coding domains

abstract:
Heterochromatin is the predominant architectural feature of genomes that ensures genomic stability across eukaryotes. It mostly functions in restricting expression of repeats, mobile elements such as transposons and other regions. The establishment, maintenance and spreading of heterochromatin requires several factors including chromatin modifiers. However, how exactly heterochromatin formation is avoided in protein-coding domains is poorly understood. Here we show that a plant specific paralogue of RNA polymerase (pol) II, named pol IV, is involved in avoidance of facultative heterochromatic marks in protein coding genes, in addition to silencing the repeats and transposons forming constitutive heterochromatin. In its absence, H3K27 trimethylation mark intrudes the protein coding genes, more profoundly in genes embedded with repeats. In a subset of genes that lack the compensatory silencing, spurious transcriptional activity results in small(s)RNA production leading to post-transcriptional gene silencing. We show that such effects are significantly pronounced in rice, a plant with larger genome with distributed heterochromatin when compared to Arabidopsis. Our results indicate the surprising division of labour among plant-specific polymerases, not just in establishing effective silencing via small RNAs and epigenetics, but also in influencing chromatin boundaries.

O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

One-line summaryRNA polymerase IV maintains chromatin states to influence regulation of protein coding genes.


title: Characterization of a novel Lbx1 loss of function mouse line

abstract: Adolescent Idiopathic Scoliosis (AIS) is the most common type of spine deformity affecting 2-3% of the population worldwide. The etiology of this disease is still poorly understood. Several GWAS studies have identified single nucleotide polymorphisms (SNPs) located near the gene LBX1 that is significantly correlated with AIS risk. LBX1 is a transcription factor with roles in myocyte precursor migration, cardiac neural crest specification, and neuronal fate determination in the neural tube. Here, we further investigated the role of LBX1 in the developing spinal cord of mouse embryos using a CRISPR-generated mouse model expressing a truncated version of LBX1 (Lbx1{Delta}). Homozygous mice died at birth, likely due to cardiac abnormalities. To further study the neural tube phenotype, we used RNA-sequencing to identify 410 genes differentially expressed between the neural tubes of E12.5 wildtype and Lbx1{Delta}/{Delta} embryos. Genes with increased expression in the deletion line were involved in neurogenesis and those with broad roles in embryonic development. Many of these genes have also been associated with scoliotic phenotypes. In comparison, genes with decreased expression were primarily involved in skeletal development. Subsequent skeletal and immunohistochemistry analysis further confirmed these results. This study aids in understanding the significance of links between Lbx1 function and AIS susceptibility.


title: Hematopoiesis at single cell resolution spanning human development and maturation

abstract:
Hematopoiesis is a process of constitutive regeneration whereby hematopoietic stem and progenitor cells (HSPCs) replenish mature blood cells. During maturation and aging, HSPCs shift their output to support the demands of prenatal development and postnatal maturation both at homeostasis and in response to stress. How HSPC ontogeny changes throughout life is unknown; studies to date have largely focused on specific individual ages, particularly at single cell resolution. Here, we performed single cell RNA-seq of human HSPCs from early prenatal development into mature adulthood. We observed shifts in HSPC transcriptional states and differentiation trajectories over time. We identified age-specific gene expression patterns throughout human maturation and developed methods for identifying, prospectively purifying, and functionally validating age-specific HSC states. Together, our findings define the temporal maturation of human HSPCs and uncover principles applicable to age-biased blood diseases.

SummarySingle cell RNA sequencing reveals that the mechanisms of human hematopoietic stem and progenitor cell (HSPC) fate commitment change over a lifetime from gestation to mature adulthood.


title: Recursive feedforward regulatory logic underlies robustness of the specification-to-differentiation transition and fidelity of terminal cell fate during C. elegans endoderm development

abstract:
Development is driven by gene regulatory networks (GRNs) that progressively dictate specification and differentiation of cell fates. The architecture of GRNs directly determines the specificity and accuracy of developmental outcomes. We report here that the core regulatory circuitry for endoderm development in C. elegans is comprised of a recursive series of interlocked feedforward modules linking a cascade of six sequentially expressed GATA-type transcription factors. This structure results in a reiterated sequential redundancy, in which removal of a single factor or alternate factors in the cascade results in no, or a mild, effect on endoderm development and gut differentiation, while elimination of any two factors that are sequentially deployed in the cascade invariably results in a strong phenotype. The strength of the observed phenotypes is successfully predicted by a computational model based on the timing and levels of transcriptional states. The feedforward regulatory logic in the GRN appears to ensure timely onset of terminal differentiation genes and allows rapid and robust lockdown of cell fate during early embryogenesis. We further found that specification-to-differentiation transition is linked through a common regulator, the END-1 GATA factor that straddles the two processes. Finally, we revealed roles for key GATA factors in establishing spatial regulatory state domains by acting as transcriptional repressors that appear to define the boundaries of the digestive tract. Our findings support a comprehensive model of the core gene network that describes how robust endoderm development is achieved during C. elegans embryogenesis.

Graphic abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/457588v2_ufig1.gif" ALT="Figure 1">
View larger version (23K):
org.highwire.dtl.DTLVardef@8515d6org.highwire.dtl.DTLVardef@15ca234org.highwire.dtl.DTLVardef@9d0fe7org.highwire.dtl.DTLVardef@17f28db_HPS_FORMAT_FIGEXP  M_FIG C_FIG


title: Feedforward regulatory logic underlies robustness of the specification-to-differentiation transition and fidelity of terminal cell fate during C. elegans endoderm development


title: Membrane Constriction by ESCRT-III Proteins Induces Structural Lipid Bilayer Asymmetries

abstract:
Lipids in biological membranes are thought to be functionally organized, but few experimental tools can probe nanoscale membrane structure. Using brominated lipids as contrast probes for cryo-EM and a model ESCRT-III membrane remodeling system, we observed leaflet-level and protein-localized lipid structural patterns within highly constricted and thinned membrane nanotubes. These nanotubes differed markedly from protein-free, flat bilayers in leaflet thickness, lipid diffusion rates, and lipid compositional and conformational asymmetries. Simulations and cryo-EM imaging of brominated SDPC showed how a pair of phenylalanine residues scored the outer leaflet with a helical hydrophobic defect where polyunsaturated docosahexaenoyl (DHA) tails accumulated at the bilayer surface. Combining cryo-EM of halogenated lipids with molecular dynamics thus enables new characterizations of the composition and structure of membranes on molecular length scales.

One-Sentence SummaryCryo-EM imaging of halogenated lipids and MD simulations provide molecular-scale insight into constricted bilayer structure.


title: Brominated Lipid Probes Expose Structural Asymmetries in Constricted Membranes


title: Mismatch repair deficiency is not sufficient to increase tumor immunogenicity

abstract: DNA mismatch repair deficiency (MMRd) in human cancer is associated with high tumor mutational burden (TMB), frameshift mutation-derived neoantigens, increased T cell infiltration, and remarkable responsiveness to immune checkpoint blockade (ICB) therapy. Nevertheless, about half of MMRd tumors do not respond to ICB for unclear reasons. While tumor cell line transplant models of MMRd have reinforced the importance of TMB in immune response, critical questions remain regarding the role of immunosurveillance in the evolution of MMRd tumors induced in vivo. Here, we developed autochthonous mouse models of lung and colon cancer with highly efficient ablation of MMR genes via in vivo CRISPR/Cas9 targeting. Surprisingly, MMRd in these models did not result in increased immunogenicity or response to ICB. Mechanistically, we showed this lack of immunogenicity to be driven by profound intratumoral heterogeneity (ITH). Studies in animals depleted of T cells further demonstrated that immunosurveillance in MMRd tumors has no impact on TMB but shapes the clonal architecture of neoantigens by exacerbating ITH. These results provide important context for understanding immune evasion in cancers with high TMB and have major implications for therapies aimed at increasing TMB.


title: TopoRoot: A method for computing hierarchy and fine-grained traits of maize roots from X-ray CT images

abstract:
Background3D imaging, such as X-ray CT and MRI, has been widely deployed to study plant root structures. Many computational tools exist to extract coarse-grained features from 3D root images, such as total volume, root number and total root length. However, methods that can accurately and efficiently compute fine-grained root traits, such as root number and geometry at each hierarchy level, are still lacking. These traits would allow biologists to gain deeper insights into the root system architecture (RSA).

ResultsWe present TopoRoot, a high-throughput computational method that computes fine-grained architectural traits from 3D X-ray CT images of field-excavated maize root crowns. These traits include the number, length, thickness, angle, tortuosity, and number of children for the roots at each level of the hierarchy. TopoRoot combines state-of-the-art algorithms in computer graphics, such as topological simplification and geometric skeletonization, with customized heuristics for robustly obtaining the branching structure and hierarchical information. TopoRoot is validated on both real and simulated root images, and in both cases it was shown to improve the accuracy of traits over existing methods. We also demonstrate TopoRoot in differentiating a maize root mutant from its wild type segregant using fine-grained traits. TopoRoot runs within a few minutes on a desktop workstation for volumes at the resolution range of 400^3, without need for human intervention.

ConclusionsTopoRoot improves the state-of-the-art methods in obtaining more accurate and comprehensive fine-grained traits of maize roots from 3D CT images. The automation and efficiency makes TopoRoot suitable for batch processing on a large number of root images. Our method is thus useful for phenomic studies aimed at finding the genetic basis behind root system architecture and the subsequent development of more productive crops.


title: A novel multifunctional role for Hsp70 in binding post-translational modifications on clients

abstract:
Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and inability of current technologies to distinguish direct vs bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the budding yeast Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multi-point interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors which can be used to probe chaperone function in cells, we have also identified a suite of PTM-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically-important PTMs on client proteins.

O_LIIn vivo confirmation of Hsp70 dimerization
C_LIO_LIComprehensive direct interactome of Hsp70
C_LIO_LIMulti-domain interactions between Hsp70 and client proteins
C_LIO_LIIdentification of novel biologically-important client protein PTMs
C_LI


title: Calpain DEK1 acts as a developmental switch gatekeeping cell fate transitions

abstract: Calpains are cysteine proteases that control cell fate transitions. Although calpains are viewed as modulatory proteases displaying severe, pleiotropic phenotypes in eukaryotes, human calpain targets are also directed to the N-end rule degradatory pathway. Several of these destabilized targets are transcription factors, hinting at a gene regulatory role. Here, we analyze the gene regulatory networks of Physcomitrium patens and characterize the regulons that are deregulated in DEK1 calpain mutants. Predicted cleavage patterns of regulatory hierarchies in the five DEK1-controlled subnetworks are consistent with the genes pleiotropy and the regulatory role in cell fate transitions targeting a broad spectrum of functions. Network structure suggests DEK1-gated sequential transition between cell fates in 2D to 3D development. We anticipate that both our method combining phenotyping, transcriptomics and data science to dissect phenotypic traits and our model explaining the calpains role as a switch gatekeeping cell fate transitions will inform biology beyond plant development.


title: Developmental timing of Drosophila pachea pupae is robust to temperature changes

abstract: Rearing temperature is correlated with the timing and speed of development in a wide range of poikiloterm animals that do not regulate their body temperature. However, exceptions exist, especially in species that live in environments with high temperature extremes or oscillations. Drosophila pachea is endemic to the Sonoran desert in Mexico, in which temperatures and temperature variations are extreme. We wondered if the developmental timing in D. pachea may be sensitive to differing rearing temperatures or if it remains constant. We determined the overall timing of the Drosophila pachea life-cycle at different temperatures. The duration of pupal development was similar at 25{degrees}C, 29{degrees}C and 32{degrees}C, although the relative progress differed at particular stages. Thus, D. pachea may have evolved mechanisms to buffer temperature effects on developmental speed, potentially to ensure proper development and individuals fitness in desert climate conditions.


title: Differential Analysis of RNA Structure Probing Experiments at Nucleotide Resolution: Uncovering Regulatory Functions of RNA Structure

abstract: RNAs perform their function by forming specific structures, which can change across cellular conditions. Structure probing experiments combined with next generation sequencing technology have enabled transcriptome-wide analysis of RNA secondary structure in various cellular conditions. Differential analysis of structure probing data in different conditions can reveal the RNA structurally variable regions (SVRs), which is important for understanding RNA functions. Here, we propose DiffScan, a computational framework for normalization and differential analysis of structure probing data in high resolution. DiffScan preprocesses structure probing datasets to remove systematic bias, and then scans the transcripts to identify SVRs and adaptively determines their lengths and locations. The proposed approach is compatible with most structure probing platforms (e.g., icSHAPE, DMS-seq). When evaluated with simulated and benchmark datasets, DiffScan identifies structurally variable regions at nucleotide resolution, with substantial improvement in accuracy compared with existing SVR detection methods. Moreover, the improvement is robust when tested in multiple structure probing platforms. Application of DiffScan in a dataset of multi-subcellular RNA structurome identified multiple regions that form different structures in nucleus and cytoplasm, linking RNA structural variation to regulation of mRNAs encoding mitochondria-associated proteins. This work provides an effective tool for differential analysis of RNA secondary structure, reinforcing the power of structure probing experiments in deciphering the dynamic RNA structurome.


title: Microplastic consumption induces inflammatory signatures in the colon and prolongs a viral arthritis

abstract: Global microplastic (MP) contamination and the effects on the environment are well described. However, the potential for MP consumption to affect human health remains controversial. Mice consuming {approx}80 {micro}g/kg/day of 1 {micro}m polystyrene MPs via their drinking water for a month showed no weight loss, nor were MPs detected in organs. The microbiome was also unchanged. MP consumption did lead to small transcriptional changes in the colon suggesting plasma membrane perturbations and mild inflammation. Mice were challenged with the arthritogenic chikungunya virus, with MP consumption leading to a significantly prolonged arthritic foot swelling that was associated with elevated Th1, NK cell and neutrophil signatures. Immunohistochemistry also showed a significant increase in the ratio of neutrophils to monocyte/macrophages. The picture that emerges is reminiscent of enteropathic arthritis, whereby perturbations in the colon are thought to activate innate lymphoid cells that can inter alia migrate to joint tissues to promote inflammation.


title: Assessment of antimicrobial activity of ethanolic and aqueous extracts of Aesculus hippocastanum L. (horse chestnut) bark against bacteria isolated from urine of patients diagnosed positive to urinary tract infections.

abstract:
The search for new antimicrobials is essential to address the worldwide issue of antibiotic resistance which affects all areas requiring the use of antibiotics including the management of diseases such as urinary tract infections (UTIs).

AimTo assess the antimicrobial activity of ethanolic and aqueous extract of Aesculus hippocastanum L. (horse chestnut) bark.

Material and MethodBioactive compounds were extracted from A. hippocastanum bark using water and ethanol as solvent. The extracts were tested against 10 clinical strains isolated from urine of patients diagnosed positive to urinary tract infections and including five Gram-positive bacteria (Kocuria rhizophila 1542, Enterococcus avium 1669, Staphylococcus simulans 5882, Conybacterium spp 1638, Enterococcus faecalis 5960) and five Gram-negative (Proteus mirabilis 1543, Morganella morganii 543, Citrobacter freundi 426, Acynetobacter baumannii 5841 and Achromobacter xylosoxidans 4892). Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 25922 were used as standard Gram+ and Gram-respectively. The susceptibility of the test strains to antibiotic was assessed using the Kirby Bauer disc diffusion method while the antibacterial activity of the extracts was evaluated using the well diffusion method. We finally determined the Minimum inhibitory concentration (MIC) and the minimum bactericidal concentrations (MBC) by the microdilution method.

ResultsA. hippocastanum bark possessed a dry matter content of 65.73%. The volume yield of the ethanolic and aqueous extract (AE) was 77,77% and 74,07% (v/v), respectively, whereas their mass yields were 13,4% and 24,3% (w/w), respectively. All the bacteria were susceptible to amoxiclav, imipenem and ceftriaxone, both standard bacteria (E. coli ATCC 25922 and S. aureus ATCC 6538) were sensitive to all antibiotics while the clinical strains were resistant to at least one antibiotic. K. rizophilia 1542 and Conybacterium spp 1638 were the most resistant bacteria both with multidrug resistance index of 0.45. Except AE on P. Mirabilis 1543 and E. faecalis 5960 (0 mm), both AE and EE were active against all the microorganisms tested with inhibition diameters (mm) which ranged from 5.5-10.0 for AE and 8.0-14.5 for EE. The MICs of EEs varied from 1-4 mg/ml while those of EAs varied from 4-16 mg/ml. Almost all the MBCs of AEs were indeterminate (>64 mg/ml) while those of EE were successfully determined. The ethanolic extracts (EE) were overall more active than the aqueous ones.

ConclusionThe A. hippocastanum bark extracts had overall weak antibacterial activity (MIC [≥]0.625 mg/ml) and bacteriostatic potential (MBC/MIC [≥]16) on both Gram-positive and Gram-negative bacteria. Therefore, studies with other solvents (such as methanol and chloroform), other extraction techniques, and synergy tests with conventional antibiotics are needed to conclude on a potential better antimicrobial activity of this plant material.


title: Survival of a threatened salmon is linked to spatial variability in river conditions

abstract: Extirpation of the Central Valley spring-run Chinook Salmon ESU (Oncorhynchus tshawytscha) from the San Joaquin River is emblematic of salmonid declines across the Pacific Northwest. Habitat restoration and fish reintroduction efforts are ongoing, but recent telemetry studies have revealed low outmigration survival of juveniles to the ocean. Previous investigations have focused on modeling survival relative to river discharge and geographic regions, but have largely overlooked the effects of habitat variability. To evaluate the link between environmental conditions and survival of juvenile spring-run Chinook Salmon, we combined high spatial resolution habitat mapping approaches with acoustic telemetry along a 150 km section of the San Joaquin River during the spring of 2019. While overall outmigration survival was low (5%), our habitat-based classification scheme described variation in survival of acoustic-tagged smolts better than other candidate models based on geography or distance. There were two regional mortality sinks evident along the longitudinal profile of the river, revealing poor survival in areas that shared warmer temperatures but that diverged in chlorophyll-, fDOM, turbidity and dissolved oxygen levels. These findings demonstrate the value of integrating river habitat classification frameworks to improve our understanding of survival dynamics of imperiled fish populations. Importantly, our data generation and modeling methods can be applied to a wide variety of fish species that transit heterogeneous and diverse habitat types.


title: Inexpensive monitoring of flying insect activity and abundance using wildlife cameras

abstract:
O_LIThe ability to measure flying insect activity and abundance is important for ecologists, conservationists and agronomists alike. However, existing methods are laborious and produce data with low temporal resolution (e.g. trapping and direct observation), or are expensive, technically complex, and require vehicle access to field sites (e.g. radar and lidar entomology).
C_LIO_LIWe propose a method called "camfi" for long-term non-invasive monitoring of the activity and abundance of low-flying insects using images obtained from inexpensive wildlife cameras, which retail for under USD$100 and are simple to operate. We show that in certain circumstances, this method facilitates measurement of wingbeat frequency, a diagnostic parameter for species identification. To increase usefulness of our method for very large monitoring programs, we have developed and implemented a tool for automatic detection and annotation of flying insect targets based on the popular Mask R-CNN framework. This tool can be trained to detect and annotate insects in a few hours, taking advantage of transfer learning.
C_LIO_LIWe demonstrate the utility of the method by measuring activity levels and wingbeat frequencies in Australian Bogong moths Agrotis infusa in the Snowy Mountains of New South Wales, and find that these moths have a mean wingbeat frequency of 48.6 Hz (SE = 1.4), undertake dusk flights in large numbers, and that the intensity of their dusk flights is modulated by daily weather factors. Validation of our tool for automatic image annotation gives baseline performance metrics for comparisons with future annotation models. The tool performs well on our test set, and produces annotations which can be easily modified by hand if required. Training completed in less than 2 h on a single machine, and inference took on average 1.15 s per image on a laptop.
C_LIO_LIOur method will prove invaluable for ongoing efforts to understand the behaviour and ecology of the iconic Bogong moth, and can easily be adapted to other flying insects. The method is particularly suited to studies on low-flying insects in remote areas, and is suitable for very large-scale monitoring programs, or programs with relatively low budgets.
C_LI


title: Genetic Influence underlying Brain Connectivity Phenotype: A Study on Two Age-Specific Cohorts

abstract:
BackgroundHuman brain structural connectivity is an important imaging quantitative trait for brain development and aging. Mapping the network connectivity to the phenotypic variation provides fundamental insights in understanding the relationship between detailed brain topological architecture, function, and dysfunction. However, the underlying neurobiological mechanism from gene to brain connectome, and to phenotypic outcomes, and whether this mechanism changes over time, remain unclear.

MethodsThis study analyzes diffusion weighted imaging data from two age-specific neuroimaging cohorts, extracts structural connectome topological network measures, performs genome-wide association studies (GWAS) of the measures, and examines the causality of genetic influences on phenotypic outcomes mediated via connectivity measures.

ResultsOur empirical study has yielded several significant findings: 1) It identified genetic makeup underlying structural connectivity changes in the human brain connectome for both age groups. Specifically, it revealed a novel association between the minor allele (G) of rs7937515 and the decreased network segregation measures of the left middle temporal gyrus across young and elderly adults, indicating a consistent genetic effect on brain connectivity across the lifespan. 2) It revealed rs7937515 as a genetic marker for body mass index (BMI) in young adults but not in elderly adults. 3) It discovered brain network segregation alterations as a potential neuroimaging biomarker for obesity. 4) It demonstrated the hemispheric asymmetry of structural network organization in genetic association analyses and outcome-relevant studies.

DiscussionThese imaging genetic findings underlying brain connectome warrant further investigation for exploring their potential influences on brain-related diseases, given the significant involvement of altered connectivity in neurological, psychiatric and physical disorders.

Impact StatementThe genetic architecture underlying brain connectivity, and whether this mechanism changes over time, remain largely unknown. To understand the inter-individual variability at different life stages, this study performed genome-wide association studies of brain network connectivity measures from two age-specific neuroimaging cohorts, and identified a common association between the minor allele (G) of rs7937515 and decreased network segregation measures of the left middle temporal gyrus. The mediation analysis further elucidated neurobiological pathway of brain connectivity mediators linking the genes FAM86C1/FOLR3 with body mass index. This study provided new insights into the genetic mechanism of inter-regional connectivity alteration in the brain.

category: genetics


title: DNA Replication-transcription conflicts do not significantly contribute to spontaneous mutations due to replication errors in Escherichia coli

abstract:
Encounters between DNA replication and transcription can cause genomic disruption, particularly when the two meet head-on. Whether these conflicts produce point mutations is debated. This paper presents detailed analyses of a large collection of mutations generated during mutation accumulation experiments with mismatch-repair (MMR) defective Escherichia coli. With MMR absent, mutations are primarily due to DNA replication errors. Overall, there were no differences in the frequencies of base-pair substitutions or small indels (insertion and deletions [≤] 4 bp) in the coding sequences or promoters of genes oriented codirectionally versus head-on to replication. Among a subset of highly expressed genes there was a 2- to 3-fold bias for indels in genes oriented head-on to replication, but this difference was almost entirely due to the asymmetrical genomic locations of tRNA genes containing mononucleotide runs, which are hotspots for indels. No additional orientation bias in mutation frequencies occurred when MMR- strains were also defective for transcription-coupled repair (TCR). However, in contrast to other reports, loss of TCR slightly increased the overall mutation rate, meaning that TCR is antimutagenic. There was no orientation bias in mutation frequencies among the stress-response genes that are regulated by RpoS or induced by DNA damage. Thus, biases in the locations of mutational targets can account for most, if not all, apparent biases in mutation frequencies between genes oriented head-on versus co-directional to replication. In addition, the data revealed a strong correlation of the frequency of base-pair substitutions with gene length, but no correlation with gene expression levels.

IMPORTANCEBecause DNA replication and transcription occur on the same DNA template, encounters between the two machines occur frequently. When these encounters are head-to-head, genomic disruption can occur. But, whether replication-transcription conflicts contribute to spontaneous mutations is debated. Analyzing in detail a large collection of mutations generated with mismatch repair defective Escherichia coli strains, we find that across the genome there are no significant differences in mutation frequencies between genes oriented co-directionally versus head-on to replication. Among a subset of highly expressed genes there was a 2- to 3-fold bias for small insertions and deletions in head-on oriented genes, but this difference was almost entirely due to the asymmetrical locations of tRNA genes containing mononucleotide runs, which are hotspots for these mutations. Thus, biases in the positions of mutational target sequences can account for most, if not all, apparent biases in mutation frequencies between genes oriented head-on and co-directionally to replication.


title: Immune and malignant cell phenotypes of ovarian cancer are determined by distinct mutational processes

abstract:
High-grade serous ovarian cancer (HGSOC) is an archetypal cancer of genomic instability patterned by distinct mutational processes, intratumoral heterogeneity and intraperitoneal spread. We investigated determinants of immune recognition and evasion in HGSOC to elucidate co- evolutionary processes underlying malignant progression and tumor immunity. Mutational processes and anatomic sites of tumor foci were key determinants of tumor microenvironment cellular phenotypes, inferred from whole genome sequencing, single-cell RNA sequencing, digital histopathology and multiplexed immunofluorescence of 160 tumor sites from 42 treatment-naive HGSOC patients. Homologous recombination-deficient (HRD)-Dup (BRCA1 mutant-like) and HRD- Del (BRCA2 mutant-like) tumors harbored increased neoantigen burden, inflammatory signaling and ongoing immunoediting, reflected in loss of HLA diversity and tumor infiltration with highly- differentiated dysfunctional CD8+ T cells. Foldback inversion (FBI, non-HRD) tumors exhibited elevated TGF{beta} signaling and immune exclusion, with predominantly naive/stem-like and memory T cells. Our findings implicate distinct immune resistance mechanisms across HGSOC subtypes which can inform future immunotherapeutic strategies.

HIGHLIGHTSO_LIMulti-region, multi-modal profiling of malignant and immune cell phenotypes in ovarian cancer
C_LIO_LIAnatomic site specificity is a determinant of cancer cell and intratumoral immune phenotypes
C_LIO_LITumor mutational processes impact mechanisms of immune control and immune evasion
C_LIO_LISpatial topology of HR-deficient tumors is defined by immune interactions absent from immune inert HR-proficient subtypes
C_LI


title: Interleukin-11 plays a key role in human and mouse alcohol-related liver disease

abstract:
BackgroundAlcoholic hepatitis (AH) reflects acute exacerbation of alcoholic liver disease (ALD) and is a growing healthcare burden worldwide with limited treatment options. Interleukin-11 (IL-11) is a pro-fibrotic, pro-inflammatory cytokine with increasingly recognized toxicities in parenchymal and epithelial cells.

AimThe aim of this study was to explore the prognostic value of IL-11 serum levels in patients suffering from AH and cirrhosis of various etiology and to understand the role of IL-11 in experimental ALD.

MethodsIL-11 serum concentration and tissue expression was determined in a cohort comprising 50 patients with AH, 110 patients with cirrhosis and 19 healthy volunteers. Findings were replicated in an independent patient cohort including 186 patients. Ethanol-fed wildtype mice were treated with a neutralizing murine IL-11 receptor-antibody (anit-IL11RA) and thereafter examined for severity signs and markers of ALD.

ResultsHuman IL-11 serum concentration and liver tissue expression increased with severity of liver disease and were most pronounced in AH. In a multivariate Cox-regression, a serum level above 6.4 picograms/milliliter was a MELD independent risk factor for transplant-free liver disease survival in patients with compensated and decompensated cirrhosis. Findings were confirmed in an independent cohort. In mice, severity of alcohol-induced liver inflammation was positively correlated to enhanced hepatic IL-11 expression. Pretreatment with a neutralizing anti-IL11RA inhibited hepatic inflammation and mice were protected from ethanol-induced liver injury. In comparison to IgG-control, ethanol-fed mice treated with anti-IL11RA showed decreased steatosis, hepatic neutrophil infiltration, and expression of pro-inflammatory cytokines.

ConclusionIL-11 plays a crucial role in the pathogenesis of ALD and could serve as an independent prognostic factor for transplant-free survival. Blocking IL-11 signaling might be a therapeutic option in human ALD, particularly AH.


title: Spatial transcriptomics reveals antiparasitic targets associated with essential behaviors in the human parasite Brugia malayi

abstract: Lymphatic filariasis (LF) is a chronic debilitating neglected tropical disease (NTD) caused by mosquito-transmitted nematodes that afflicts over 60 million people. Control of LF relies on routine mass drug administration with antiparasitics that clear circulating larval parasites but are ineffective against adults. The development of effective adulticides is hampered by a poor understanding of the processes and tissues driving parasite survival in the host. The adult filariae head region contains essential tissues that control parasite feeding, sensory, secretory, and reproductive behaviors, which express promising molecular substrates for the development of antifilarial drugs, vaccines, and diagnostics. We have adapted spatial transcriptomic approaches to map gene expression patterns across these prioritized but historically intractable head tissues. Spatial and tissue-resolved data reveal distinct biases in the origins of known drug targets and secreted antigens. These data were used to identify potential new drug and vaccine targets, including putative hidden antigens expressed in the alimentary canal, and to spatially associate receptor subunits belonging to druggable families. Spatial transcriptomic approaches provide a powerful resource to aid gene function inference and seed antiparasitic discovery pipelines across helminths of relevance to human and animal health.

category: microbiology


title: Structure-based screening of drug candidates targeting the SARS-CoV-2 envelope protein

abstract: The COVID-19 (coronavirus disease 2019) pandemic is caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). SARS-CoV-2 produces a small hydrophobic envelope (E) protein which shares high homology with SARS-CoV E protein. By patch-clamp recording, the E protein is demonstrated to be a cation-selective ion channel. Furthermore, the SARS-CoV-2 E protein can be blocked by a SARS-CoV E protein inhibitor hexamethylene amiloride. Using structural model and virtual screening, another E protein inhibitor AZD5153 is discovered. AZD5153 is a bromodomain protein 4 inhibitor against hematologic malignancies in clinical trial. The E protein amino acids Phe23 and Val29 are key determinants for AZD5153 sensitivity. This study provides two promising lead compounds and a functional assay of SARS-CoV-2 E protein for the future drug candidate discovery.


title: Direct RNA sequencing reveals SARS-CoV-2 m6A sites and possible differential DRACH motif methylation among variants

abstract: The causative agent of COVID-19 pandemic, the SARS-CoV-2 coronavirus, has a 29,903 bases positive-sense single-stranded RNA genome. RNAs exhibit about 100 modified bases that are essential for proper function. Among internal modified bases, the N6-methyladenosine, or m6A, is the most frequent, and is implicated in SARS-CoV-2 immune response evasion. Although the SARS-CoV-2 genome is RNA, almost all genomes sequenced so far are in fact, reverse transcribed complementary DNAs. This process reduces the true complexity of these viral genomes because incorporation of dNTPs hides RNA base modifications. Here, in this perspective paper, we present an initial exploration of the Nanopore direct RNA sequencing to assess the m6A residues in the SARS-CoV-2 sequences of ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N, ORF10 and the 3-untranslated region. We identified 15 m6A methylated positions, of which, 6 are in ORF N. Also, because m6A is associated with the DRACH motif, we compared its distribution in major SARS-CoV-2 variants. Although DRACH is highly conserved among variants we show that variants Beta and Eta have a fourth position C>U change in DRACH at 28,884b that could affect methylation. The Nanopore technology offers a unique opportunity for the study of viral epitranscriptomics. This technique is PCR-free and is not sequencing-by-synthesis, therefore, no PCR bias and synthesis errors are introduced. The modified bases are preserved and assessed directly with no need for chemical treatments or antibodies. This is the first report of direct RNA sequencing of a Brazilian SARS-CoV-2 sample coupled with the identification of modified bases.