Applications of the indole-alkaloid gramine modulate the assembly of individual members of the barley rhizosphere microbiota Microbial communities proliferating at the root-soil interface, collectively referred to as the rhizosphere microbiota, represent an untapped beneficial resource for plant growth, development and health. Integral to a rational manipulation of the microbiota for sustainable agriculture is the identification of the molecular determinants of these communities. In plants, biosynthesis of allelochemicals is centre stage in defining inter-organismal relationships in the environment. Intriguingly, this process has been moulded by domestication and breeding selection. The indole-alkaloid gramine, whose occurrence in barley (Hordeum vulgare L.) is widespread among wild genotypes but has been counter selected in several modern varieties, is a paradigmatic example of this phenomenon. This prompted us to investigate how exogenous applications of gramine impacted on the rhizosphere microbiota of two, gramine-free, elite barley varieties grown in a reference agricultural soil. High throughput 16S rRNA gene amplicon sequencing revealed that applications of gramine interfere with the proliferation of a subset of soil microbes with a relatively broad phylogenetic assignment. Strikingly, growth of these bacteria appeared to be rescued by barley plants in a genotype- and dosage-independent manner. In parallel, we discovered that host recruitment cues can interfere with the impact of gramine application in a host genotype-dependent manner. Interestingly, this latter effect displayed a bias for members of the phyla Proteobacteria. These initial observations indicate that gramine can act as a determinant of the prokaryotic communities inhabiting the root-soil interface.
Plant biomechanics and resilience to environmental changes are controlled by specific lignin chemistries in each vascular cell type and morphotype The biopolymer lignin, deposited in the cell walls of vascular cells, is essential for long-distance water conduction and structural support of plants. Independently of the species, each different vascular cell type contains a conserved lignin chemistry with specific aromatic and aliphatic substitutions. Yet, the biological role of this conserved and specific lignin chemistry for each cell type remained unclear. Herein, we investigate the role of specific lignin chemistries for cellular function by producing single cell analyses on vascular cell morphotypes, all enabling sap conduction but differing in morphology. We found that specific lignin chemistries accumulate in each morphotype. Moreover, lignin accumulates dynamically, increasing in quantity and changing composition, to alter the cell wall biomechanics of each morphotype during their maturation. For similar aromatic substitution, residues with alcohol aliphatic functions increased stiffness whereas aldehydes increased flexibility. Modifying this specific lignin chemistry impairs the cell wall biomechanics of each morphotype and consequently reduces their capacity to optimally conduct water in normal conditions, and to recover from drought. Altogether, lignin chemistry is differently controlled for each sap conducting cell types during their maturation to dynamically adjust their biomechanics and hydraulic properties to adapt to developmental and environmental constraints.
Material-mediated histogenesis using mechano-chemically microstructured cell niches Stem-cell derived tissue models are commonly cultured under globally-delivered stimuli that trigger histogenesis via self-organizing activity. However, the culture of such tissue models is prone to stochastic behavior, limiting the reproducibility of cellular composition and resulting in non-physiological architectures. To overcome these shortcomings, we developed a method for printing cell niche microenvironments with microstructured cues that mediate local histogenic processes, including mechanosensing and differentiation of selected cell types. Microstructured cues include independently tunable mechano-chemical properties, with conjugated peptides, proteins, and morphogens across a range of Youngs moduli. By rationally designing niches, we mediate the structure of tissues derived from stem-cell-progenitor sources, including a bone-fat assembly from stromal mesenchyme, and embryonic tissues derived from hiPSC. We show that microstructured cues can recapitulate mechano-chemical signals resembling early embryonic histogenesis. This outcome includes a role for niche mechanics in human embryonic organization, where soft niche mechanics bias markers of mesendodermal differentiation and epithelial-to-mesenchymal-transition (EMT), as well as a demonstration of a material-mediated morphogen signaling centers able to induce foci of mesenchymal and EMT differentiation. Thus, microstructured materials can mediate local histogenic processes to enhance the structure and composition of tissue models. Balancing sensitivity and specificity in preclinical research The success of scientific discovery in preclinical drug development is based on the different roles of exploration and confirmation. Exploration requires sensitivity while confirmation needs to be specific. Based on published empirical data, we simulated two preclinical research trajectories to examine their effectiveness to detect true effects while keeping animal numbers low. We compare a conventional p-value based approach with a method based on an a priori determined smallest effect size of interest (SESOI). Using a SESOI increases transition rates from exploration to confirmation and results in higher detection rates across the trajectory. Specificity in the SESOI trajectory increases if the prior probability of true effects is low. We conclude that employing a SESOI is superior to a p-value based approach. Based on our findings, we propose a reconsideration of planning and conducting preclinical experiments, especially when the prior probability of true hypotheses is low. From fitting the average to fitting the individual: A cautionary tale for mathematicalmodelers An outstanding challenge in the clinical care of cancer is moving from a one-size-fits-all approach that relies on population-level statistics towards personalized therapeutic design. Mathematical modeling is a powerful tool in treatment personalization, as it allows for the incorporation of patient-specific data so that treatment can be tailor-designed to the individual. Herein, we work with a mathematical model of murine cancer immunotherapy that has been previously-validated against the average of an experimental dataset. We ask the question: what happens if we try to use this same model to perform personalized fits, and therefore make individualized treatment recommendations? Typically, this would be done by choosing a single fitting methodology, and a single cost function, identifying the individualized best-fit parameters, and extrapolating from there to make personalized treatment recommendations. Our analyses show the potentially problematic nature of this approach, as predicted personalized treatment response proved to be sensitive to the fitting methodology utilized. We also demonstrate how a small amount of the right additional experimental measurements could go a long way to improve consistency in personalized fits. Finally, we show how quantifying the robustness of the average response could also help improve confidence in personalized treatment recommendations.

Author summaryAs we enter the era of healthcare where personalized medicine becomes a more common approach to treating cancer patients, harnessing the power of mathematical models will only become more essential. Using a preclinical dataset on cancer immunotherapy, we explore the challenges and limitations that arise when trying to move from fitting and making predictions for the population-level average, to fitting and making predictions for an individual. We find that the standard of approach of picking a single fitting methodology and a single cost function may end up having limited predictive value when applied to individual data. We also show how having a small amount of the right additional experimental data, and establishing the robustness of average treatment response, can help improve confidence in personalized model predictions. Conserved organ-specific microbial assemblages in different populations of a terrestrial crab Brachyuran crabs originated in the oceans and evolved specific morphological and physiological adaptations to live in freshwater, intertidal and even terrestrial habitats but the role of a selection mechanism involving symbiotic microorganisms long these colonization processes are not known. In this work we investigated the associated microbiota of three populations of a terrestrial brachyuran crab, Chiromantes haematocheir, to find evidence of a conserved crab-specific microbiome unrelated to the population of origin and dissimilar from environmental microbial assemblages. Bacterial 16S rRNA gene and fungal ITS sequences were obtained from selected crab organs and environmental matrices to profile microbial communities. In spite of the presence of truly marine larval stages and no gregarious behaviour, favouring microbiota exchanges, we found common, organ-specific microbiota, associated to the gut and the gills of the crabs (with more than 15% of the genera detected specifically enriched only in one organ). Our results suggest an early establishment of a new common, stable microbiota in the transition from water to land. Bulk-assembly of monodisperse coacervates and giant unilamellar vesicles with programmable hierarchical complexity Compartmentalization is an essential step for the emergence of protocellular life on the early earth and the endeavor to bottom-up construct synthetic cells in the lab. Among manifold natural strategies for compartmentalization, assembly of lipid vesicles or coacervate droplets with resemblance to cell envelope or cytoplasm have emerged as two dominant paradigms. However, the spontaneous assembly of lipid vesicles or coacervates generally results in polydisperse microcompartments and lacks the ability to efficiently integrate functional building blocks towards defined complexity, which give rise to the difficulty to achieve the order and complexity towards the scope of that of life via spontaneous assembly. Herein, we show that the interplay of coacervates and colloidal particles provides a bulk-assembly approach to form monodisperse coacervates and giant unilamellar vesicles (GUVs) with programmable hierarchical complexity. The hierarchical complexity of the synthetic cell microcompartments can be delicately engineered from the control of the spatial or temporal organization of components in entity unit to manifold forms of interconnected synthetic cell consortia, achieving a compartment hierarchy analogous to the organelle-cell-tissue structure. The increase in complexity gives rise to emergent properties, for instance, collective morphology evolution of coacervate assemblages, gated permeability of GUVs without sophisticated protein machinery, and remarkable structural and functional stability of GUVs at extreme conditions. This work paves an unprecedented step to form monodisperse, hierarchical coacervates and GUVs via bulk-assembly, which provides a prebiotically plausible approach for the proper integration of inanimate matter towards the order and complexity of life and monodisperse, hierarchical microdroplets for follow-up scientific and technological applications. Modulation of P2X4 pore closure by magnesium,potassium, and ATP The P2X4 receptor plays a prominent role in cellular responses to extracellular ATP. Through classical all-atom molecular dynamics (MD) simulations totaling 24 {micro}s we have investigated how metal-complexed ATP stabilizes the channels open state and prevents its closing. We have identified two metal-binding sites, magnesium (Mg2+) and potassium (K+), one at the intersection of the three subunits in the ectodomain (MBS1) and the second one near the ATP binding site (MBS2), similar to those characterized in Gulf coast P2X. Our data indicate that when Mg2+ and K+ ions are complexed with ATP, the channel is locked into an open state. Interestingly, irrespective of the number of bound ATP molecules, Mg2+ ions bound to the MBS2 resisted collapsing of the open state protein to a closed state by stabilizing the ATP-protein interactions. However, when Mg2+ in the MBS2 was replaced with K+ ions, as might be expected when in equilibrium with an extracellular solution, the interactions between the subunits were weakened and we found evidence of pore collapse. This collapse was apparent when fewer than two ATP were bound to MBS2 in the presence of K+. Therefore, the different capacities of common cations to stabilize the channel may underlie a mechanism governing P2X4 channel gating in physiological systems. This study provides structural insights into the differential modulation of ATP activation of P2X4 by Mg2+ and K+. Estrogen-related receptor alpha and Rplp1-dependent translation coordinately regulate starvation response and decrease NASH progression Although general translation declines during fasting, maintaining the translation of a subset or proteins is necessary for metabolic homeostasis and cell viability. Using unbiased proteome analysis of hepatic cells during starvation, we identified a novel pathway in which Esrra-mediated transcription of Rplp1-dependent translation of lysosomal proteins declined during early starvation and recovered after prolonged starvation to restore autophagy-lysosome function. Interestingly, hepatic Esrra-Rplp1-dependent translation rate of lysosomal proteins also was impaired in patients and mice with non-alcoholic steatohepatitis (NASH), and translational response to starvation was dysregulated in mice with NASH. Remarkably, activation of Esrra pharmacologically, genetically, or by alternate day fasting restored protein translation, increased expression of lysosomal proteins, induced autophagy, and reduced lipotoxicity, inflammation, and fibrosis in cell culture and in vivo models of NASH. Thus, hepatic Esrra is essential for ribosome-dependent translation of lysosomal proteins during starvation, and prevention of lipotoxicity and progression in NASH.

Lay summaryFasting for weight loss improves non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH); however, the mechanism is not well understood. Our study shows that a nuclear protein, estrogen related receptor alpha (Esrra), increases ribosome-mediated translation of autophagy and lysosome proteins during chronic starvation to maintain essential metabolic pathways for cell survival. Surprisingly, this translational pathway is impaired during NASH with reduced lysosome-autophagy activity accompanied by increased inflammation and fibrosis gene expression in the liver. Pharmacologic, genetic and dietary activation of Esrra decreases lipid-mediated toxicity in liver cells as well as inflammation and fibrosis in livers from mice with NASH. These findings suggest that the Esrra-ribosome-lysosome pathway is important for liver response to fasting and NASH and thus may be a good therapeutic target for the treatment of NASH. A 3D transcriptomics atlas of the mouse olfactory mucosa The sense of smell helps us navigate the environment, but its molecular architecture and underlying logic remain unknown. The spatial location of odorant receptor genes (Olfrs) in the nose is widely thought to be independent of the structural diversity of the odorants they detect. Using spatial transcriptomics, we created a genome-wide 3D atlas of the mouse olfactory mucosa (OM). Topographic maps of genes differentially expressed in space reveal that both Olfrs and non-Olfrs are distributed in a continuous and overlapping fashion over five broad zones in the OM. The spatial locations of Olfrs correlate with the mucus solubility of the odorants they recognize, providing direct evidence for the chromatographic theory of olfaction. This resource resolved the molecular architecture of the mouse OM, and will inform future studies on mechanisms underlying Olfr gene choice, axonal pathfinding, patterning of the nervous system, and basic logic for the peripheral representation of smell. The first genotypic fitness network in a vertebrate reveals that hybridization increases access to novel fitness peaks Estimating the complex relationship between fitness and genotype or phenotype (i.e. the adaptive landscape) is one of the central goals of evolutionary biology. However, adaptive walks connecting genotypes to organismal fitness, speciation, and novel ecological niches are still poorly understood and processes for surmounting fitness valleys remain controversial. One outstanding system for addressing these connections is a recent adaptive radiation of ecologically and morphologically novel pupfishes (a generalist, molluscivore, and scale-eater) endemic to San Salvador Island, Bahamas. We leveraged whole-genome sequencing of 139 hybrids from two independent field fitness experiments to identify the genomic basis of fitness, estimate genotypic fitness networks, and measure the accessibility of adaptive walks on the fitness landscape. We identified 132 SNPs that were significantly associated with fitness in field enclosures. Six out of the 13 regions most strongly associated with fitness contained differentially expressed genes and fixed SNPs between trophic specialists; one gene (mettl21e) was also misexpressed in lab-reared hybrids, suggesting a potential intrinsic genetic incompatibility. We then constructed genotypic fitness networks from adaptive alleles and show that scale-eating specialists are the most isolated of the three species on these networks. Intriguingly, introgressed and de novo variants reduced fitness landscape ruggedness as compared to standing variation, increasing the accessibility of genotypic fitness paths from generalist to specialists. Our results suggest that adaptive introgression and de novo mutations alter the shape of the fitness landscape, providing key connections in adaptive walks circumventing fitness valleys and triggering the evolution of novelty during adaptive radiation. Limitations of composability of cis-regulatory elements in messenger RNA Genes are commonly abstracted into a coding sequence and cis-regulatory elements (CREs), such as promoters and terminators, and short sequence motifs within these regions. Modern cloning techniques allow easy assembly of synthetic genetic constructs from discrete cis-regulatory modules. However, it is unclear how much the contributions of CREs to gene expression depend on other CREs in the host gene. Using budding yeast, we probe the extent of composability, or independent effects, of distinct CREs. We confirm that the quantitative effect of a terminator on gene expression depends on both promoter and coding sequence. We then explore whether individual cisregulatory motifs within terminator regions display similar context dependence, using transcriptomewide datasets of mRNA decay. To test the extent of composability, we construct reporter genes consisting of combinations of motifs within various terminator contexts, paired with different promoters. Our results show that the effect of a motif on RNA abundance depends both on its host terminator, and also on the associated promoter sequence. This emphasises the need for improved motif inference that includes both local and global context effects, which in turn could aid in the accurate use of CREs for the engineering of synthetic genetic constructs. Cell Cycle Delay, Pro-metaphase Arrest and C-metaphase Inducing Effects of Petroleum Ether Fraction of Leaf Aqueous Extract of Clerodendrum viscosum Vent. Clerodendrum viscosum is a traditionally used medicinal plant. The present study aimed to analyze cell cycle delay, pro-metaphase arrest, and c-metaphase inducing effects of the petroleum ether fraction (AQPEF) of leaf aqueous extract of C. Viscosum (LAECV). The LAECV was fractionated with petroleum ether and its metaphase arrest, cell cycle delay, and c-metaphase inducing activities were tested on A. cepa root tip cells. The AQPEF induced cell cycle delay, and colchicine like metaphase, c-metaphase, in A. cepa root tip cells. Thus, the present study explores AQPEF as an active fraction of LAECV having metaphase arresting, cell cycle delay, and c-metaphase inducing potentials. Between two walls: Modeling the adsorption behavior of Beta-glucosidase A on bare and SAM-functionalised gold surfaces The efficient immobilization of enzymes on surfaces remains a complex but central issue in the biomaterials field, which requires us to understand this process at the atomic level. Using a multi-scale approach combining all-atom molecular dynamics and coarse-grain Brownian dynamics simulations, we investigated the adsorption behavior of {beta}-glucosidase A ({beta}GA) on bare and SAM-functionalized gold surfaces. We monitored the enzyme position and orientation during the MD trajectories, and measured the contacts it forms with both surfaces. While the adsorption process has little impact on the protein conformation, it can nonetheless perturb its mechanical properties and catalytic activity. Our results show that compared to the SAM-functionalized surface, the adsorption of {beta}GA on bare gold is more stable, but also less specific, and more likely to disrupt the enzymes function. This observation emphasizes the fact that the structural organization of proteins at the solid interface is a keypoint when designing devices based on enzyme immobilization, as one must find an acceptable stability-activity trade-off.

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org.highwire.dtl.DTLVardef@d3d0d7org.highwire.dtl.DTLVardef@3403e4org.highwire.dtl.DTLVardef@eb804dorg.highwire.dtl.DTLVardef@13469c2_HPS_FORMAT_FIGEXP  M_FIG C_FIG Parallel Characterization of cis-Regulatory Elements for Multiple Genes Using CRISPRpath Current pooled CRISPR screens for cis-regulatory elements (CREs) can only accommodate one gene based on its expression level. Here, we describe CRISPRpath, a scalable screening strategy for parallelly characterizing CREs of genes linked to the same biological pathway and converging phenotypes. We demonstrate the ability of CRISPRpath for simultaneously identifying functional enhancers of six genes in the 6-thioguanine-induced DNA mismatch repair pathway using both CRISPR interference (CRISPRi) and CRISPR nuclease (CRISPRn) approaches. 60% of the identified enhancers are known promoters with distinct epigenomic features compared to other active promoters, including increased chromatin accessibility and interactivity. Furthermore, by imposing different levels of selection pressure, CRISPRpath can distinguish enhancers exerting strong impact on gene expression from those exerting weak impact. Our results offer a nuanced view of cis-regulation and demonstrate that CRISPRpath can be leveraged for understanding the complex gene regulatory program beyond transcriptional output at scale. Estimation of cell cycle kinetics in higher plant root meristem links organ position with cellular fate and chromatin structure Plant root development is a complex spatial-temporal process that originates in the root apical meristem (RAM). To shape the organs structure signaling between the different cells and cell files must be highly coordinated. Thereby, diverging kinetics of chromatin remodeling and cell growth in these files need to be integrated and balanced by differential cell growth and local differences in cell proliferation frequency. Understanding the local differences in cell cycle duration in the RAM and its correlation with chromatin organization is crucial to build a holistic view on the different regulatory processes and requires a quantitative estimation of the chromatin geometry and underlying mitotic cell cycle phases timing at every cell file and every position. Unfortunately, so far precise methods for such analysis are missing.

This study presents a robust and straightforward pipeline to determine in parallel the duration of cell cycles key stages in all cell layers of a plants root and their nuclei organization. The methods combine marker-free techniques based on the detection of the nucleus, deep analysis of the chromatin phase transition, incorporation of 5-ethynyl-2'-deoxyuridine (EdU), and mitosis with a deep-resolution plant phenotyping platform to analyze all key cell cycle events kinetics.

In the Arabidopsis thaliana L. RAM S-phase duration was found to be as short as 20-30 minutes in all cell files. The subsequent G2-phase duration however depends on the cell type/position and varies from 3.5 hours in the pericycle to more than 4.5 hours in the epidermis. Overall, S+G2+M duration in Arabidopsis under our condition is 4 hours in the pericycle and up to 5.5 hours in the epidermis.

Endocycle duration was determined as the time required to achieve 100% EdU index in the transition zone and estimated to be in the range of 3-4 hours.

Besides Arabidopsis, we show that the presented technique is applicable also to root tips of other dicot and monocot plants (tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum L.) and wheat (Triticum aestivum L.). Heterogeneous recruitment abilities to RNA polymerases generate nonlinear scaling of gene expression level with cell volume While most genes expression levels are proportional to cell volumes, some genes exhibit nonlinear scaling between their expression levels and cell volume. Therefore, their mRNA and protein concentrations change as the cell volume increases, which often have crucial biological functions such as cell-cycle regulation. However, the biophysical mechanism underlying the nonlinear scaling between gene expression and cell volume is still unclear. In this work, we show that the nonlinear scaling is a direct consequence of the heterogeneous recruitment abilities of promoters to RNA polymerases based on a gene expression model at the whole-cell level. Those genes with weaker (stronger) recruitment abilities than the average ability spontaneously exhibit superlinear (sublinear) scaling with cell volume. Analysis of the promoter sequences and the nonlinear scaling of Saccharomyces cerevisiaes mRNA levels shows that motifs associated with transcription regulation are indeed enriched in genes exhibiting nonlinear scaling, in concert with our model. Characterising ontogeny of numerosity discrimination in zebrafish reveals the use of multiple, numerical and non- numerical mechanisms. A sense of non-symbolic numerical magnitudes is widespread in the animal kingdom and has been documented in adult zebrafish. Here we investigated the ontogeny of this ability using a group size preference task in juvenile zebrafish. Fish showed group size preference from 21 days post fertilization (dpf) and reliably chose the larger group when presented with discriminations of between 1 vs. 3, 2 vs. 5 and 2 vs. 3 conspecifics but not 2 vs. 4 conspecifics. When the ratio between the number of conspecifics in each group was maintained at 1:2, fish could discriminate between 1 vs. 2 individuals and 3 vs. 6, but again, not when given a choice between 2 vs. 4 individuals. These findings are in agreement with studies in other species suggesting the systems involved in quantity representation do not operate separately from other cognitive mechanisms. Rather they suggest quantity processing in fish may be the result of an interplay between attentional, cognitive and memory-related mechanisms as in humans and other animals. Our results emphasise the potential of the use of zebrafish to explore the genetic and neural processes underlying the ontogeny and function of number cognition. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Intestinal GCN2 controls Drosophila systemic growth in response to Lactiplantibacillus plantarum symbiotic cues encoded by r/tRNA operons Symbiotic bacteria interact with their host through the release of symbiotic cues. Here, we took advantage of the mutualism between Drosophila and Lactiplantibacillus plantarum (Lp) to investigate a novel mechanism of host-symbiont interaction. Using chemically-defined holidic diets, we found that association with Lp improves the growth of larvae fed on amino acid-imbalanced diets, even though Lp cannot produce the limiting amino acid. We show that in this context Lp supports its hosts growth through a molecular dialog that requires functional operons encoding ribosomal and transfer RNAs (r/tRNAs) in Lp and the GCN2 kinase in Drosophilas enterocytes. Our data indicate that products of Lp r/tRNAs loci activate GCN2 in a subset of larval enterocytes, a mechanism necessary to remodel the intestinal transcriptome and ultimately to support anabolic growth. Our findings unravel a novel beneficial molecular dialog between host and microbes, which relies on a non-canonical role of GCN2 as a mediator of non-nutritional symbiotic cues encoded by r/tRNA operons. Beyond Spike: Identification of nine highly prevalent SARS-CoV-2-specific CD8 T-cell epitopes in a large Norwegian cohort T-cell epitopes with broad population coverage may form the basis for a new generation of SARS-CoV-2 vaccines. However, published studies on immunoprevalence are limited by small test cohorts, low frequencies of antigen-specific cells and lack of data correlating eluted HLA ligands with T-cell responsiveness. As the protective role of pre-existing cross-reactivity to homologous peptides is unclear, we aimed to identify SARS-CoV-2-specific minimal epitopes recognized by CD8 T-cells among 48 peptides eluted from prevalent HLA alleles, and an additional 84 predicted binders, in a large cohort of convalescents (n=83) and pre-pandemic control samples (n=19). We identified nine conserved SARS-CoV-2-specific epitopes restricted by four of the most prevalent HLA class I alleles in the Norwegian study cohort, to which responding CD8 T cells were detected in 70-100% of convalescents expressing the relevant HLA allele. Only two of these were derived from the Spike protein, included in current vaccines. We found a strong correlation between immunoprevalence and immunodominance. Thus, the CD8 T-cell response to SARS-CoV-2 is more focused than previously believed. Using a new algorithm, we predict that a vaccine including these epitopes could induce a T-cell response in 83% of Caucasians. Are narrow-ranging species doomed to extinction? Projected dramatic decline in future climate suitability of two highly threatened species Narrow-ranging species are usually omitted from Species distribution models (SDMs) due to statistical constraints, while they are predicted to be particularly vulnerable to climate change. The recently available high-resolution environmental predictors, along with recently developed methods enable to increase the eligibility of narrow-ranging species for SDMs, provided their distribution is well known. We fill a gap of knowledge on the effect of predicted climate change on narrow-ranging species. We modelled the distribution of the golden mantella frog Mantella aurantiaca and the Manapany day gecko Phelsuma inexpectata, for which the distribution of their occurrence records is well documented. Our modelling scheme included a range of processes susceptible to address statistical issues related to narrow-ranging species. We predict an alarming decline in climate suitability in the whole current distribution area of both species by 2070, potentially leading to a complete extinction in most scenarios. We identified the areas with the best climate suitability in the future, but these remain largely suboptimal regarding species climatic niche. The high level of habitat fragmentation suggests that both species likely need to be at least partly translocated. Climate change may not only drive range contractions or distribution shifts in narrow-ranging species, but may lead to the complete extirpation of suitable environments across their entire region. This study suggests that the level of threats of narrow-ranging species already identified as threatened may be underestimated, especially in heterogeneous tropical areas. We stress the need to develop sampling campaigns and implement proactive actions for narrow-ranging species in the tropics. Polycystins are upregulated in fibrotic kidneys and promote renal fibrosis Autosomal polycystic kidney disease (ADPKD) is caused by mutations in PKD1or PKD2 gene, which encodes polycystin-1 or polycystin-2 respectively. Paradoxically, polycystins are up-regulated in human ADPKD kidneys and overexpression of polycystin-1 even induced renal cyst formation in mice, suggesting that up-regulation of polycystin is pathogenic. In this study, we determined the role of polycystin in renal fibrosis and explored its underlying mechanisms. We found that the expression of polycystin-1 or polycystin-2 was positively correlated with the expression of collagen-I in the kidney of three different fibrotic mouse models. Pharmaceutical inhibition or genetic knockout of polycystin-2 reduced the expression of pro-fibrotic markers in fibrotic kidneys of two animal models. Moreover, conditional knockout of Pkd1 gene attenuated renal fibrosis in two mouse models. Enhancer of Zeste homolog 2 (EZH2) is a methyltransferase which plays important role in renal fibrosis. We found that EZH2 was positively correlated with the expression of polycystins in fibrotic kidneys. Conditional knockout of the Ezh2 attenuated the anti-fibrotic responses induced by Pkd1 deletion in fibrotic kidneys.

In conclusion, polycystins are up-regulated in injured kidneys and polycystins promote renal fibrosis through up-regulation of EZH2 in fibrotic kidneys, suggesting that renal fibrosis in polycystic kidneys is not the direct effect of PKD gene mutations.

SignificanceMutations of polycystins cause autosomal polycystic kidney disease (ADPKD) which is associated with increased renal fibrosis in cystic kidneys. However, the role of polycystin in renal fibrosis remains unclear. Here, we showed that the expression of polycystins is positively correlated with the development of renal fibrosis in injured kidneys. Pharmaceutical inhibition or genetic deletion of polycystin-2 attenuated renal fibrosis in fibrotic kidneys. Moreover, genetic deletion of polycystin-1 inhibited renal fibrosis in two different animal models. Finally, we showed that ablation of the methyltransferase EZH2 mitigated the anti-fibrotic effect of polycystin-1 deletion in fibrotic kidneys. We conclude that polycystin promotes through EZH2 and renal fibrosis in cystic kidney is not the direct effect of PKD gene mutations. SUMM4 biochemically complex couples insulator function and DNA replication timingcontrol The mechanisms that establish DNA replication timing programs in eukaryotes remain incompletely understood. Drosophila SNF2-related factor SUUR imparts under-replication (UR) of late-replicating intercalary heterochromatin (IH) in polytene chromosomes. We developed a proteomics technique termed MERCI to isolate a native complex SUMM4 comprising SUUR and chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates the ATPase activity of SUUR and is required for its normal spatiotemporal distribution in vivo. Both SUMM4 subunits mediate the activities of gypsy insulator disrupting enhancer-promoter interactions and establishing chromatin barriers. Furthermore, SuUR or mod(mdg4) mutations reverse UR of IH. Our findings uncover a critical role for architectural proteins in attenuating replication fork progression and suggest an alternative mechanism for DNA replication timing that does not depend on an asynchronous firing of replication origins.

One-Sentence SummaryA stable protein complex comprising an insulator factor and a SNF2-like ATPase imparts late replication of heterochromatin. The C. elegans gonadal sheath Sh1 cells extend asymmetrically over a differentiating germ cell population in the proliferative zone The C. elegans adult hermaphrodite germ line is surrounded by a thin tube formed by somatic sheath cells that support germ cells as they mature from the stem-like mitotic state through meiosis, gametogenesis and ovulation. Recently, we discovered that the distal-most Sh1 sheath cells associate with mitotic germ cells as they exit the niche. Here we report that these distal sheath-associated germ cells differentiate first in animals with temperature-sensitive mutations affecting germ cell state, and stem-like germ cells are maintained distal to the Sh1 boundary. We analyze several markers of the distal sheath, which is best visualized with endogenously tagged membrane proteins, as overexpressed fluorescent proteins fail to localize to distal membrane processes and can cause gonad morphology defects. However, such reagents with highly variable expression can be used to determine the relative positions of the two Sh1 cells, one of which often extends further distal than the other. Group-walk, a rigorous approach to group-wise false discovery rate analysis by target-decoy competition Target-decoy competition (TDC) is a commonly used method for false discovery rate (FDR) control in the analysis of tandem mass spectrometry data. This type of competitionbased FDR control has recently gained significant popularity in other fields after Barber and Candes laid its theoretical foundation in a more general setting that included the feature selection problem. In both cases, the competition is based on a head-to-head comparison between an (observed) target score and a corresponding decoy (knockoff) score. However, the effectiveness of TDC depends on whether the data is homogeneous, which is often not the case: in many settings, the data consists of groups with different score profiles or different proportions of true nulls. In such cases, applying TDC while ignoring the group structure often yields imbalanced lists of discoveries, where some groups might include relatively many false discoveries and other groups include relatively very few. On the other hand, as we show, the alternative approach of applying TDC separately to each group does not rigorously control the FDR.

We developed Group-walk, a procedure that controls the FDR in the target-decoy / knockoff setting while taking into account a given group structure. Group-walk is derived from the recently developed AdaPT -- a general framework for controlling the FDR with sideinformation. We show using simulated and real datasets that when the data naturally divides into groups with different characteristics Group-walk can deliver consistent power gains that in some cases are substantial. These groupings include the precursor charge state (4% more discovered peptides at 1% FDR threshold), the peptide length (3.6% increase) and the mass difference due to modifications (26% increase).

Group-walk is available at https://cran.r-project.org/web/packages/groupwalk/index.html