ITS-based assessment of Madagascar's fungal diversity and arrival of ectomycorrhizal fungi to the island Madagascar is known for its high diversity and endemism of Fauna and Flora, which makes it particularly interesting for research on diversity and evolution. Fungi, however, have been largely overlooked, and whether fungi exhibit the same patterns as animals and plants has yet to be further examined.

We collected fungal sporocarps and ectomycorrhizal (EcM) roots during opportunistic surveys in five forests in Madagascar and generated a dataset of fungal Internal Transcribed Spacer (ITS) DNA sequences. We analysed them together with all publicly available fungal ITS DNA sequences. We identified 620 Operational Taxonomic Units (OTUs) from Madagascar, 10% of which contained only sequences from our surveys. We found 292 OTUs belonging to EcM species with /russula-lactarius, /boletus, /tomentella-telephora, /cortinarius and /amanita as the most abundant EcM lineages. Overall, sixty percent of all the fungi and 81% of the EcM species found are endemic to Madagascar. Lastly, we conducted a phylogenetic analysis using all the OTUs in Amanitaceae, Boletaceae and Russulaceae families to elucidate their relative timing of arrival in Madagascar. We found that the EcM species from Madagascar in the three families diverged recently (less than 34 mya), long after the separation of India and Madagascar (88 mya), which is consistent with a dispersal mediated process of arrival on the island. Our study provides the first comprehensive view of the current state of knowledge of EcM fungi in Madagascar represented by molecular data useful for ecological and evolutionary studies.
Lowland plant arrival in alpine ecosystems facilitates a decrease in soil carbon content under experimental climate warming Climate warming is releasing carbon from soils around the world1-3, constituting a positive climate feedback. Warming is also causing species to expand their ranges into new ecosystems4-9. Yet, in most ecosystems, whether range expanding species will amplify or buffer expected soil carbon loss is unknown10. Here we used two whole-community transplant experiments and a follow-up glasshouse experiment to determine whether the establishment of herbaceous lowland plants in alpine ecosystems influences soil carbon content under warming. We found that warming (transplantation to low elevation) led to a negligible decrease in alpine soil carbon content, but its effects became significant and 52% {+/-} 31% (mean {+/-} 95% CIs) larger after lowland plants were introduced at low density into the ecosystem. We present evidence that decreases in soil carbon content likely occurred via lowland plants increasing rates of root exudation, soil microbial respiration and CO2 release under warming. Our findings suggest that warming-induced range expansions of herbaceous plants have the potential to alter climate feedbacks from this system, and that plant range expansions among herbaceous communities may be an overlooked mediator of warming effects on carbon dynamics.
Frequency modulated timer regulates mammalian hibernation Mammalian hibernators decrease basal metabolism and body temperature (Tb) to minimize energy expenditure in harsh seasons. During hibernation, Tb drops to low temperature (<10 {degrees}C) and remains constant for days, known as deep torpor in small mammalian hibernators. Spontaneous interbout arousals interrupt torpor bouts, when Tb recovers to euthermic state ~37 {degrees}C. Torpor-interbout arousal event repeats during several months of hibernation. However, little is known about mechanisms governing Tb fluctuation across torpor-interbout arousal cycles during hibernation. Recent improvement in data-logging techniques enables us to monitor Tb for more than hundred days with high precision, opening up new avenues for quantitative analysis to address the principle governing Tb fluctuation. Here, we analyzed Tb fluctuation across torpor-interbout arousal cycle of Syrian hamster, which can hibernate in response to chronic cold and short photoperiod under a laboratory condition, using generalized harmonic analysis and discovered a model with frequency modulation quantitatively reproducing Tb fluctuation. This analysis also identified an unexpectedly longer period of 120-430 days as the period that modulates another period of several days, generating Tb fluctuation for Syrian hamster. We propose that concerted action of two endogenous periods governs torpor-interbout arousal cycles during hibernation. 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. Naive human B cells engage the receptor binding domain of SARS-CoV-2, variants of concern, and related sarbecoviruses Exposure to a pathogen elicits an adaptive immune response aimed to control and eradicate. Interrogating the abundance and specificity of the naive B cell repertoire contributes to understanding how to potentially elicit protective responses. Here, we isolated naive B cells from 8 seronegative human donors targeting the SARS-CoV-2 receptor-binding domain (RBD). Single B cell analysis showed diverse gene usage with no restricted complementarity determining region lengths. We show that recombinant antibodies engage SARS-CoV-2 RBD, circulating variants, and pre-emergent coronaviruses. Representative antibodies signal in a B cell activation assay and can be affinity matured through directed evolution. Structural analysis of a naive antibody in complex with spike shows a conserved mode of recognition shared with infection-induced antibodies. Lastly, both naive and affinity-matured antibodies can neutralize SARS-CoV-2. Understanding the naive repertoire may inform potential responses recognizing variants or emerging coronaviruses enabling the development of pan-coronavirus vaccines aimed at engaging germline responses.

One Sentence SummaryIsolation of antibody germline precursors targeting the receptor binding domain of coronaviruses. Cooperation of two opposite flagella allows high-speed swimming and active turning in zoospores Phytophthora species cause diseases in a large variety of plants and represent a serious agricultural threat, leading, every year, to multibillion dollar losses. Infection occurs when these biflagellated zoospores move across the soil at their characteristic high speed and reach the roots of a host plant. Despite the relevance of zoospore spreading in the epidemics of plant diseases, characteristics of individual swimming of zoospores have not been fully investigated. It remains unknown about the characteristics of two opposite beating flagella during translation and turning, and the roles of each flagellum on zoospore swimming. Here, combining experiments and modeling, we show how these two flagella contribute to generate thrust when beating together, and identify the mastigonemes-attached anterior flagellum as the main source of thrust. Furthermore, we find that turning involves a complex active process, in which the posterior flagellum temporarily stops, while the anterior flagellum keeps on beating and changes its pattern from sinusoidal waves to power and recovery strokes, similar to Chlamydomonass breaststroke, to reorient its body to a new direction. Our study is a fundamental step towards a better understanding of the spreading of plant pathogens motile forms, and shows that the motility pattern of these biflagellated zoospores represents a distinct eukaryotic version of the celebrated "run-and-tumble" motility class exhibited by peritrichous bacteria. Endosomal cargo recycling mediated by Gpa1 and Phosphatidylinositol-3-Kinase is inhibited by glucose starvation Cell surface protein trafficking is regulated in response to nutrient availability, with multiple pathways directing surface membrane proteins to the lysosome for degradation in response to suboptimal extracellular nutrients. Internalised protein and lipid cargoes recycle back to the surface efficiently in glucose replete conditions, but this trafficking is attenuated following glucose starvation. We find cells with either reduced or hyperactive phosphatidylinositol 3-kinase (PI3K) activity are defective for recycling. Furthermore, we find the yeast G subunit Gpa1, an endosomal PI3K effector, is required for surface recycling of cargoes. Following glucose starvation, mRNA and protein levels of a distinct G subunit Gpa2 are elevated following nuclear translocation of Mig1, which inhibits recycling of various cargoes. As Gpa1 and Gpa2 interact at the surface where Gpa2 concentrates during glucose starvation, we propose this disrupts PI3K activity required for recycling, potentially diverting Gpa1 to the surface and interfering with its endosomal role in recycling. In support of this model, glucose starvation and over-expression of Gpa2 alters PI3K endosomal phosphoinositide production. Glucose deprivation therefore triggers a survival mechanism to increase retention of surface cargoes in endosomes and promote their lysosomal degradation. Cellular feedback to organotelluranes displays modulation of antioxidant proteins gene expression Organotelluranes RT3 and RT4 are thiol reagents that induce mitochondrial transition pore (MTP) opening in a sensitive and insensitive manner to cyclosporin A. Although RT3 and RT4 promote glutathione depletion, paradoxically, they are also an efficient antioxidant for membrane lipids. These compounds antagonistic effects elicited the challenging question of how the gene expression of antioxidant enzymes would respond to treatment with these compounds. The influence of RT3 and RT4 on antioxidant enzyme expression was investigated in cultured aortic smooth muscle cells (ASMC). RT3 and RT4 promoted disruption of ionic calcium homeostasis, mitochondrial transmembrane potential ({Delta}{Psi}), and cell death in a dose-dependent manner. The cell death mechanisms responded qualitatively to the increase of the organotellurane concentration and changed from apoptosis to necrosis. RT3 and RT4 increased the expression of thioredoxin significantly. RT3 also increased the expression of glutaredoxin and glutathione peroxidase, slightly the catalase expression without significant effects on SOD expression. The results are consistent with GSH and protein thiol depletion and discussed based on the cell toxicity mechanism exhibited by these compounds.



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org.highwire.dtl.DTLVardef@52bb82org.highwire.dtl.DTLVardef@19476d9org.highwire.dtl.DTLVardef@6aa99org.highwire.dtl.DTLVardef@1aa605e_HPS_FORMAT_FIGEXP  M_FIG C_FIG The obstacles and potential clues of prime editing applications in tomato, a dicot plant Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a "priming" extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. Modifying the pegRNA sequences by shortening or introducing mismatches to the primer binding sequences (PBS) in order to reduce their melting temperatures (Tm) did not enhance the PE efficiency at the SlBMP21, SlALC and SlALS1 loci. Our data show challenges of PE approach in tomato, indicating a further improvement of the PE system for the successful applications such as use of improved expression systems. Our work provides an important clue for the successful application of the PE approach in crop improvement. Action at a distance: Defects in division plane positioning in the root meristematic zone affect cell organization in the differentiation zone Cell division plane orientation is critical for plant and animal development and growth. TANGLED1 (TAN1) and AUXIN-INDUCED-IN-ROOT-CULTURES9 (AIR9) are division-site localized microtubule-binding proteins required for division plane positioning. tan1 and air9 Arabidopsis thaliana single mutants have minor or no noticeable phenotypes but the tan1 air9 double mutant has synthetic phenotypes including stunted growth, misoriented divisions, and aberrant cell-file rotation in the root differentiation zone. These data suggest that TAN1 plays a role in nondividing cells. To determine whether TAN1 is required in elongating and differentiating cells in the tan1 air9 double mutant, we limited its expression to actively dividing cells using the G2/M-specific promoter of the syntaxin KNOLLE (pKN:TAN1-YFP). Unexpectedly, in addition to rescuing division plane defects, pKN:TAN1-YFP rescued root growth and the root differentiation zone cell file rotation defects in the tan1 air9 double mutant. This suggests that defects that occur in the meristematic zone later affect the organization of elongating and differentiating cells.

Summary StatementExpression of TAN1 in the root meristematic zone rescues cell file rotation defects in tan1 air9 mutants, suggesting defects that occur in mitosis may influence organization of nondividing cells. Citation needed? Wikipedia and the COVID-19 pandemic BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. Online single-cell data integration through projecting heterogeneous datasets into a common cell-embedding space Computational tools for integrative analyses of diverse single-cell experiments are facing formidable new challenges including dramatic increases in data scale, sample heterogeneity, and the need to informatively cross-reference new data with foundational datasets. Here, we present SCALEX, a deep-learning method that integrates single-cell data by projecting cells into a batch-invariant, common cell-embedding space in a truly online manner (i.e., without retraining the model). SCALEX substantially outperforms online iNMF and other state-of-the-art non-online integration methods on benchmark single-cell datasets of diverse modalities, (e.g., scRNA-seq, scATAC-seq), especially for datasets with partial overlaps, accurately aligning similar cell populations while retaining true biological differences. We showcase SCALEXs advantages by constructing continuously expandable single-cell atlases for human, mouse, and COVID-19 patients, each assembled from diverse data sources and growing with every new data. The online data integration capacity and superior performance makes SCALEX particularly appropriate for large-scale single-cell applications to build-upon previously hard-won scientific insights. Beta-cell specific Insr deletion promotes insulin hypersecretion and improves glucose tolerance prior to global insulin resistance Abstract

Insulin receptor (Insr) protein can be found at higher levels in pancreatic {beta}-cells than in most other tissues, but the consequences of {beta}-cell insulin resistance remain enigmatic. Ins1cre allele was used to delete Insr specifically in {beta}-cells of both female and male mice. Experimental mice were compared to Ins1cre-containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined {beta}-cells revealed transcriptomic consequences of Insr loss, which differed between female and male mice. Action potential and calcium oscillation frequencies were increased in Insr knockout {beta}- cells from female, but not male mice, whereas only male {beta}InsrKO mice had reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female {beta}InsrKO and {beta}InsrHET mice exhibited elevated insulin release in perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr did not alter {beta}-cell area up to 9 months of age, nor did it impair hyperglycemia-induced proliferation. Based on our data, we adapted a mathematical model to include {beta}-cell insulin resistance, which predicted that {beta}-cell Insr knockout would improve glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance was significantly improved in female {beta}InsrKO and {beta}InsrHET mice when compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We did not observe improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of {beta}-cell specific insulin resistance. The propensity for hyperinsulinemia was associated with mildly reduced fasting glucose and increased body weight. We further validated our main in vivo findings using the Ins1-CreERT transgenic line and found that male mice had improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that loss of {beta}-cell Insr contributes to glucose-induced hyperinsulinemia, thereby improving glucose homeostasis in otherwise insulin sensitive dietary and age contexts. Effect of Adolescent Stress on Adult Morphine-Induced Behavioral Sensitization is Dependent Upon Genetic Background 1Deaths related to opioid use have skyrocketed in the United States, leading to a public health epidemic. Research has shown that both biological (genes) and environmental (stress) precursors are linked to opioid use. In particular, stress during adolescence - a critical period of frontal lobe development - influences the likelihood of abusing drugs. However, little is known about the biological mechanisms through which adolescent stress leads to long-term risk of opioid use, or whether genetic background moderates this response. Male and female C57BL/6J and BALB/cJ mice were exposed to chronic variable social stress (CVSS) or control conditions throughout adolescence and then tested for morphine locomotor sensitization or morphine consumption in adulthood. To examine possible mechanisms that underlie stress-induced changes in morphine behaviors, we assessed physiological changes in response to acute stress exposure and prefrontal cortex miRNA gene expression. Adolescent stress did not influence morphine sensitization or consumption in BALB/cJ animals, and there was limited evidence of stress effects in female C57BL/6J mice. In contrast, male C57BL/6J mice exposed to adolescent CVSS had blunted morphine sensitization compared to control animals; no differences were observed in the acute locomotor response to morphine administration or morphine consumption. Physiologically, C57BL/6J mice exposed to CVSS had an attenuated corticosterone recovery following an acute stressor and downregulation of twelve miRNA in the prefrontal cortex compared to control mice. The specificity of the effects for C57BL/6J versus BALB/cJ mice provides evidence of a gene by environmental interaction influencing opioid behaviors. Long-term differences in stress reactivity or miRNA expression suggests two possible biological mechanisms to evaluate in future research. Allopurinol Blocks The Formation And Progression Of Aortic Aneurysm In A Mouse Model Of Marfan Syndrome BackgroundThe pathogenesis and progression of aortic aneurysm in Marfan syndrome (MFS) involves dysregulated TGF-{beta} and nitric oxide signaling, altered hemodynamics, and biomechanical forces. Increasing evidence indicates that redox stress participates in MFS aortopathy, though its contribution is not well established. We reported elevated reactive oxygen species (ROS) formation and NADPH oxidase NOX4 upregulation in MFS patients and mice aortic samples. Here we address the contribution of xanthine oxidoreductase (XOR), which is transcribed as dehydrogenase (XDH) and then post-translationally modified to oxidase (XO), catabolizing mainly purines into uric acid and ROS.

Methods and ResultsWe report that in MFS patients, XOR protein levels increased in aortic samples. In MFS mice (Fbn1C1041G/+), XOR mRNA transcripts augmented in the aorta of young but not older MFS mice. Moreover, the enzymatic activity of XO form increased with respect to XDH. The administration of the XOR inhibitor allopurinol (ALO) blocked the progression of aortic root aneurysm in MFS mice. ALO was also protective when administrated before the appearance of aneurysm. MFS-induced elastic fiber fragmentation, fibrotic remodeling, nuclear translocation of pNRF2, and increased 3-nitrotyrosine levels in the aortic tunica media as well as endothelial dysfunction were all prevented by allopurinol. The large overproduction of H2O2 in MFS aorta did not occur in allopurinol-treated mice whose uric acid plasma levels remained unaltered.

ConclusionsThis study strengthens the concept that redox stress is an important determinant of aortic aneurysm formation and progression in MFS and supports a clinical trial for ALO in the pharmacological treatment of MFS aortopathy.

GRAPHICAL ABSTRACT

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org.highwire.dtl.DTLVardef@eab02aorg.highwire.dtl.DTLVardef@1d3b39eorg.highwire.dtl.DTLVardef@1b54d0forg.highwire.dtl.DTLVardef@1d61ad0_HPS_FORMAT_FIGEXP  M_FIG C_FIG Construction of a new chromosome-scale, long-read reference genome assembly for the Syrian hamster, Mesocricetus auratus BackgroundThe Syrian hamster (Mesocricetus auratus) has been suggested as a useful mammalian model for a variety of diseases and infections, including infection with respiratory viruses such as SARS-CoV-2. The MesAur1.0 genome assembly was generated in 2013 using whole-genome shotgun sequencing with short-read sequence data. Current more advanced sequencing technologies and assembly methods now permit the generation of near-complete genome assemblies with higher quality and greater continuity.

FindingsHere, we report an improved assembly of the M. auratus genome (BCM_Maur_2.0) using Oxford Nanopore Technologies long-read sequencing to produce a chromosome-scale assembly. The total length of the new assembly is 2.46 Gbp, similar to the 2.50 Gbp length of a previous assembly of this genome, MesAur1.0. BCM_Maur_2.0 exhibits significantly improved continuity with a scaffold N50 that is 6.7 times greater than MesAur1.0. Furthermore, 21,616 protein coding genes and 10,459 noncoding genes are annotated in BCM_Maur_2.0 compared to 20,495 protein coding genes and 4,168 noncoding genes in MesAur1.0. This new assembly also improves the unresolved regions as measured by nucleotide ambiguities, where approximately 17.11% of bases in MesAur1.0 were unresolved compared to BCM_Maur_2.0 in which the number of unresolved bases is reduced to 3.00%.

ConclusionsAccess to a more complete reference genome with improved accuracy and continuity will facilitate more detailed, comprehensive, and meaningful research results for a wide variety of future studies using Syrian hamsters as models. Innovative GenExpA software for selecting suitable reference genes for reliable normalization of gene expression in melanoma The algorithms commonly used to select the best stable reference gene in RT-qPCR data analysis have their limitations. We showed that simple selection of the reference gene or pair of genes with the lowest stability value from the pool of potential reference genes - a commonly used approach - is not sufficient to accurately and reliably normalize the target gene transcript and can lead to biologically incorrect conclusions. For reliable assessment of changes in a target gene expression level, we propose our innovative GenExpA software, which works in a manner independent of the experimental model and the normalizer used. GenExpA software selects the best reference by combining the NormFinder algorithm with progressive removal of the least stable gene from the candidate genes in a given experimental model and in the set of daughter models assigned to it. The reliability of references is validated based on the consistency of the statistical analyses of normalized target gene expression levels through all models, described by the coherence score (CS). The use of the CS value imparts a new quality to qPCR analysis because it clarifies how low the stability value of reference must be in order for biologically correct conclusions to be drawn. We tested our method on qPCR data for the B4GALT genes family in melanoma, which is characterized by a high mutation rate, and in melanocytes. GenExpA is available at https://github.com/DorotaHojaLukowicz/GenExpA or https://www.sciencemarket.pl/baza-programow-open-source#oferty.

HighlightsGenExpA - next-generation software for normalizer selection and validation

The GenExpA tool defines how low the stability value of the reference should be

The GenExpA tool determines the level of gene expression analysis robustness

The GenExpA tool increases the speed of analysis and reduces cost Getting more out of FLAG-Tag co-immunoprecipitation mass spectrometry experiments using FAIMS. Co-immunoprecipitation of proteins coupled to mass spectrometry is critical for the understanding of protein interaction networks. In instances where a suitable antibody is not available, it is common to graft synthetic tags onto target protein sequences and allowing the use of commercially available antibodies for affinity purification. A common approach is through FLAG-Tag co-immunoprecipitation. To allow the selective elution of protein complexes, competitive displacement using a large molar excess of the tag peptides is often carried out. Yet, this creates downstream challenges for the mass spectrometry analysis due to the presence of large quantities of these peptides. Here, we demonstrate that Field Asymmetric Ion Mobility Spectrometry (FAIMS), a gas phase ion separation device prior to mass spectrometry analysis can be applied to FLAG-Tag co-immunoprecipitation experiment to increase the depth of protein coverage. By excluding these abundant tag peptides, we were able to observe deeper coverage of interacting proteins and as a result, deeper biological insights, without the need for additional sample handling or altering sample preparation protocols. Constraints on the evolution of toxin-resistant Na,K-ATPases have limited dependence on sequence divergence A growing body of theoretical and experimental evidence suggests that intramolecular epistasis is a major determinant of rates and patterns of protein evolution and imposes a substantial constraint on the evolution of novel protein functions. Here, we examine the role of intramolecular epistasis in the recurrent evolution of resistance to cardiotonic steroids (CTS) across tetrapods, which occurs via specific amino acid substitutions to the -subunit family of Na,K-ATPases (ATP1A). After identifying a series of recurrent substitutions at two key sites of ATP1A that are predicted to confer CTS resistance in diverse tetrapods, we then performed protein engineering experiments to test the functional consequences of introducing these substitutions onto divergent species backgrounds. In line with previous results, we find that substitutions at these sites can have substantial background-dependent effects on CTS resistance. Globally, however, these substitutions also have pleiotropic effects that are consistent with additive rather than background-dependent effects. Moreover, the magnitude of a substitutions effect on activity does not depend on the overall extent of ATP1A sequence divergence between species. Our results suggest that epistatic constraints on the evolution of CTS-resistant forms of Na,K-ATPase likely depend on a small number of sites, with little dependence on overall levels of protein divergence. We propose that dependence on a limited number sites may account for the observation of convergent CTS resistance substitutions observed among taxa with highly divergent Na,K-ATPases.

Significance StatementIndividual amino acid residues within a protein work in concert to produce a functionally coherent structure that must be maintained even as orthologous proteins in different species diverge over time. Given this dependence, we expect identical mutations to have more similar effects on protein function in more closely related species. We tested this hypothesis by performing protein-engineering experiments on ATP1A, an enzyme mediating target-site insensitivity to cardiotonic steroids (CTS) in diverse animals. These experiments reveal that the phenotypic effects of substitutions can sometimes be background-dependent, but also that the magnitude of these phenotypic effects does not correlate with overall levels of ATP1A sequence divergence. Our results suggest that epistatic constraints are determined by states at a small number of sites, potentially explaining the frequent convergent CTS resistance substitutions among Na,K-ATPases of highly divergent taxa. Predicting lapses of attention with sleep-likeslow waves. Attentional lapses are ubiquitous and can negatively impact performance. They correlate with mind wandering, or thoughts that are unrelated to ongoing tasks and environmental demands. In other cases, the stream of consciousness itself comes to a halt and the mind goes blank. What is happening in the brain that leads to these mental states? To understand the neural mechanisms underlying attentional lapses, we cross-analysed the behaviour, subjective experience and neural activity of healthy participants performing a task. Random interruptions prompted participants to indicate their mental states as task-focused, mind-wandering or mind-blanking. High-density electroencephalography revealed the occurrence of spatially and temporally localized slow waves, a pattern of neural activity characteristic of the transition toward sleep. These slow waves accompanied behavioural markers of lapses and preceded reports of mind wandering and mind blanking. Furthermore, the location of slow waves distinguished sluggish versus impulsive behaviours, mind wandering versus mind blanking. Our results suggest attentional lapses share a common physiological origin: the emergence of local sleep-like activity within the awake brain. Bespoke data augmentation and network construction enable developmental morphological classification on limited microscopy datasets Deep learning is a powerful tool for image classification, yet training deep learning models often requires large datasets, which are not always available in biological research. Here we use a small dataset to train an accurate classifier by employing data augmentation regimes and Bayesian optimisation. We establish a new staging system for the Hamburger-Hamilton stage 10 chick embryo brain, based on morphology, identifying three sub-stages uncoupled from somite number, the usual metric used to stage chick embryos. Incorporating biologically informed, data-driven preprocessing, we train a deep convolutional neural network to classify our sub-stages. We find that augmenting our images with a combination of rotation, blur, shear, and cutout transformations is effective in training an accurate classifier on a small microscopy dataset. We then apply our approach to an unrelated small microscopy dataset comprising images of limb buds, finding good agreement with the results from brain classification. Through saliency analysis, we ensure that relevant features are being used by our classifier, gain insight into the efficacy of different data augmentations, and identify sub-stage specific features. In summary, we apply biological domain expertise to design effective data augmentation regimes for training a neural network-based image classifier on small, specialised datasets, classifying the developing brains and limbs with up to 90.9% and 94.4% accuracy respectively. These reliable classifiers can inform future experiments. Identification of the three zinc-binding sites on Tau protein Tau protein has been extensively studied due to its key roles in microtubular cytoskeleton regulation and in the formation of aggregates found in some neurodegenerative diseases. Recently it has been shown that zinc is able to induce tau aggregation by interacting with several binding sites. However, the precise location of these sites and the molecular mechanism of zinc-induced aggregation remain unknown. Here we used Nuclear Magnetic Resonance (NMR) to identify zinc binding sites on hTau40 isoform. These experiments revealed three distinct zinc binding sites on tau, located in the N-terminal part (H14, H32, H94, and H121), the repeat region (H299, C322, H329 and H330) and the C-terminal part (H362, H374, H388 and H407). Further analysis enabled us to show that the C-terminal and the N-terminal sites are independent of each other. Using molecular simulations, we modeled the structure of each site in a complex with zinc. Given the clinical importance of zinc in tau aggregation, our findings pave the way for designing potential therapies for tauopathies.

HighlightsO_LIZinc is known to induce tau aggregation in neurodegenerative diseases
C_LIO_LIZinc binding locations and mechanism are not yet clear
C_LIO_LIUsing NMR we localized 3 zinc binding site on tau
C_LIO_LIBy molecular simulations, we proposed a modeled structure of each site
C_LIO_LIOur findings pave the way for designing potential therapies for tauopathies
C_LI Distribution and molecular evolution of the anti-CRISPR family AcrIF7 Anti-CRISPRs are proteins capable of blocking CRISPR-Cas systems and typically encoded in mobile genetic elements. Since their discovery, numerous anti-CRISPR families have been identified. However, little is known about the distribution and sequence diversity of members within a family, nor how these traits influence the anti-CRISPRs function and evolution. Here we use AcrIF7 to explore the dissemination and molecular evolution of an anti-CRISPR family. We uncovered five sub-clusters and prevalent anti-CRISPR variants within the group. Remarkably, AcrIF7 homologs display high similarity despite their broad geographical, ecological and temporal distribution. Although mainly associated with Pseudomonas aeruginosa, AcrIF7 was identified in distinct genetic backgrounds indicating horizontal dissemination, primarily by phages. Using mutagenesis, we recreated variation observed in databases but also extended the sequence diversity of the group. Characterisation of the variants identified residues key for the anti-CRISPR function and other contributing to its mutational tolerance. Moreover, molecular docking revealed that variants with affected function lose key interactions with its CRISPR-Cas target. Analysis of publicly available data and the generated variants suggests that the dominant AcrIF7 variant corresponds to the minimal and optimal anti-CRISPR selected in the family. Our study provides a blueprint to investigate the molecular evolution of anti-CRISPR families. Non-Newtonian Blood Rheology Impacts Left Atrial Stasis in Patient-Specific Simulations The lack of mechanically effective contraction of the left atrium (LA) during atrial fibrillation (AF) disturbs blood flow, increasing the risk of thrombosis and ischemic stroke. Thrombosis is most likely in the left atrial appendage (LAA), a small narrow sac where blood is prone to stagnate. Slow flow promotes the formation of erythrocyte aggregates in the LAA, also known as rouleaux, causing viscosity gradients that are usually disregarded in patient-specific simulations. To evaluate these non-Newtonian effects, we built atrial models derived from 4D computed tomography scans of patients and carried out computational fluid dynamics simulations using the Carreau-Yasuda constitutive relation. We examined six patients, three of whom had AF and LAA thrombosis or a history of transient ischemic attacks (TIAs). We modeled the effects of hematocrit and rouleaux formation kinetics by varying the parameterization of the Carreau-Yasuda relation and modulating non-Newtonian viscosity changes based on residence time. Comparing non-Newtonian and Newtonian simulations indicates that slow flow in the LAA increases blood viscosity, altering secondary swirling flows and intensifying blood stasis. While some of these effects can be subtle when examined using instantaneous metrics like shear rate or kinetic energy, they are manifested in the blood residence time, which accumulates over multiple heartbeats. Our data also reveal that LAA blood stasis worsens when hematocrit increases, offering a potential new mechanism for the clinically reported correlation between hematocrit and stroke incidence. In summary, we submit that hematocrit-dependent non-Newtonian blood rheology should be considered in calculating patient-specific blood stasis indices by computational fluid dynamics. The maize gene maternal derepression of r1 (mdr1) encodes a DNA glycosylase with maternal and paternal fertility functions Demethylation of transposons can activate expression of nearby genes and cause imprinted gene expression in endosperm, and it is hypothesized to lead to expression of transposon siRNAs that reinforce silencing in the next generation through transfer either into egg or embryo. Here we describe maternal derepression of r1 (mdr1), which encodes a DNA glycosylase with homology to Arabidopsis DEMETER and which is partially responsible for demethylation of thousands of regions in endosperm. Instead of promoting siRNA expression in endosperm, MDR1 activity inhibits it. Methylation of most repetitive DNA elements in endosperm is not significantly affected by MDR1, with an exception of Helitrons. While maternally-expressed imprinted genes preferentially overlap with MDR1 demethylated regions, the majority of genes that overlap demethylated regions are not imprinted. Double mutant megagametophytes lacking both MDR1 and its close homolog DNG102 result in early seed failure, and double mutant microgametophytes fail pre-fertilization. These data establish DNA demethylation by glycosylases as essential in maize endosperm and pollen and suggest that neither transposon repression nor genomic imprinting are its main function in endosperm. Activation of the type 3 secretion system of enteropathogenic E. coli leads to remodeling of its membrane composition and function The cell envelope of gram-negative bacteria is a complex structure, essential for bacterial survival and for resistance to many antibiotics. Channels that cross the bacterial envelope and the host cell membrane form secretion systems that are activated upon attachment to host, enabling bacteria to inject effector molecules into the host cell, required for bacterial-host interaction. The type III secretion system (T3SS) is critical for the virulence of several pathogenic bacteria, including enteropathogenic E. coli (EPEC). The EPEC T3SS activation is associated with repression of carbon storage regulator (CsrA), resulting in gene expression remodeling, which is known to affect EPEC central carbon metabolism and contributes to the adaptation to a cell-adherent lifestyle in a poorly understood manner. We reasoned that the changes in bacterial envelope upon attachment to host and the activation of a secretion system may involve a modification of the lipid composition of bacterial envelope. Accordingly, we performed a lipidomics analysis on mutant strains that simulate T3SS activation. We saw a shift in glycerophospholipid metabolism towards the formation of lysophospholipids, attributed to corresponding upregulation of the phospholipase pldA and the acyltransferase ygiH upon T3SS activation in EPEC. We also detected a shift from menaquinones and ubiquinones to undecaprenyl lipids, concomitant to abnormal synthesis of O-antigen. The remodeling of lipid metabolism is mediated by CsrA and associated with increased bacteria cell size and Zeta potential, and a corresponding alteration in EPEC permeability to vancomycin, increasing the sensitivity of T3SS-activated strains and of adherent wild type EPEC to the antibiotic.

ImportanceThe characterization of EPEC membrane lipid metabolism upon attachment to host is an important step towards a better understanding the shift of EPEC, a notable human pathogen, from a planktonic to adherent life style. It may also apply to other pathogenic bacteria that use this secretion system. We predict that upon attachment to host cells the lipid remodeling upon T3SS activation contributes to bacterial fitness and promotes host colonization, and show that it is associated with increased cell permeability and higher sensitivity to vancomycin.

To the best of our knowledge, this is the first demonstration of a bacterial lipid remodeling due to activation of a secretion system.