Tardigrade CAHS Proteins Act as Molecular Swiss Army Knives to Mediate Desiccation Tolerance Through Multiple Mechanisms Tardigrades, also known as water bears, make up a phylum of small but extremely hardy animals, renowned for their ability to survive extreme stresses, including desiccation. How tardigrades survive desiccation is one of the enduring mysteries of animal physiology. Here we show that CAHS D, an intrinsically disordered protein belonging to a unique family of proteins possessed only by tardigrades, undergoes a liquid-to-gel phase transition in a concentration dependent manner. Unlike other gelling proteins, such as gelatin, our data support a mechanism in which gel formation of CAHS D is driven by intermolecular {beta}-{beta} interactions. We find that gel formation corresponds with strong coordination of water and slowing of water diffusion. The degree of water coordination correlates with the ability of CAHS D to protect lactate dehydrogenase from unfolding when dried. This implies that the mechanism for unfolding protection can be attributed to a combination of hydration and slowed molecular motion. Conversely, rapid diffusion leading to efficient molecular shielding appears to be the predominant mechanism preventing protein aggregation. Our study demonstrates that distinct mechanisms are required for holistic protection during desiccation, and that protectants, such as CAHS D, can act as molecular  Swiss Army Knives capable of providing protection through several different mechanisms simultaneously.
Phenotypic Heterogeneity Facilitates Survival While Hindering the Evolution of Drug Resistance Due to Intraspecific Competition Rising rates of resistance to antimicrobial drugs threatens the effective treatment of infections across the globe. Drug resistance has been established to emerge from non-genetic mechanisms, such as "persistence" in quiescent microbes and fluctuations in gene expression in actively replicating cells, as well as from genetic mutations. However, it is still unclear how non-genetic drug resistance affects the evolution of genetic drug resistance. We develop deterministic and stochastic population models that incorporate resource competition to quantitatively investigate the transition from non-genetic to genetic resistance during the exposure to static and cidal drugs. We find that non-genetic resistance facilitates the survival of cell populations during drug treatment, but that it hinders the development of genetic resistance due to the competition between the non-genetically and genetically resistant subpopulations. Non-genetic drug resistance in the presence of subpopulation competition is found to increase the first-appearance and fixation times of drug resistance mutations, while increasing the probability of mutation before population extinction during cidal drug treatment. Intense intraspecific competition during drug treatment leads to extinction of the susceptible and non-genetically resistant subpopulations. These findings advance our fundamental understanding of the evolution of drug resistance and may guide novel treatment strategies for patients with drug-resistant infections.

SIGNIFICANCEDrug resistance is predicted to kill as many as 10 million people per year and cost over 100 trillion USD in cumulative lost production globally by 2050. To mitigate these socio-economic costs, we need to fundamentally understand the drug resistance process. We investigate the effect that different forms of resistance have on the evolution of drug resistance using mathematical modeling and computer simulations. We find that the presence of non-genetically drug-resistant cells (whose resistance is temporary and not encoded in a genetic mutation) allows the population to survive drug treatment, while competition between these subpopoulations simultaneously slows down the evolution of permanent genetic drug resistance and in some cases drives them extinct. These findings have important implications for advancing evolutionary theory and for developing effective "resistance-proof" treatments.
"Neighbourhood watch" model: embryonic epiblast cells assess positional information in relation to their neighbours In many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret these. Using experimental manipulation of early chick embryos including misexpression of an inducer (VG1 or ACTIVIN) and an inhibitor (BMP4), we test two alternative models for their ability to explain how the site of primitive streak formation is positioned relative to the rest of the embryo. In one model, cells read morphogen concentrations cell-autonomously. In the other, cells sense changes in morphogen status relative to their neighbourhood. We find that only the latter model can account for the experimental results, including some counter-intuitive predictions. This mechanism (which we name "neighbourhood watch" model) illuminates the classic "French Flag Problem" and how positional information is interpreted by a sheet of cells in a large developing system.

Summary statementIn a large developing system, the chick embryo before gastrulation, cells interpret gradients of positional signals relative to their neighbours to position the primitive streak, establishing bilateral symmetry. 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 An AI-guided signature reveals the nature of the shared proximal pathways of host immune response in MIS-C and Kawasaki disease A significant surge in cases of multisystem inflammatory syndrome in children (MIS-C, also called Pediatric Inflammatory Multisystem Syndrome - PIMS) has been observed amidst the COVID-19 pandemic. MIS-C shares many clinical features with Kawasaki disease (KD), although clinical course and outcomes are divergent. We analyzed whole blood RNA sequences, serum cytokines, and formalin fixed heart tissues from these patients using a computational toolbox of two gene signatures, i.e., the 166-gene viral pandemic (ViP) signature, and its 20-gene severe (s)ViP subset that were developed in the context of SARS-CoV-2 infection and a 13-transcript signature previously demonstrated to be diagnostic for KD. Our analyses revealed that KD and MIS-C are on the same continuum of the host immune response as COVID-19. While both the pediatric syndromes converge upon an IL15/IL15RA-centric cytokine storm, suggestive of shared proximal pathways of immunopathogenesis, they diverge in other laboratory parameters and cardiac phenotypes. The ViP signatures also revealed unique targetable cytokine pathways in MIS-C, place MIS-C farther along in the spectrum in severity compared to KD and pinpoint key clinical (reduced cardiac function) and laboratory (thrombocytopenia and eosinopenia) parameters that can be useful to monitor severity. Outbreak.info Research Library: A standardized, searchable platform to discover and explore COVID-19 resources and data To combat the ongoing COVID-19 pandemic, scientists have been conducting research at breakneck speeds, producing over 52,000 peer reviewed articles within the first 12 months. In contrast, a little over 1,000 peer reviewed articles were published within the first 12 months of the SARS-CoV-1 pandemic starting in 2002. In addition to publications, there has also been an upsurge in clinical trials to develop vaccines and treatments, scientific protocols to study SARS-CoV-2, methodology for epidemiological modeling, and datasets spanning molecular studies to social science research. One of the largest challenges has been keeping track of the vast amounts of newly generated disparate data and research that exist in independent repositories. To address this issue, we developed outbreak.info, which provides a standardized, searchable interface of heterogeneous resources on COVID-19 and SARS-CoV-2. Unifying metadata from twelve repositories, we have assembled a collection of over 260,000 publications, clinical trials, datasets, protocols, and other resources as of March 2022. We used a rigorous schema to enforce a consistent format across different data sources and resource types, and linked related resources where possible. This enables users to quickly retrieve information across data repositories, regardless of resource type or repository location. Outbreak.info also combines the combined research library with spatiotemporal genomics data on SARS-CoV-2 variants. The web interface provides interactive visualizations and reports to explore the unified data and generate hypotheses. In addition to providing a web interface, we also publish the data we have assembled and standardized in a high performance public API and an R package. Finally, we discuss the challenges inherent in combining metadata from scattered and heterogeneous resources and provide recommendations to streamline this process to aid scientific research. Heterogeneous recruitment abilities to RNA polymerases generate nonlinear scaling of gene expression 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. Somatic mutations of activated signaling genes, transcription factors, or tumor suppressors are a precondition for leukemic transformation from myelodysplastic syndromes: a sequencing analysis of 64 paired samples The transformation biology of secondary AML from MDS is still not fully understood. Here, we performed a large cohort of paired sequences including target, whole-exome and single cell sequencing to search AML transformation-related mutations (TRM). The results showed that fifty-five out of the 64 (85.9%) patients presented presumptive TRM involving activated signaling, transcription factors, or tumor suppressors. Most of TRM (63.6%, 35 cases) emerged at the leukemia transformation point. All five of the remaining nine patients analyzed by paired whole exome sequencing showed TRM which are not included in the reference targets. Single-cell sequencing indicated that the activated cell signaling route was related to TRM which take place prior to phenotypic development. Of note, defined TRM was limited to a small set of genes (less than ten, in the order: NRAS/KRAS, CEBPA, TP53, FLT3, RUNX1, CBL, PTPN11 and WT1, accounted for 91.0% of the mutations). In conclusion, somatic mutations involving in activated signaling, transcription factors, or tumor suppressors appeared to be a precondition for AML transformation from myelodysplastic syndromes. The TRM may be considered as new therapy targets. The Shigella type III effector protein OspB is a cysteine protease The type III secretion system is required for virulence of many pathogenic bacteria. Bacterial effector proteins delivered into target host cells by this system modulate host signaling pathways and processes in a manner that promotes infection. Here, we define the activity of the effector protein OspB of the human pathogen Shigella spp., the etiological agent of shigellosis and dysenteric disease. Using the yeast Saccharomyces cerevisiae as a model organism, we show that OspB sensitizes cells to inhibition of TORC1, the central regulator of growth and metabolism. In silico analyses reveal that OspB bears structural homology to bacterial cysteine proteases, and we define a conserved cysteine-histidine catalytic dyad required for OspB function. Using yeast genetic screens, we identify a crucial role for the arginine N-degron pathway in the growth inhibition phenotype and show that inositol hexakisphosphate is an OspB cofactor. We find that a yeast substrate for OspB is the TORC1 component Tco89p, proteolytic cleavage of which generates a C-terminal fragment that is targeted for degradation via the arginine N-degron pathway; processing and degradation of Tco89p is required for the OspB phenotype. In all, we demonstrate that the Shigella T3SS effector OspB is a cysteine protease and decipher its interplay with eukaryotic cell processes.

ImportanceShigella spp. are important human pathogens and one of the leading causes of diarrheal mortality worldwide, especially in children. Virulence depends on the Shigella type III secretion system (T3SS), and definition of the roles of the bacterial effector proteins secreted by the T3SS is key to understanding Shigella pathogenesis. The effector protein OspB has been shown to contribute to a range of phenotypes during infection, yet the mechanism of action is unknown. Here, we show that OspB possesses cysteine protease activity in both yeast and mammalian cells, and that enzymatic activity of OspB depends on a conserved cysteine-histidine catalytic dyad. We determine how its protease activity sensitizes cells to TORC1 inhibition in yeast, finding that OspB cleaves a component of yeast TORC1, and that the degradation of the C-terminal cleavage product is responsible for OspB mediated hypersensitivity to TORC1 inhibitors. Thus, OspB is a cysteine protease that depends on a conserved cysteine-histidine catalytic dyad. Cell-specific Bioorthogonal Tagging of Glycoproteins Altered glycosylation is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function. Intramolecular 13C signals inform about carbon allocation in plants Stable isotopes at natural abundance are key tools to study physiological processes occurring outside the temporal scope of manipulation and monitoring experiments. Whole-molecule carbon isotope ratios (13C/12C) enable assessments of plant carbon uptake yet conceal information about carbon allocation. Here, we identify an intramolecular 13C/12C signal at treering glucose C-5 and C-6 and develop experimentally testable theories on its origin. More specifically, we assess the potential of processes within C3 metabolism for signal introduction based (inter alia) on constraints on signal propagation posed by metabolic networks. We propose that the intramolecular signal reports carbon allocation into major metabolic pathways in actively photosynthesising leaf cells including the anaplerotic, shikimate, and non-mevalonate pathway. We support our theoretical framework by linking it to previously reported whole-molecule 13C/12C increases in cellulose of ozone-treated Betula pendula and a highly significant relationship between the intramolecular signal and tropospheric ozone concentration. Our theory postulates a pronounced preference of leaf-cytosolic triose-phosphate isomerase to catalyse the forward reaction in vivo (dihydroxyacetone phosphate to glyceraldehyde 3-phosphate). In conclusion, intramolecular 13C/12C analysis resolves information about carbon uptake and allocation enabling more comprehensive assessments of carbon metabolism than whole-molecule 13C/12C analysis.

HighlightIntramolecular 13C/12C analysis resolves information about carbon uptake and allocation (and associated environmental controls) enabling more comprehensive assessments of carbon metabolism, plant-environment interactions, and environmental variability than whole-molecule 13C/12C analysis. Accuracy and Power Analysis of Social Networks Built From Count Data O_LIPower analysis is used to estimate the probability of correctly rejecting a null hypothesis for a given statistical model and dataset. Conventional power analyses assume complete information, but the stochastic nature of behavioural sampling can mean that true and estimated networks are poorly correlated. Power analyses do not currently take the effect of sampling into account. This could lead to inaccurate estimates of statistical power, potentially yielding misleading results.
C_LIO_LIHere we develop a method for computing network correlation: the correlation between an estimated social network and its true network, using a Gamma-Poisson model of social event rates for networks constructed from count data. We use simulations to assess how the level of network correlation affects the power of nodal regression analyses. We also develop a generic method of power analysis applicable to any statistical test, based on the concept of diminishing returns.
C_LIO_LIWe demonstrate that our network correlation estimator is both accurate and moderately robust to its assumptions being broken. We show that social differentiation, mean social event rate, and the harmonic mean of sampling times positively impacts the strength of network correlation. We also show that the required level of network correlation to achieve a given power level depends on many factors, but that 0.80 network correlation usually corresponds to around 0.80 power for nodal regression in ideal circumstances.
C_LIO_LIWe provide guidelines for using our network correlation estimator to verify the accuracy of networks built from count data, and to conduct power analysis. This can be used prior to data collection, in post hoc analyses, or even for subsetting networks in dynamic network analysis. The network correlation estimator and custom power analysis methods have been made available as an R package.
C_LI Tackling polypharmacology of kinase inhibitors and selecting chemical kinase probes by transcription factor activity profiling. Protein kinase inhibitors (PKI) evolved as promising drugs for multiple diseases. However, PKIs promiscuity causes polypharmacological effects challenging therapeutic development. We show that the polypharmacology of PKIs can be inferred from their impact on transcription factors (TF) lying at the apexes of signaling pathways. Using a high-content reporter assay, we evaluated TF activity profiles (TFAP) in cells treated with Akt, CDK, Aurora, Raf, MEK, and ERK inhibitors. For each kinase family, we found kinase-specific consensus TFAP signatures signifying the on-target PKI activity. Remarkably, proximal kinases had the highest similarity of their PKIs consensus signatures. Furthermore, we show that individual PKIs exhibited the consensus, "on-target signatures" within certain "specificity windows" and distinct "off-target signatures" at other concentrations. We also show that the TFAP signatures permit pinpointing the off-target PKI activity at irrelevant kinases and non-kinase effectors. Therefore, the TFAP approach provides clear-cut quantitative metrics for assessing PKI activity and concentration ranges where the on-target activity dominates cell response. By illuminating PKI biology invisible to target-based techniques, this effect-based approach streamlines the selection of kinase chemical probes and PKI drug prioritization. Versatile Cell-Based Assay for Measuring DNA Alkylation Damage and its repair DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair. The relative speed of the glucorticoid stress response varies independently of scope and is predicted by environmental variability and longevity across birds. The acute glucocorticoid response is a key mediator of the coordinated vertebrate response to unpredictable challenges. Rapid glucocorticoid increases initiate changes that allow animals to cope with stressors. The scope of the glucocorticoid response -- defined here as the absolute increase in glucocorticoids -- is associated with individual differences in performance and varies across species with environment and life history. In addition to varying in scope, responses can differ enormously in speed; however, relatively little is known about whether speed and absolute glucocorticoid levels covary, how selection shapes speed, or what aspects of speed are important. We used corticosterone samples collected at 5 time points from 1,750 individuals of 60 species of birds to ask i) how the speed and scope of the glucocorticoid response covary and ii) whether variation in absolute or relative speed is predicted by environmental context or life history. As predicted by a recent model, faster absolute glucocorticoid responses were strongly associated with a larger scope. Despite this covariation, the relative speed of the glucocorticoid response (standardized within species) varied independently of absolute scope, suggesting that selection could operate on both features independently. Species with faster relative glucocorticoid responses lived in locations with more variable temperature and had shorter lifespans. Our results suggest that rapid changes associated with the speed of the glucocorticoid response, such as those occurring through non-genomic receptors, might be an important determinant of coping ability and we emphasize the need for studies designed to measure speed independently of absolute glucocorticoid levels. Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP proteins The human proteome is replete with short linear motifs (SLiMs) of 4-6 residues that are critical for protein-protein interactions, yet the importance of the sequence surrounding such motifs is underexplored. We devised a proteomic screen to systematically examine the influence of SLiM sequence context on protein-protein interactions. Focusing on the EVH1 domain of ENAH, an actin regulator that is upregulated in invasive cancers, we screened 36-residue proteome-derived peptides for binding. We discovered a pocket on the ENAH EVH1 domain that diverged from its orthologs to recognize extended SLiMs, and we found that proteins with two EVH1-binding SLiMs can wrap around a single domain. We also found that the ciliary protein PCARE uses an extended 23-residue region to obtain higher affinity than any known ENAH EVH1-binding motif. Our screen provides a way to uncover the effects of broader proteomic context on motif-mediated interactions, revealing diverse mechanisms of contextual control over EVH1 interactions and establishing that SLiMs cant be fully understood outside of their native context. No evidence of human genome integration of SARS-CoV-2 found by long-read DNA sequencing A recent study proposed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks the LINE-1 (L1) retrotransposition machinery to integrate into the DNA of infected cells. If confirmed, this finding could have significant clinical implications. Here, we applied deep (>50x) long-read Oxford Nanopore Technologies (ONT) sequencing to HEK293T cells infected with SARS-CoV-2, and did not find the virus integrated into the genome. By examining ONT data from separate HEK293T cultivars, we completely resolved 78 L1 insertions arising in vitro in the absence of L1 overexpression systems. ONT sequencing applied to hepatitis B virus (HBV) positive liver cancer tissues located a single HBV insertion. These experiments demonstrate reliable resolution of retrotransposon and exogenous virus insertions via ONT sequencing. That we found no evidence of SARS-CoV-2 integration suggests such events are, at most, extremely rare in vivo, and therefore are unlikely to drive oncogenesis or explain post-recovery detection of the virus. Alteration of gut microbiota with a broad-spectrum antibiotic does not impair maternal care in the European earwig. AO_SCPLOWBSTRACTC_SCPLOWThe microbes residing within the gut of an animal host often increase their own fitness by modifying their hosts physiological, reproductive, and behavioural functions. Whereas recent studies suggest that they may also shape host sociality and therefore have critical effects on animal social evolution, the impact of the gut microbiota on maternal care remains unexplored. This is surprising, as this behaviour is widespread among animals, often determines the fitness of both juveniles and parents, and is essential in the evolution of complex animal societies. Here, we tested whether life-long alterations of the gut microbiota with rifampicin - a broad-spectrum antibiotic - impair pre- and post-hatching maternal care in the European earwig. Our results first confirm that rifampicin altered the mothers gut microbial communities and indicate that the composition of the gut microbiota differs before and after egg care. Contrary to our predictions, however, the rifampicin-induced alterations of the gut microbiota did not modify pre- or post-hatching care. Independent of maternal care, rifampicin increased the females feces production and resulted in lighter eggs and juveniles. By contrast, rifampicin altered none of the other 21 physiological, reproductive and longevity traits measured over the 300 days of a females lifetime. Overall, these findings reveal that altering the gut microbiota with a large spectrum antibiotic such as rifampicin does not necessarily affect host sociality. They also emphasize that not all animals have evolved a co-dependence with their microbiota and call for caution when generalizing the central role of gut microbes in host biology. Influence of delayed density and ultraviolet radiation on caterpillar baculovirus infection and mortality O_LIInfectious disease is an important potential driver of population cycles, but this must occur through delayed density-dependent infection and resulting fitness effects. Delayed density-dependent infection by baculoviruses can be caused by environmental persistence of viral occlusion bodies, which can be influenced by environmental factors. In particular, ultraviolet radiation is potentially important in reducing the environmental persistence of viruses by inactivating viral occlusion bodies.
C_LIO_LIDelayed density-dependent viral infection has rarely been observed empirically at the population level although theory predicts that it is necessary for these pathogens to drive population cycles. Similarly, field studies have not examined the potential effects of ultraviolet radiation on viral infection rates in natural animal populations. We tested if viral infection is delayed density-dependent with the potential to drive cyclic dynamics and if ultraviolet radiation influences viral infection levels.
C_LIO_LIWe censused 18 Ranchmans tiger moth (Arctia virginalis) populations across nearly 9{degrees} of latitude over two years and quantified the effects of direct and delayed density and ultraviolet radiation on baculovirus infection rates, infection severity, and survival to adulthood. Caterpillars were collected from each population in the field and reared in the laboratory. Baculovirus has not previously been described infecting Arctia virginalis, and we used genetic methods to confirm the identity of the virus.
C_LIO_LIWe found that infection rate, infection severity, and survival to adulthood exhibited delayed density-dependence. Ultraviolet radiation in the previous summer decreased infection severity, and increased survival probability of the virus. Structural equation modelling indicated that the effect of lagged density on moth survival was mediated through infection rate and infection severity, and was 2.5 fold stronger than the effect of ultraviolet radiation on survival through infection severity. We successfully amplified polh, lef-8, and lef-9 viral genes from caterpillar samples, and BLAST search results confirmed that the virus was a nucleopolyhedrovirus.
C_LIO_LIOur findings provide clear evidence that delayed density dependence can arise through viral infection rate and severity in insects, which supports the role of viral disease as a potential mechanism, among others, that may drive insect population cycles. Furthermore, our findings support predictions that ultraviolet radiation can modify viral disease dynamics in insect populations, most likely through attenuating viral persistence in the environment.
C_LI Changes in Behavior and Salivary Serum Amyloid A in cows with Subclinical Mastitis The aim of this study was to identify detailed changes in behavior, and in salivary serum amyloid A (SAA), associated with subclinical mastitis. This included standard sickness behaviors (which form part of an adaptive response to conserve energy, minimize heat loss and facilitate recovery following infection and inflammation) and lesser-studied behaviors, that are biologically predicted to change at low-level infection, and therefore particularly relevant for detecting subclinical mastitis (here labelled  luxury behaviors). SAA is a physiological marker of systemic inflammation, with levels in milk and serum already known to increase during subclinical mastitis. We investigated whether the same was true for SAA in cow saliva. Data were collected for n = 34 commercial barn-housed dairy cows, tested in 17 matched-pairs. Each pair comprised a cow with subclinical mastitis (SCM) and a healthy control (CTRL), identified using somatic cell count (SCC) (SCM: SCC >200 x1000 cells/ml; CTRL: SCC <100 x1000 cells/ml). SCM cows were selected for study ad-hoc, at which point they were paired with a CTRL cow, based upon parity and calving date; consequently, the full data set was accrued over several months. Data were collected for each pair over 3 days: SCC (l4:00-l5:00h) Day 1; behavior (24h from 00:00h) Day 2; salivary serum amyloid-A (SAA) Day 3. We report, for the first time, that an increase in salivary SAA occurs during subclinical mastitis; SAA was higher in SCM cows and demonstrated a positive (weak) correlation with SCC. The behavioral comparisons revealed that SCM cows had reductions in activity (behavioral transitions and distance moved), social exploration, social reactivity (here: likelihood to move away/be displaced following receipt of agonism), performance of social grooming and head butts, and the receipt of agonistic non-contact challenges. In addition, SCM cows received more head swipes, and spent a greater proportion of time lying with their head on their flank than CTRL cows. SCM cows also displayed an altered feeding pattern; they spent a greater proportion of feeding time in direct contact with two conspecifics, and a lower proportion of feeding time at self-locking feed barriers, than CTRL cows. Behavioral measures were found to correlate, albeit loosely, with serum SAA in a direction consistent with predictions for sickness behavior. These included positive correlations with both lying duration and the receipt of all agonistic behavior, and negative correlations with feeding, drinking, the performance of all social and all agonistic behavior, and social reactivity. We conclude that changes in salivary SAA, social behavior, and activity offer potential in the detection of subclinical mastitis and recommend further investigation to substantiate and refine our findings. 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. CandyCollect: At-home saliva sampling for capture of respiratory pathogens Streptococcus pyogenes is a major human-specific bacterial pathogen and a common cause of a wide range of symptoms from mild infection such as pharyngitis (commonly called strep throat) to lifethreatening invasive infection. Traditional methods for diagnosis include collecting a sample using a pharyngeal swab, which can cause discomfort and even discourage adults and children from seeking proper testing and treatment in the clinic. Saliva samples are an alternative to pharyngeal swabs. To improve the testing experience for strep throat, we developed a novel lollipop-inspired sampling platform (called CandyCollect) to capture bacteria in saliva. The device can be used in clinics or in the home and shipped back to a lab for analysis, integrating with telemedicine. CandyCollect is designed to capture bacteria on an oxygen plasma treated polystyrene surface embedded with flavoring substances to enhance the experience for children and inform the required time to complete the sampling process. In addition, the open channel structure prevents the tongue from scraping and removing the captured bacteria. Flavoring substances did not affect bacterial capture and the device has a shelf life of at least 2 months (with experiments ongoing to extend the shelf life). We performed a usability study with 17 participants who provided feedback on the device design and the dissolving time of the candy. This technology and advanced processing techniques, including polymerase chain reaction (PCR), will enable user-friendly and effective diagnosis of streptococcal pharyngitis. Comparative analysis of cattle breeds as satellite cell donors for cultured beef BackgroundCultured meat is a promising new field with the potential for considerable environmental and animal welfare benefits. One technological approach to cultured meat production utilises the proliferative and differentiative capacity of muscle-derived satellite cells (SCs) to produce large volumes of cultured muscle tissue from small biopsies of donor animals. Differing genotypes between cattle breeds lead to predictable phenotypic traits, resulting in breeds being favoured for their respective meat or milk production characteristics in the livestock industry. However, whilst these breeds show significant differences in muscle growth, it is unclear whether the physiological differences observed between them in vivo are reflected in differences in SC behaviour in vitro, particularly with respect to proliferation, differentiation and cellular longevity, and hence whether particular breeds might represent preferred SC donors for a cultured beef bioprocess.

ResultsComparing SCs isolated from five breeds (Belgian Blue, Holstein Friesian, Galloway, Limousin and Simmental), we found that the proliferation rates were largely unaffected by the donor breed. In contrast, potentially meaningful differences were observed in the kinetics and extent of myogenic differentiation. Furthermore, whilst differentiation dropped for all breeds with increasing population doublings (PDs), SCs from Belgian Blue and Limousin cattle showed significantly longer retention of differentiation capacity over long-term passaging.

ConclusionSCs from all breeds were able to proliferate and differentiate, although Limousin and (particularly) Belgian Blue cattle, both breeds commonly used for traditional meat production, may represent preferred donors for cultured beef production. A tool for assessing alignment of biomedical data repositories with open, FAIR, citation and trustworthy principles Increasing attention is being paid to the operation of biomedical data repositories in light of efforts to improve how scientific data is handled and made available for the long term. Multiple groups have produced recommendations for functions that biomedical repositories should support, with many using requirements of the FAIR data principles as guidelines. However, FAIR is but one set of principles that has arisen out of the open science community. They are joined by principles governing open science, data citation and trustworthiness, all of which are important aspects for biomedical data repositories to support. Together, these define a framework for data repositories that we call OFCT: Open, FAIR, Citable and Trustworthy. Here we developed an instrument using the open source PolicyModels toolkit that attempts to operationalize key aspects of OFCT principles and piloted the instrument by evaluating eight biomedical community repositories listed by the NIDDK Information Network (dkNET.org). Repositories included both specialist repositories that focused on a particular data type or domain, in this case diabetes and metabolomics, and generalist repositories that accept all data types and domains. The goal of this work was both to obtain a sense of how much the design of current biomedical data repositories align with these principles and to augment the dkNET listing with additional information that may be important to investigators trying to choose a repository, e.g., does the repository fully support data citation? The evaluation was performed from March to November 2020 through inspection of documentation and interaction with the sites by the authors. Overall, although there was little explicit acknowledgement of any of the OFCT principles in our sample, the majority of repositories provided at least some support for their tenets. Ecological interactions in breast cancer: Cell facilitation promotes growth and survival under drug pressure The interplay of positive and negative interactions between drug sensitive and resistant cells determines the efficacy of treatment in heterogeneous cancer cell populations. We developed isogenic estrogen receptor positive (ER+) breast cancer cell lineages sensitive and resistant to ribociclib-induced CDK4/6 inhibition to investigate cell interactions under selective pressure. During treatment with ribociclib alone or in combination with endocrine therapies, sensitive cells survive and proliferate better when grown together with resistant cells than when grown in monoculture. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays detected estrone, a less active metabolite, in medium taken from each lineage but detected highly active estradiol only from resistant cells. Because sensitive cells are more dependent on estrogen signaling than resistant cells, they benefit from the increase in local estradiol from resistant cells, leading to facilitated growth when grown under treatment in coculture. We developed a suite of mathematical models to quantify the strength of competition and facilitation between cell lines and evaluate the effects of treatments that modify these interactions. These analyses support the conclusion that resistant cell lineages facilitate growth of sensitive lineages during therapy through the increased production and sharing of local estradiol and themselves suffer from increased competition and suppression as a result. These models show that blocking facilitation has the potential to control both resistant and sensitive cell populations and inhibit the emergence of a refractory population. This combination of experiments and models shows that interactions within heterogeneous cancer cell populations can combine positive and negative interactions to shape the dynamics of resistance. Exogenous attention generalizes location transfer of perceptual learning in adults with amblyopia Visual perceptual learning (VPL) is a behavioral manifestation of brain neuroplasticity. However, its practical effectiveness is limited because improvements are often specific to the trained conditions and require significant time and effort. It is critical to understand the conditions that promote learning and transfer. Covert endogenous (voluntary) and exogenous (involuntary) spatial attention help overcome VPL location specificity in neurotypical adults, but whether they also do so for people with atypical visual development is unknown. This is the first study to investigate the role of exogenous attention during VPL in adults with amblyopia, an ideal population given their asymmetrically developed, but highly plastic, visual cortex. Here we show that training on a discrimination task leads to improvements in foveal contrast sensitivity, acuity, and stereoacuity. Remarkably, exogenous attention helps generalize learning beyond trained spatial locations. Our findings reveal that attention can enhance the effectiveness of perceptual learning during rehabilitation of visual disorders.