Heritable variation in locomotion, reward sensitivity and impulsive behaviors in a genetically diverse inbred mouse panel Drugs of abuse, including alcohol and stimulants like cocaine, produce effects that are subject to individual variability, and genetic variation accounts for at least a portion of those differences. Notably, research in both animal models and human subjects point towards reward sensitivity and impulsivity as being trait characteristics that predict relatively greater positive subjective responses to stimulant drugs. Here we describe use of the eight Collaborative Cross (CC) founder strains and multiple CC strains to examine the heritability of reward sensitivity and impulsivity traits, as well as genetic correlations between these measures and existing addiction-related phenotypes. Methods. Strains were all tested for activity in an open field and reward sensitivity (intake of chocolate BOOST(R)). Mice were then divided into two counterbalanced groups and underwent reversal learning (impulsive action and waiting impulsivity) or delay discounting (impulsive choice). Results. CC and founder mice demonstrate significant heritability for impulsive action, impulsive choice, waiting impulsivity, locomotor activity, and reward sensitivity, with each impulsive phenotype determined to be non-correlating, independent traits. This research was conducted within the broader, inter-laboratory effort of the Center for Systems Neurogenetics of Addiction (CSNA) to characterize CC and DO mice for multiple, cocaine abuse related traits. These data will facilitate the discovery of genetic correlations between predictive traits, which will then guide discovery of genes and genetic variants that contribute to addictive behaviors.
A Practical Guide to Sparse k-means Clustering for Studying Molecular Development of the Human Brain Studying the molecular development of the human brain presents unique challenges for selecting a data analysis approach. The rare and valuable nature of human postmortem brain tissue, especially for developmental studies, means the sample sizes are small (n), but the use of high throughput genomic and proteomic methods measure the expression levels for hundreds or thousands of variables (e.g. genes or proteins (p)) for each sample. This leads to a data structure that is high dimensional (p >> n) and introduces the curse of dimensionality, which poses a challenge for traditional statistical approaches. In contrast, high dimensional analyses, especially cluster analyses developed for sparse data, have worked well for analyzing genomic datasets where p >> n. Here we explore applying a lasso-based clustering method developed for high dimensional genomic data with small sample sizes. Using protein and gene data from the developing human visual cortex, we compared clustering methods. We identified an application of sparse K-means clustering (Robust Sparse K-means Clustering (RSKC)) that partitioned samples into age-related clusters that reflect lifespan stages from birth to aging. RSKC adaptively selects a subset of the genes or proteins contributing to partitioning samples into age-related clusters that progress across the lifespan. This approach addresses a problem in current studies that could not identify multiple postnatal clusters. Moreover, clusters encompassed a range of ages like a series of overlapping waves illustrating that chronological- and brain-age have a complex relationship. In addition, a recently developed workflow to create plasticity phenotypes (Balsor et al., 2020) was applied to the clusters and revealed neurobiologically relevant features that identified how the human visual cortex changes across the lifespan. These methods can help address the growing demand for multimodal integration, from molecular machinery to brain imaging signals, to understand the human brains development.
Colicin E1 opens its hinge to plug TolC The double membrane architecture of Gram-negative bacteria forms a barrier that is effectively impermeable to extracellular threats. Bacteriocin proteins evolved to exploit the accessible, surface-exposed proteins embedded in the outer membrane to deliver cytotoxic cargo. Colicin E1 is a bacteriocin produced by, and lethal to, Escherichia coli that hijacks the outer membrane proteins TolC and BtuB to enter the cell. Here we capture the colicin E1 translocation domain inside its membrane receptor, TolC, by high-resolution cryoEM, the first reported structure of a bacteriocin bound to TolC. Colicin E1 binds stably to TolC as an open hinge through the TolC pore--an architectural rearrangement from colicin E1s unbound conformation. This binding is stable in live E. coli cells as indicated by single-molecule fluorescence microscopy. Finally, colicin E1 fragments binding to TolC plugs the channel, inhibiting its native efflux function as an antibiotic efflux pump and heightening susceptibility to three antibiotic classes. In addition to demonstrating that these protein fragments are useful starting points for developing novel antibiotic potentiators, this method could be expanded to other colicins to inhibit other outer membrane protein functions. Limited selection towards lighter seeds in Danish grasslands over an eight-year period Time-series analysis of cover data from 236 Danish grassland sites demonstrated community selection on seed mass. Across four grassland habitat types and during an eight-year period there was significant community selection towards lighter seeds. In the analysis, the continuous seed size variable was used to group plant species into functional types. This method was chosen in order to account for the sampling process of the abundance data, and is in contrast to most other analyses of trait selection, where the community weighted mean of the traits is used as the dependent variable. Generally, little is known on the underlying causes of possible plant trait community selection in semi-natural grasslands that are not subjected to successional processes. Development of an in-situ printing system with human platelet lysate-based bio-adhesive to treat corneal perforation PurposeCorneal perforation is a clinical emergency. Tissue glue to seal the perforation, and supplementary topical medication represents existing standard treatment. Previously, our group developed a transparent human platelet lysate (hPL)-based biomaterial that showed good cell compatibility and accelerated corneal epithelial cells healing in-vitro. This study aims to develop a novel treatment method for corneal perforation using this biomaterial.

MethodsRheometry was used to measure the hPL-based biomaterial behaviour at room and corneal surface temperatures. Its adhesiveness to porcine skin and burst pressure limit were also measured. Based on rheological behaviour, a hand-held biopen was developed to extrude it onto the cornea. An animal trial (5 New Zealand white rabbits) to compare impact of the biomaterial and cyanoacrylate glue (control group) on a 2mm perforation was conducted to evaluate safety and efficacy.

ResultsThe hPL-based biomaterial showed higher adhesiveness compared to commercial fibrin glue and withstood burst pressure approximately 6.4x higher than routine intraocular pressure. Treatment rabbits had lower pain scores and faster recovery, despite generating similar scar-forming structure compared to controls. No secondary corneal ulcer was generated in rabbits treated with the bio-adhesive.

ConclusionsThis study reports a novel in-situ printing system capable of delivering a hPL-based, transparent bio-adhesive and successfully treating small corneal perforations. Bio-adhesive-treated rabbits recovered faster and required no additional analgesia. Both groups showed scarred corneal tissue after healing, however no infection and inflammation was observed by 3 weeks. The delivery system was easy to use and may represent an alternative treatment for corneal perforation.

HighlightsO_LIThis study presents a novel in situ printing system to treat corneal perforation
C_LIO_LIThe system is comprised of a human platelet lysate-based bio-adhesive and a pen-like hand held delivery system
C_LIO_LIMechanical tests showed our transparent bio-adhesive has a higher adhesiveness compared to existing treatments and burst pressure threshold approximately 6.4 times higher than normal intraocular pressure.
C_LIO_LIIn vivo rabbit trial showed that compared to cyanoacrylate glue, the bio-adhesive was safer, faster healing and led to less pain in rabbits.
C_LI Toxoplasma bradyzoites exhibit physiological plasticity of calcium and energy stores controlling motility and egress. Toxoplasma gondii has evolved different developmental stages for disseminating during acute infection (i.e. tachyzoites) and for establishing chronic infection (i.e. bradyzoites). Calcium ion (Ca2+) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress and cell invasion. However, the roles of Ca2+ signaling pathways in bradyzoites remain largely unexplored. Here we show that Ca2+ responses are highly restricted in bradyzoites and that they fail to egress in response to agonists. Development of dual-reporter parasites revealed dampened calcium responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca2+ imaging demonstrated lower Ca2+ basal levels, reduced magnitude, and slower Ca2+ kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with down-regulation of calcium ATPases involved in intracellular Ca2+ storage in the endoplasmic reticulum (ER) and acidocalcisomes. Once liberated from cysts by trypsin digestion, bradyzoites incubated in glucose plus calcium rapidly restored their intracellular Ca2+ and ATP stores leading to enhanced gliding. Collectively, our findings indicate that intracellular bradyzoites exhibit dampened Ca2+ signaling and lower energy levels that restrict egress, and yet upon release they rapidly respond to changes in the environment to regain motility. The impact of housing conditions on mesenchymal stromal/stem cell populations differ between adipose tissue and skeletal muscle BackgroundIn pigs, the ratio between lean mass and fat mass in the carcass determines production efficiency and is strongly influenced by the number and size of cells in tissues. During growth, the increase in the number of cells results from the recruitment of different populations of multipotent mesenchymal stromal/stem cells (MSCs) residing in the tissues. We hypothesized that the impact of hygiene of housing conditions during growth on the proportions of MSCs in adipose tissue and skeletal muscle may differ between pigs with different residual feed intake (RFI), a measure of feed efficiency.

MethodsAt the age of 12 weeks, Large White pigs from two lines divergently selected for low and high RFI were housed in two contrasting hygiene conditions (good vs poor). After six weeks, pigs were slaughtered (n = 30; 5-9/group). Samples of subcutaneous adipose tissue and longissimus skeletal muscle were collected, and cells from the stromal vascular fraction (SVF), which includes mesenchymal stromal/stem cell populations, were isolated from each tissue. Adipose and muscle cell populations from the SVF were phenotyped by flow cytometry using antibodies that targeted different cell surface markers (CD45 to separate hematopoietic cells from MSCs; CD34, CD38, CD56 and CD140a to identify MSC populations with adipogenic and/or myogenic potential).

ResultsAdipose tissue and muscle shared some common MSC populations although MSC diversity was higher in muscle than in adipose tissue. In muscle, the CD45-CD56+CD34-CD140a+ and CD45-CD56+CD34+CD140a+ cell populations were abundant. Of these two cell populations, only the proportions of CD45-CD56+CD34+CD140a+ cells increased (P < 0.05) in pigs housed in poor hygiene as compared with pigs in good hygiene conditions. For the CD45-CD56-CD34- cell population, present in low proportion, there was an interaction between hygiene condition and genetic line (P < 0.05) with a decrease in low RFI pigs housed in poor hygiene conditions. In adipose tissue, the two abundant MSC populations were CD45-CD56-CD34- and CD45-CD56+CD34-. The proportion of CD45-CD56-CD34- cells increased (P < 0.05) whereas the proportion of CD45-CD56+CD34- tended to decrease (P < 0.1) in pigs housed in poor conditions. This study shows that the proportions of some MSC populations were affected by hygiene of housing conditions in a tissue-dependent manner in pigs of both RFI lines. It suggests that MSCs may play a significant role in adipose tissue and skeletal muscle homeostasis and may influence later growth and body composition in growing animals. Mendelian Randomization for causal inference accounting for pleiotropy and sample structure using genome-wide summary statistics Mendelian Randomization (MR) is a valuable tool for inferring causal relationships among a wide range of traits using summary statistics from genome-wide association studies (GWASs). Existing summary-level MR methods often rely on strong assumptions, resulting in many false positive findings. To relax MR assumptions, ongoing research has been primarily focused on accounting for confounding due to pleiotropy. Here we show that sample structure is another major confounding factor, including population stratification, cryptic relatedness, and sample overlap. We propose a unified MR approach, MR-APSS, which (i) accounts for pleiotropy and sample structure simultaneously by leveraging genome-wide information; and (ii) allows to include more genetic variants with moderate effects as instrument variables (IVs) to improve statistical power without inflating type I errors. We first evaluated MR-APSS using comprehensive simulations and negative controls, and then applied MR-APSS to study the causal relationships among a collection of diverse complex traits. The results suggest that MR-APSS can better identify plausible causal relationships with high reliability. In particular, MR-APSS can perform well for highly polygenic traits, where the IV strengths tend to be relatively weak and existing summary-level MR methods for causal inference are vulnerable to confounding effects. Spatial proteomics reveals subcellular reorganization in human keratinocytes exposed to UVA light The effects of UV light on the skin have been extensively investigated. However, systematic information about how exposure to UVA light, the least energetic but the most abundant UV radiation reaching the Earth, shapes the subcellular organization of proteins is lacking. Using subcellular fractionation, mass spectrometry-based proteomics, machine learning algorithms, immunofluorescence, and functional assays, we mapped the subcellular reorganization of the proteome of human keratinocytes in response to UVA light. Our workflow quantified and assigned subcellular localization for over 1600 proteins, of which about 200 were found to redistribute upon UVA exposure. Reorganization of the proteome affected modulators of signaling pathways, cellular metabolism, and DNA damage response. Strikingly, mitochondria were identified as one of the main targets of UVA-induced stress. Further investigation demonstrated that UVA induces mitochondrial fragmentation, up-regulates redox-responsive proteins and attenuates respiratory rates. These observations emphasize the role of this radiation as a potent metabolic stressor in the skin. Wolbachia load variation in Drosophila is more likely caused by drift than by host genetic factors Symbiosis is a continuum of long-term interactions ranging from mutualism to parasitism, according to the balance between costs and benefits for the protagonists. The density of endosymbionts is, in both cases, a key factor that determines both the transmission of symbionts and the host extended phenotype, and is thus tightly regulated within hosts. However, the evolutionary and molecular mechanisms underlying bacterial density regulation are currently poorly understood. In this context, the symbiosis between the fruit fly and its intracellular bacteria Wolbachia (wMelPop strain) is particularly interesting to study. Although vertically transmitted, the symbiont is pathogenic, and a positive correlation between virulence and wMelPop density is observed. In addition, the number of repeats of a bacterial genomic region -Octomom-is positively correlated with Wolbachia density, underlying a potential genetic mechanism that controls bacterial density. Interestingly, the number of repeats varies between host individuals, but most likely also within them. Such genetic heterogeneity within the host could promote conflicts between bacteria themselves and with the host, notably by increasing within-host competition between symbiont genotypes through a process analogous to the tragedy of the commons. To characterize the determinisms at play in the regulation of bacterial density, we first introgressed wMelPop in different genetic backgrounds of D. melanogaster, and found different density levels and Octomom copy numbers in each host lineage. To determine whether such variations reflect a host genetic determinism on density regulation through Octomom copy number selection, we replicated the introgressions and performed reciprocal crosses on the two Drosophila populations with the most extreme density levels. In both experiments, we detected an absence of directionality in the patterns of infection, associated with a strong instability of these patterns across generations. Given that bacterial density was highly correlated with Octomom copy numbers in all experiments, these results rather suggest a strong influence of drift and a random increase in the frequency of certain bacterial variants. We then discuss how drift, both on the symbiont population during transmission and on the host population, could limit the efficiency of selection in such a symbiotic system, and the consequences of drift on the regulation of density and composition of bacterial populations. Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of mitochondrial TCA cycle enzyme, succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect in PC tumors, triggers dysregulation of Ca2+ homeostasis, and aberrantly activates calpain and the protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a cascade of phospho- signaling where GSK3 inhibition inactivates energy sensing by AMP-kinase through dephosphorylation of the AMP-kinase {gamma} subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC tumor formation. A novel Cdk5 inhibitor, MRT3-007, reversed this phospho-cascade, invoking an anti- Warburg effect, cell cycle arrest, and senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important novel mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.

HighlightsO_LILoss of SDHB function in pheochromocytoma causes Ca2+ dysregulation, calpain activation, and aberrant activation of the protein kinase Cdk5.
C_LIO_LIHyperactive Cdk5 deregulates a GSK3/PRKAG2/AMPK signaling cascade.
C_LIO_LIp25 overexpression and consequent aberrant Cdk5 activity in chromaffin cells causes pheochromocytoma.
C_LIO_LIInhibition of Cdk5 activates the PRKAG2/AMPK/p53 signaling to rescue cell senescence and block PC tumor progression.
C_LI

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/475410v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@16f0a48org.highwire.dtl.DTLVardef@10c1c7aorg.highwire.dtl.DTLVardef@21b100org.highwire.dtl.DTLVardef@1eb8674_HPS_FORMAT_FIGEXP  M_FIG C_FIG Outer hair cell electro-mechanical power conversion Outer hair cells are the cellular motors in the mammalian inner ear responsible for sensitive high-frequency hearing. Motor function requires expression of the protein prestin in the OHC lateral membrane and mechano-electrical transduction in the apical hair bundle. In the present report, electrical power consumption and mechanical power output are determined using previously published voltage clamp data from isolated OHCs and membrane patches. Results reveal that power output by the prestin-motor complex peaks at a best frequency much higher than implied by the low-pass character of nonlinear capacitance, and much higher than the whole-cell resistive-capacitive corner frequency. High frequency power output is enabled by a -90{degrees} shift in the phase of prestin-dependent electrical charge displacement, manifested electrically as emergence of imaginary-valued nonlinear capacitance. 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. Exploratory analysis of multiple traits co-adaptations in the population history During the history of range expansion, the populations encounter with variety of environments. They respond to the local environments by modifying the mutually interacting traits. Therefore, to understand the whole life history of the populations, it is ideal to capture the history of their range expansion with reference to the series of surrounding environments and to infer the coadaptation of the multiple traits. Toward this end, we provide an exploratory analysis based on the features of populations: site frequency spectra of populations, population-specific FST, association between genes and environments, positive selections on traits mapped on the admixture graph, and GWAS results. Correspondence analysis of genes, environments, and traits provides a birds-eye view of the history of population differentiation and range expansion and various types of environmental selections at the times. Principal component analysis of the estimated trait-specific polygenic adaptations mapped on the admixture graph enables to understand the coadaptation of multiple traits. The potential usefulness was confirmed by analyzing a public dataset of wild poplar in northwestern America. In response to the northern cold temperature and longer daylength, the populations increased the photosynthetic activity and nutrient use efficiency at the expense of the risk of pathogen invasion, and in response to warm temperature, they increased the growth. At higher altitude, they shifted the maximum activity to earlier period in spring to reduce the activity in dry summer. The R codes for our representation method and simulations of population colonization used in this study are available as supplementary script. Dynamic modulation of enhancer responsiveness by core promoter elements in living Drosophila embryo Regulatory interactions between enhancers and core promoters are fundamental for the temporal and spatial specificity of gene expression in development. The central role of core promoters is to initiate productive transcription in response to enhancers activation cues. However, it has not been systematically assessed how individual core promoter elements affect the induction of transcriptional bursting by enhancers. Here, we provide evidence that each core promoter element dynamically alters "enhancer responsiveness" by changing functional parameters of transcriptional bursting in developing Drosophila embryos. Quantitative live imaging analysis revealed that the timing and the continuity of burst induction are common regulatory steps on which core promoter elements impact. We also show that core promoters use the upstream TATA box as an amplifier of transcriptional bursting upon interaction with enhancers. Genome editing analysis of the pair-rule gene fushi tarazu further revealed that the endogenous TATA box and the downstream core promoter element are both essential for the correct expression and the function of key developmental genes in early embryos. We suggest that core promoter elements serve as a key regulatory module in converting enhancer activity into transcription dynamics during animal development. A chromosome-level genome assembly of the European Beech (Fagus sylvatica) reveals anomalies for organelle DNA integration, repeat content and distribution of SNPs The European Beech is the dominant climax tree in most regions of Central Europe and valued for its ecological versatility and hardwood timber. Even though a draft genome has been published recently, higher resolution is required for studying aspects of genome architecture and recombination. Here we present a chromosome-level assembly of the more than 300 year-old reference individual, Bhaga, from the Kellerwald-Edersee National Park (Germany). Its nuclear genome of 541 Mb was resolved into 12 chromosomes varying in length between 28 Mb and 73 Mb. Multiple nuclear insertions of parts of the chloroplast genome were observed, with one region on chromosome 11 spanning more than 2 Mb of the genome in which fragments up to 54,784 bp long and covering the whole chloroplast genome were inserted randomly. Unlike in Arabidopsis thaliana, ribosomal cistrons are present in Fagus sylvatica only in four major regions, in line with FISH studies. On most assembled chromosomes, telomeric repeats were found at both ends, while centromeric repeats were found to be scattered throughout the genome apart from their main occurrence per chromosome. The genome- wide distribution of SNPs was evaluated using a second individual from Jamy Nature Reserve (Poland). SNPs, repeat elements and duplicated genes were unevenly distributed in the genomes, with one major anomaly on chromosome 4. The genome presented here adds to the available highly resolved plant genomes and we hope it will serve as a valuable basis for future research on genome architecture and for understanding the past and future of European Beech populations in a changing climate. Admp regulates tail bending by controlling ventral epidermal cell polarity via phosphorylated myosin translocation. The tails transient but pronounced ventral bending is found in many chordate embryos and provides an interesting model of how tissue interactions contribute to the overall shape (Lu et al., 2020). Here, we identify one of the upstream regulators of tail bending. Admp knock-down embryo (Admp MO) completely inhibited ventral tail bending. Admp MO showed specific inhibition of the cell-cell intercalation ventral epidermis. In the early stages of the intercalation, each ventral epidermal cell exhibited a boat-shaped morphology (boat cell) with a narrow apical surface in which phosphorylated myosin (pMLC) accumulated. In the Admp MO, the number of the boat cells reduced and pMLC was observed at the basal side in the Admp MO embryo. Finally, laser cutter experiments showed a pMLC-dependent relaxation of the ventral tail bending, which was observed when each ventral midline epidermal cell was cut along the apico-basal axis. Based on these results, we propose a novel function for Admp, in which Admp controls the pMLC translocation to the apical side of the ventral epidermal cells and leads the tail bending by ventral resistance to the notochord extension.

Summary StatementAdmp is an upstream regulator of the tail bending in the protochordate Ciona tailbud embryo, regulating tissue polarity of the ventral midline epidermis by the translocation of the phosphorylated myosin. Multispecific DARPin(R) therapeutics demonstrate very high potency against SARS-CoV-2 variants in vitro SARS-CoV-2 has infected millions of people globally and continues to undergo evolution. Emerging variants can be partially resistant to vaccine induced immunity and therapeutic antibodies, emphasizing the urgent need for accessible, broad-spectrum therapeutics. Here, we report a comprehensive study of ensovibep, the first trispecific clinical DARPin candidate, that can simultaneously engage all three units of the spike protein trimer to potently inhibit ACE2 interaction, as revealed by structural analyses. The cooperative binding of the individual modules enables ensovibep to retain inhibitory potency against all frequent SARS-CoV-2 variants, including Omicron BA.1 and BA.2, as of February 2022. Moreover, viral passaging experiments show that ensovibep, when used as a single agent, can prevent development of escape mutations comparably to a cocktail of monoclonal antibodies (mAb). Finally, we demonstrate that the very high in vitro antiviral potency also translates into significant therapeutic protection and reduction of pathogenesis in Roborovski dwarf hamsters infected with either the SARS-CoV-2 wild-type or the Alpha variant. In this model, ensovibep prevents fatality and provides substantial protection equivalent to the standard of care mAb cocktail. These results support further clinical evaluation and indicate that ensovibep could be a valuable alternative to mAb cocktails and other treatments for COVID-19. Library-free BoxCarDIA solves the missing value problem in label-free quantitative proteomics The last decade has seen significant advances in the application of quantitative mass spectrometry-based proteomics technologies to tackle important questions in plant biology. The current standard for quantitative proteomics in plants is the use of data-dependent acquisition (DDA) analysis with or without the use of chemical labels. However, the DDA approach preferentially measures higher abundant proteins, and often requires data imputation due to quantification inconsistency between samples. In this study we systematically benchmarked a recently developed library-free data-independent acquisition (directDIA) method against a state-of-the-art DDA label-free quantitative proteomics workflow for plants. We next developed a novel acquisition approach combining MS1-level BoxCar acquisition with MS2-level directDIA analysis that we call BoxCarDIA. DirectDIA achieves a 33% increase in protein quantification over traditional DDA, and BoxCarDIA a further 8%, without any changes in instrumentation, offline fractionation, or increases in mass-spectrometer acquisition time. BoxCarDIA, especially, offers wholly reproducible quantification of proteins between replicate injections, thereby addressing the long-standing missing-value problem in label-free quantitative proteomics. Further, we find that the gains in dynamic range sampling by directDIA and BoxCarDIA translate to deeper quantification of key, low abundant, functional protein classes (e.g., protein kinases and transcription factors) that are underrepresented in data acquired using DDA. We applied these methods to perform a quantitative proteomic comparison of dark and light grown Arabidopsis cell cultures, providing a critical resource for future plant interactome studies. Our results establish BoxCarDIA as the new method of choice in quantitative proteomics using Orbitrap-type mass-spectrometers, particularly for proteomes with large dynamic range such as that of plants. Morphology of immatures of the thelytokous ant, Monomorium triviale Wheeler (Formicidae: Myrmicinae: Solenopsidini) with descriptions of the extraordinary last-instar queen larvae The ant genus Monomorium is one of the most species-rich but taxonomically problematic groups in the hyperdiverse subfamily Myrmicinae. An East Asian species, M. triviale Wheeler, produces both reproductive queens and sterile workers via obligate thelytokous parthenogenesis. Here, we describe the immature forms of M. triviale based on light and scanning electron microscopy observations, with a note on the striking caste dimorphism in the last larval instar. The last-instar queen larvae were easily recognized by their large size, "aphaenogastroid" body shape, and rows of doorknob-like tubercles on the lateral and dorsal body surface. This type of queen-specific structure has not been found in ants in general, let alone congeneric species found in Japan. In stark contrast to the queen larvae, worker larvae showed a "pheidoloid" body shape and a body surface similar to other ants. The worker larvae were estimated to have three instars, consistent with previously described congeners. The pupae of both castes had no cocoon, a characteristic commonly described in other Myrmicinae species. In total, the developmental period from egg to adult worker averaged 59 days under 25{degrees}C. We discuss possible functions of the tubercles of queen larvae based on previous studies. 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. The initial response of females towards congeneric males matches the propensity to hybridise in Ophthalmotilapia. AO_SCPLOWBSTRACTC_SCPLOWCichlid radiations often harbour closely related species with overlapping niches and distribution ranges. Such species sometimes hybridise in nature, which raises the question how can they coexist. This also holds for the Tanganyika mouthbrooders Ophthalmotilapia ventralis and O. nasuta. Earlier studies found indications of asymmetrical hybridisation with females of O. ventralis accepting males of O. nasuta, but not the other way around. We hypothesised that this was due to differences in the capacity for species recognition. Given the higher propensity of O. ventralis females towards hybridisation, we expect a reduced ability for species recognition in O. ventralis females, compared to O. nasuta females. We staged two experiments, one focusing on 22 female O. nasuta and one on 21 female O. ventralis. These fish were placed in one half of a tank and briefly exposed to a conspecific or a heterospecific male, a conspecific female, or nothing (control). Female response was evaluated by scoring six tracking parameters and by noting the occurrence of ten discrete behaviours before and during the encounter. Females always responded to the presence of another fish by approaching it. Remarkably, for both O. nasuta and O. ventralis, we did not find a different response between encounters with conspecific males and females. However, in agreement with our hypothesis, O. nasuta females behaved differently towards conspecific or heterospecific males, whereas O. ventralis females did not. When presented with a heterospecific male, O. nasuta females performed a lower number of  ram behaviours. Additionally, they never displayed the  flee behaviour, a component of the species mating repertoire that was seen in all but one of the presentations with a conspecific male. Our findings show that differences in species recognition at first encounter predict to a large degree the outcome of the mating process, even in the absence of mating behaviour. SARS-CoV-2 infection induces soluble platelet activation markers and PAI-1 in the early moderate stage of COVID-19 IntroductionCoagulation dysfunction and thromboembolism emerge as strong comorbidity factors in severe COVID-19. However, it is unclear when particularly platelet activation markers and coagulation factors dysregulated during the pathogenesis of COVID-19. Here, we sought to assess the levels of coagulation and platelet activation markers at moderate and severe stages of COVID-19 to understand the pathogenesis.

MethodsTo understand this, hospitalized COVID-19 patients with (severe cases that required intensive care) or without pneumonia (moderate cases) were recruited. Phenotypic and molecular characterizations were performed employing basic coagulation tests including PT, APTT, D-Dimer and TFPI. The flow cytometry-based multiplex assays were performed to assess FXI, anti-thrombin, prothrombin, fibrinogen, FXIII, P-selectin, sCD40L, plasminogen, tissue-plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and D-Dimer.

ResultsThe investigations revealed induction of plasma P-selectin and CD40 ligand (sCD40L) in moderate COVID-19 cases which were significantly abolished with the progression of COVID-19 severity. Moreover, a profound reduction in plasma tissue factor pathway inhibitor (TFPI) and FXIII were identified particularly in the severe COVID-19. Further analysis revealed fibrinogen induction in both moderate and severe patients. Interestingly, an elevated PAI-1 more prominently in moderate, and tPA particularly in severe COVID-19 cases were observed. Particularly, the levels of fibrinogen and tPA directly correlated with the severity of the disease.

ConclusionsIn summary, induction of soluble P-selectin, sCD40L, fibrinogen and PAI-1 in moderate COVID-19 cases suggests the activation of platelets and coagulation system before patients require intensive care. These findings would help in designing better thromboprophylaxis to limit the COVID-19 severity. 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. Comparative toxicogenomics of glyphosate and Roundup herbicides by mammalian stem cell-based genotoxicity assays and molecular profiling in Sprague-Dawley rats Whether glyphosate-based herbicides (GBHs) are more potent than glyphosate alone at activating cellular mechanisms, which drive carcinogenesis remains controversial. As GBHs are more cytotoxic that glyphosate, we reasoned they may also be more capable of activating carcinogenic pathways. We tested this hypothesis by comparing the effects of glyphosate with Roundup GBHs both in vitro and in vivo. First, glyphosate was compared with representative GBHs namely MON 52276 (EU), MON 76473 (UK) and MON 76207 (USA) using the mammalian stem cell-based ToxTracker system. Here, MON 52276 and MON 76473, but not glyphosate and MON 76207, activated oxidative stress and unfolded protein responses. Second, molecular profiling of liver was performed in female Sprague-Dawley rats exposed to glyphosate or MON 52276 (both at 0.5, 50, 175 mg/kg bw/day glyphosate) for 90 days. MON 52276 but not glyphosate increased hepatic steatosis and necrosis. MON 52276 and glyphosate altered the expression of genes in liver reflecting TP53 activation by DNA damage and circadian rhythm regulation. Genes most affected in liver were similarly altered in kidneys. Small RNA profiling in liver showed decreased amounts of miR-22 and miR-17 from MON 52276 ingestion. Glyphosate decreased mir-30 while miR-10 levels were increased. DNA methylation profiling of liver revealed 5,727 and 4,496 differentially methylated CpG sites between the control and glyphosate and MON 52276 exposed animals respectively. Apurinic/apyrimidinic DNA damage formation in liver was increased with glyphosate exposure. Altogether, our results show that Roundup formulations cause more biological changes linked with carcinogenesis than glyphosate. Pan-cancer HLA Gene-mediated Tumor Immunogenicity and Immune Evasion Human Leukocyte Antigen (HLA) expression contributes to the activation of anti-tumor immunity through interactions with T cell receptors. However, pan-cancer HLA-mediated immunogenicity and immunoediting mechanisms has not been systematically studied. In a retrospective analysis of 33 tumor types from the Cancer Genome Atlas, we uncovered HLA class I and class II differential expression, which outperformed traditional clinical metrics in predicting patient survival. We also characterized the distribution of HLA supertypes across cancers and showed that patients with high HLA allelic diversity and gene expression had better prognosis. Immune microenvironments with varied survival outcomes could be predicted using a neural network model trained on HLA expression data. Furthermore, we identified a subset of tumors which upregulated HLA class I but not class II genes and exploited HLA-mediated escape strategies. Our results suggest the potential of using HLA expression data to predict patient prognosis. Taken together, we emphasize the crucial role of HLA upregulation in shaping prolonged anti-tumor immunity.

SynopsisIn a retrospective analysis of 11080 patients of 33 TCGA cancer types, we showed HLA class I and class II differential expression shape various immune microenvironments and tumor immunoediting mechanisms, predicting tumor immunogenicity and patient survival.