A protein microarray-based in vitro transglutaminase assay platform for epitope mapping and peptide discovery: implication for transglutaminase-mediated immunodominant determination Transglutaminases (TGs) are a family of crosslinking enzymes catalyzing the formation of intra- and inter-molecular glutamine-lysine isopeptide bonds in a calcium dependent manner. Activation of transglutaminases is pathogenically associated with severe human diseases including neurodegenerations, cardiovascular diseases, and autoimmune diseases. Although continuous efforts determining the enzymes substrate preference have been witnessed, a high-throughput assay platform with the "omic" efficiency is still missing for the global identification of substrate-specific TG modification sites. In this study we report a protein microarray-based in vitro TG assay platform for rapid and large-scale (up to 30000 reactions per chip) determination of the glutamine (Q)-bearing TG modification motifs. With this platform we identified the Q16 in superoxide dismutase 1 and Q109 in alpha-synuclein as the primary modification sites for tissue transglutaminase (TG2), the most ubiquitous member of the enzyme family. Of particular interest, we found a close match between the modification motifs and published vaccine epitope sequences in alpha-synuclein, implying an essential and intrinsic role transglutaminase might play in the determination of immunodominant epitopes. Our data collectively suggest the glutamine and its follow-up five residues on the C terminal of a protein compose a minimal determinant motif for TG2 modification and the TG2 modification motifs determined by our platform could finally correspond to the substrates epitope sequences in antigen processing. To screen for site-specific interfering peptides and assist gene editing-based protein engineering, we optimized through onchip amino-acid scanning the TG2 modification motif QQIV in the extracellular matrix protein fibronectin and obtained 14 variants with significantly higher TG2 reactivity that might serve as the competitive inhibitor peptides and 1 with lower reactivity. We further confirmed this optimization approach with 12-mer peptides, the longest ones that could be synthesized on the chip. Taken together, our synthetic transglutaminase assay platform might be able to deliver a precise epitope blueprint for immunotherapeutic targeting and provide proof-of-concept and directional studies for TG-based peptide discovery and protein design.
Ensovibep, a novel trispecific DARPin candidate that protects against SARS-CoV-2 variants 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.
Multifunctional RNA-binding proteins influence mRNA abundance and translational efficiency of distinct sets of target genes RNA-binding proteins (RBPs) can regulate more than a single aspect of RNA metabolism. We searched for such previously undiscovered multifunctionality within a set of 143 RBPs, by defining the predictive value of RBP abundance for the transcription and translation levels of known RBP target genes across 80 human hearts. This led us to newly associate 27 RBPs with cardiac translational regulation in vivo. Of these, 21 impacted both RNA expression and translation, albeit for virtually independent sets of target genes. We highlight a subset of these, including G3BP1, PUM1, UCHL5, and DDX3X, where dual regulation is achieved through differential affinity for target length, by which separate biological processes are controlled. Similar to the RNA helicase DDX3X, the known splicing factors EFTUD2 and PRPF8 - all identified as multifunctional RBPs by our analysis - selectively influence target translation rates depending on 5 UTR structure. Our analyses identify dozens of RBPs as being multifunctional and pinpoint potential novel regulators of translation, postulating unanticipated complexity of protein-RNA interactions at consecutive stages of gene expression. Spatial and Texture Analysis of Root System Distribution with Earth Mover's Distance (STARSEED) PurposeRoot system architectures are complex, multidimensional, and challenging to characterize effectively for agronomic and ecological discovery.

MethodsWe propose a new method, Spatial and Texture Analysis of Root SystEm distribution with Earth movers Distance (STARSEED), for comparing root system distributions that incorporates spatial information through a novel application of the Earth Movers Distance (EMD).

ResultsWe illustrate that the approach captures the response of sesame root systems for different genotypes and soil moisture levels. STARSEED provides quantitative and visual insights into changes that occur in root architectures across experimental treatments.

ConclusionSTARSEED can be generalized to other plants and provides insight into root system architecture development and response to varying growth conditions not captured by existing root architecture metrics and models. The code and data for our experiments are publicly available: https://github.com/GatorSense/STARSEED. Host phenology can drive the evolution of intermediate virulence strategies in some obligate-killer parasites AO_SCPLOWBSTRACTC_SCPLOWThe traditional mechanistic trade-offs resulting in a negative correlation between transmission and virulence are the foundation of nearly all current theory on the evolution of parasite virulence. Several ecological factors have been shown to modulate the optimal virulence strategies predicted from mechanistic trade-off models, but these ecological factors have not yet been shown to be sufficient to explain the intermediate virulence strategies observed in any natural system. The timing of seasonal activity, or phenology, is a common factor that influences the types and impact of many ecological interactions but is difficult to incorporate into virulence evolution studies. We develop a mathematical model of a disease system with seasonal host activity to study the evolutionary consequences of host phenology on the virulence of obligate-killer parasite. Results from this model demonstrated that seasonal host activity is sufficient to drive the evolution of intermediate parasite virulence in some types of natural disease systems, even when a traditional mechanistic trade-off between transmission and virulence is not assumed in the modeling framework. The optimal virulence strategy in these systems can be determined by both the duration of the host activity period as well as the variation in the host emergence timing. Parasites with low virulence strategies are favored in environments with long host activity periods and in environments in which hosts emerge synchronously. The results demonstrate that host phenology can be sufficient to select for intermediate optimal virulence strategies, providing an alternative mechanism to account for virulence evolution in some natural systems. Dynamic tracing using ultra-bright labelling and multi-photon microscopy identifies endothelial uptake of poloxamer-188 coated poly(lactic-co-glycolic acid)nano-carriers in vivo Poly(lactic-co-glycolic acid) (PLGA)-based drug formulations are approved for the use in humans, however, the potential of PLGA to design nanoparticles (NPs) and target the central nervous system remains to be exploited.

The aim of the current study was design PLGA NPs, loading them with bulky fluorophores thereby increasing single particle fluorescence to a level visible by in vivo microscopy, and investigate their brain biodistribution. We developed, highly fluorescent 70 nm PLGA NPs significantly brighter then quantum dots enabling their visualization by intravital real-time 2-photon microscopy. We found that PLGA NPs coated with pluronic F-68 (PF-68) had a substantially longer plasma half-life than uncoated NPs and were taken up by cerebro-vascular endothelial cells. High resolution confocal microscopy revealed that coated PLGA NPs were present in late endothelial endosomes of cerebral vessels within 1 hour after systemic injection and were more readily taken up by endothelial cells in peripheral organs.

The current data suggest that PF-68 coated PLGA NPs are taken up by mouse cerebral and peripheral endothelial cells in vivo. The combination of ultra-bright NPs and in vivo imaging may thus represent a promising approach to reduce the gap between development and clinical application of nanoparticle-based drug carriers. A terrain treadmill to study animal locomotion through large obstacles A major challenge to understanding locomotion in complex 3-D terrain with large obstacles is to create tools for controlled, systematic lab experiments. Existing terrain arenas only allow observations at small spatiotemporal scales ([~]10 body length, [~]10 stride cycles). Here, we create a terrain treadmill to enable high-resolution observations of animal locomotion through large obstacles over large spatiotemporal scales. An animal moves through modular obstacles on an inner sphere, while a rigidly-attached, concentric, transparent outer sphere rotated with the opposite velocity via closed-loop feedback to keep the animal on top. During sustained locomotion, a discoid cockroach moved through pillar obstacles for 25 minutes ({approx} 2500 strides) over 67 m ({approx} 1500 body lengths), and was contained within a radius of 4 cm (0.9 body length) for 83% of the duration, even at speeds of up to 10 body length/s. The treadmill enabled observation of diverse locomotor behaviors and quantification of animal-obstacle interaction.

Summary StatementA device keeps a small animal on top of a sphere while it traverses obstacles, creating a "terrain treadmill" to study locomotion over a long time and distance at high-resolution. Obesity affects mitochondrial metabolism and proliferative potential of equine endometrial progenitor cells. The study aimed to investigate the influence of obesity on cellular features of equine endometrial progenitor cells (Eca EPCs), including viability, proliferation capacity, mitochondrial metabolism, and oxidative homeostasis. Eca EPCs derived from non-obese (Non-OB) and obese (OB) mares were characterized by cellular phenotype and multipotency. Obesity-induced changes in the activity of Eca EPCs include the decline of their proliferative activity, clonogenic potential, mitochondrial metabolism and enhanced oxidative stress. Eca EPCs isolated from obese mares were characterized by an increased occurrence of early apoptosis, loss of mitochondrial dynamics, and senescence-associated phenotype. Attenuated metabolism of Eca EPCs OB was related to increased expression of pro-apoptotic markers (CASP9, BAX, P53, P21), enhanced expression of OPN, PI3K and AKT, simultaneously with decreased signaling stabilizing cellular homeostasis (including mitofusin, SIRT1, FOXP3). Obesity alters functional features and the self-renewal potential of endometrial progenitor cells. The impaired cytophysiology of progenitor cells from obese endometrium predict lower regenerative capacity if used as autologous transplants. A cell-based screen identifies HDAC inhibitors as activators of RIG-I signaling Enhancing the immune microenvironment in cancer by targeting the nucleic acid sensors is becoming a potent therapeutic strategy. Among the nucleic acid sensors, activation of the RNA sensor Retinoic Acid-inducible Gene (RIG-I) using small hairpin RNAs has been shown to elicit powerful innate and adaptive immune responses. Given the challenges inherent in pharmacokinetics and delivery of RNA based agonists, we set out to discover small molecule agonists of RIG-I using a cell-based assay. To this end, we established and validated a robust high throughput screening assay based on a commercially available HEK293 reporter cell line with a luciferase reporter downstream of tandem interferon stimulated gene 54 (ISG54) promoter elements. We first confirmed that the luminescence in this cell line is dependent on RIG-I and the interferon receptor using a hairpin RNA RIG-I agonist. We established a 96-well and a 384-well format HTS based on this cell line and performed a proof-of-concept screen using an FDA approved drug library of 1200 compounds. Surprisingly, we found two HDAC inhibitors Entinostat, Mocetinostat and the PLK1 inhibitor Volasertib significantly enhanced ISG-luciferase activity. This luminescence was substantially diminished in the null reporter cell line indicating the increase in signaling was dependent on RIG-I expression. Combination treatment of tumor cell lines with Entinostat increased RIG-I induced cell death in a mammary carcinoma cell line that is resistant to either Entinostat or RIG-I agonist alone. Taken together, our data indicates an unexpected role for HDAC1,-3 inhibitors in enhancing RIG-I signaling and highlight potential opportunities for therapeutic combinations. Improving Martini 3 for disordered and multidomain proteins Coarse-grained molecular dynamics simulations are a useful tool to determine conformational ensembles of proteins. Here, we show that the coarse-grained force field Martini 3 underestimates the global dimensions of intrinsically disordered proteins (IDPs) and multidomain proteins when compared with small angle X-ray scattering (SAXS) data, and that increasing the strength of protein-water interactions favours more expanded conformations. We find that increasing the strength of interactions between protein and water by ca. 10% results in improved agreement with the SAXS data for IDPs and multi-domain proteins. We also show that this correction results in a more accurate description of self-association of IDPs and folded proteins and better agreement with paramagnetic relaxation enhancement data for most IDPs. While simulations with this revised force field still show deviations to experiments for some systems our results suggest that it is overall a substantial improvement for coarse-grained simulations of soluble proteins. Brain deletion of Serf2 shifts amyloid conformation in a mouse model Loss of protein homeostasis accelerates ageing and contributes to age-related brain diseases like Parkinsons and Alzheimers diseases. Disease-specific proteins become aggregation-prone and form amyloid deposits that characterize disease, but the biological mechanisms driving their transition in cells and tissues are poorly understood. MOAG-4/SERF accelerates aggregation and toxicity of disease-related proteins in worm- and cell models. Whether MOAG-4/SERF affects amyloid pathology in brain, however, has remained unknown. Here we show that brain-specific knockout of SERF2 is viable and shifts aggregation in a mouse model for amyloid-beta aggregation. Without affecting amyloid-beta levels, knockout of SERF2 redirects its assembly into structurally and morphologically distinct amyloid fibrils and deposits. Our results show that SERF modifies the structural outcome of the aggregation process in mammalian brain. With structural polymorphisms recently being associated with disease severity and progression, our results may help to understand the molecular basis for structural diversity and find possibilities for structure-based interventions.

HighlightsO_LILoss of SERF2 slows embryonic development and causes perinatal lethality
C_LIO_LISERF2 affects proliferation in a cell-autonomous fashion
C_LIO_LIBrain-specific Serf2 knockout does not affect viability or A{beta} production
C_LIO_LIBrain deletion of Serf2 shifts the amyloid conformation of A{beta}
C_LI Identifying and correcting for misspecifications in GWAS summary statistics and polygenic scores Publicly available genome-wide association studies (GWAS) summary statistics exhibit uneven quality, which can impact the validity of follow-up analyses. First, we present an overview of possible misspecifications that come with GWAS summary statistics. Then, in both simulations and real data analyses, we show that additional information such as imputation INFO scores, allele frequencies, and per-variant sample sizes in GWAS summary statistics can be used to detect possible issues and correct for misspecifications in the GWAS summary statistics. One important motivation for us is to improve the predictive performance of polygenic scores built from these summary statistics. Unfortunately, due to the lack of reporting standards for GWAS summary statistics, this additional information is not systematically reported. We also show that using well-matched LD references can improve model fit and translate into more accurate prediction. Finally, we discuss how to make polygenic score methods such as lassosum and LDpred2 more robust to these misspecifications to improve their predictive power. Revisiting regulatory decoherence: Accounting for temporal bias in a co-expression analysis reveals novel candidates controlling environmental response Environment can alter the degree of phenotypic variation and covariation, potentially influencing evolutionary trajectories. However, environment-driven changes in phenotypic variation remain understudied. In an effort to exploit the abundance of RNASequencing data now available, an increasing number of ecological studies rely on population-level correlation to characterize the plastic response of the entire transcriptome and to identify environmentally responsive molecular pathways. These studies are fundamentally interested in identifying groups of genes that respond in concert to environmental shifts. We show that population-level differential co-expression exhibits biases when capturing changes of regulatory activity and strength in rice plants responding to elevated temperature. One possible cause of this bias is regulatory saturation, the observation that detectable co-variance between a regulator and its target may be low as their transcript abundances are induced. This phenomenon appears to be particularly acute for rapid-onset environmental stressors. However, our results suggest that temporal correlations may be a reliable means to detect transient regulatory activity following rapid onset environmental perturbations such as temperature stress. Such temporal bias is likely to confound the studies of phenotypic integration, where high-order organismal traits are hypothesized to be more integrated with strong correlation under stressful conditions, while recent transcriptome studies exhibited weaker coexpression between genes under stressful conditions. Collectively, our results point to the need to account for the nuances of molecular interactions and the possibly confounding effects that these can introduce into conventional approaches to study transcriptome datasets. Atrazine induced In vivo immunotoxicity in Bivalves Atrazine is ubiquitously used broad-spectrum herbicide to control the weeds in agriculture. The present study aimed to evaluate the acute toxicity and immunotoxicity of Atrazine in two ecologically and economically important bivalves. Acute toxicity of atrazine evaluated in triplicates by taking control and six experimental groups each comprising of 30 animals and treated with a range of atrazine from 2 PPM to 12 PPM for 96 hours. Mortalities were recorded for every 24 hours until 96 hours and data analyzed by one-way ANOVA and Dunnett T-test. The results indicated a significant increase in mortalities with increase in dose and time of exposure in both species. The values of LC50 were determined as 6.10 PPM and 4.90 PPM respectively for Perna viridis and Paphia malabarica. Furthermore, the immunotoxic potential of atrazine assessed by treating mussels and clams with the five sub-lethal doses of atrazine for 14 days and quantifying the viability of hemocytes by using simple yet reliable Tryphan blue exclusion assay. The results of the present study suggest atrazine-induced immunotoxicity by decreasing the number of viable hemocytes in bivalves. Hemocytes with phagocytic function are indispensable to confer innate immunity in bivalves, decreased viability of these cells leads to compromised immunity. This study is first of its kind to implicate atrazine with the immunotoxicity in bivalves. Frontal Cortex Gates Distractor Stimulus Encoding in Sensory Cortex Suppressing behavioral responses to distractor stimuli is a fundamental cognitive process, essential for performing goal-directed tasks. A common framework for the neuronal implementation of distractor suppression is the attenuation of distractor stimuli from early sensory to higher-order processing. However, details of the localization and mechanisms of attenuation are poorly understood. In this study, we trained mice to selectively respond (lick) to whisker deflections from one whisker field and ignore identical deflections from the opposite whisker field. We found that suppression of frontal cortex, aligned to the target stimulus or the distractor stimulus, increases responses to distractors (false alarms). By simultaneously recording spiking activity in the mouse whisker region of primary somatosensory cortex (S1), we found that frontal cortex selectively suppresses the neuronal encoding of distractor stimuli in target-aligned S1. Frontal cortex decorrelates the encoding of target and distractor stimuli in single units, thereby increasing stimulus selectivity across the population of target-aligned S1 neurons. Even before stimulus onset, effects of frontal cortex on S1 were robust and context-dependent, indicating proactive modulation. Overall, our study provides important mechanistic details about the roles of frontal cortex in gating sensory responses in sensory cortex.

Major findingsO_LIBehavior: Suppressing either target-aligned or distractor-aligned frontal cortex increases behavioral responses to distractor stimuli.
C_LIO_LIPopulation neuronal encoding: Suppressing target-aligned frontal cortex increases distractor stimulus encoding in target-aligned S1.
C_LIO_LISingle unit neuronal encoding: Frontal cortex decorrelates encoding of target and distractor stimuli in target-aligned sensory cortex.
C_LIO_LIProactive modulation: Frontal cortex modulation of sensory cortex pre-stimulus is robust and context-dependent.
C_LI The Telomere Length Landscape of Prostate Cancer Replicative immortality is a hallmark of cancer, and can be achieved through telomere lengthening and maintenance. We report telomere lengths (TLs) of 392 localized prostate cancer tumours and characterize their relationship to genomic, transcriptomic and proteomic features. Shorter tumour TLs were associated with elevated genomic instability, including single-nucleotide variants, indels and structural variants. Genes involved in cell proliferation and signaling were correlated with tumour TL at all levels of the central dogma. TL was also associated with multiple clinical features of a tumour. Longer TLs in non-tumour samples were associated with a lower rate of biochemical relapse after definitive local therapy. Our analysis integrates multi-omics data to illuminate the relationship of specific genomic alterations in a tumour and TL in prostate cancer. Although the role of telomere length in cancer has been well studied, its association to genomic features is less well known. We describe the multi-level integration of telomere length, genomics, transcriptomics and proteomics in localized prostate cancer. Patient Summary We examined the association between telomere length and multiple omics-level data in prostate cancer. We observed that traditional telomere mutations are rare in prostate cancer and that telomere length is associated with multiple measure of genomic instability. Robust and Remote Measurement of Heart Rate Based on A Surveillance Camera Arrhythmia is a marked symptom of many cardiovascular diseases (CVDs). The accurate and timely detection of heart rate can greatly reduce the harm of arrhythmia to people. However, it is still a challenge to robustly and remotely measure heart rate in daily life due to the changing environmental conditions during measurement, such as the varying light intensity, the movement of people, and the uncertain distance between the sensor to people. In this study, we propose a method to accurately measure human heart rate within a distance of 4.5 meters under different light intensities by simply using a surveillance camera. After a 20-second color video of a persons hand is captured by the camera, a method based on the Fast Fourier Transform (FFT) algorithm is designed to extract the blood volume pulse wave to calculate the heart rate. According to the comparison between the real heart rate and results measured by electrocardiography (ECG), the proposed method achieves an accuracy of 95.8% when the measurement is performed within a distance of 4.5 meters and 90% when within 5.0 meters. Our experiments show that when the illuminance varies between 100-1000 lux (lighting level indoor), we still get the correct results. Our experiments also demonstrate that the proposed method accurately obtain heart rate even when the light intensity is below 32 lux (300-500 lux in a workplace environment). The methods strong adaptability to changing environmental conditions makes it applicable to many scenarios, such as homes of the elderly, classrooms, and other public spaces. Naming Human Diseases: Ethical Principles of Curating Exclusive Substitute for Inopportune Nosology BackgroundIn the medical sphere, understanding naming conventions strengthen the integrity of naming human diseases remains nominal rather than substantial yet. Since the current nosology-based standard for human diseases could not offer a one-size-fits-all corrective mechanism, many idiomatic but flawed names frequently appear in scientific literature and news outlets at the cost of sociocultural impacts.

ObjectiveWe attempt to examine the ethical oversights of current naming practices and propose heuristic rationales and approaches to determine a pithy name instead of an inopportune nosology.

MethodsFirst, we examined the compiled global online news volumes and emotional tones on some inopportune nosology like German measles, Middle Eastern Respiratory Syndrome, Spanish flu, Hong Kong flu, and Huntingtons disease in the wake of COVID-19. Second, we prototypically scrutinize the lexical dynamics and pathological differentials of German measles and common synonyms by leveraging the capacity of the Google Books Ngram Corpus. Third, we demonstrated the empirical approaches to curate an exclusive substitute for an anachronistic nosology German measles based on deep learning models and post-hoc explanations.

ResultsThe infodemiological study shows that the public informed the offensive names with extremely negative tones in textual and visual narratives. The findings of the historiographical study indicate that many synonyms of German measles did not survive, while German measles became an anachronistic usage, and rubella has taken the dominant place since 1994. The PubMedBERT model could identify rubella as a potential substitution for German measles with the highest semantic similarity. The results of the semantic drift experiments further indicate that rubella tends to survive during the ebb and flow of semantic drift.

ConclusionsOur findings indicate that the nosological evolution of anachronistic names could result in sociocultural impacts without a corrective mechanism. To mitigate such impacts, we introduce some ethical principles for formulating an improved naming scheme. Based on deep learning models and post-hoc explanations, our illustrated experiments could provide hallmark references to the remedial mechanism of naming practices and pertinent credit allocations. Time-lapse imaging of bacterial morphology facilitates classification of antimicrobial agents Antimicrobial resistance is a major threat to human health. Basic knowledge of antimicrobial mechanism of action (MoA) is imperative for patient care and for identification of novel antimicrobials. However, the process of antimicrobial MoA identification is relatively laborious. Here, we developed a simple, quantitative time-lapse fluorescence imaging method, Dynamic Bacterial Morphology Imaging (DBMI), to facilitate this process. It uses a membrane dye and a nucleoid dye to track the morphological changes of single Bacillus subtilis cells in response to antimicrobials for up to 60 min. DBMI of bacterial cells facilitated assignment of the MoAs of 14 distinct, known antimicrobial compounds to the five main classes. Using this method, we found that the poorly studied antimicrobial, harzianic acid, a secondary metabolite that we purified from the fungal culture of Oidiodendron flavum, targets the cell envelope. We conclude that DBMI is a simple method, which facilitates rapid classification of the MoA of antimicrobials in functionally distinct classes. 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. Co-infection of Candidatus Piscichlamydia trichopodus (order Chlamydiales) and Henneguya sp (Myxosporea, Myxobolidae) in snakeskin gourami Trichopodus pectoralis (Regan 1910) The present study describes a simultaneous infection of a novel Chlamydia-like organism (CLO) with a Myxozoa parasite, Henneguya sp. in snakeskin gourami Trichopodus pectoralis in Thailand. A new CLO is proposed "Candidatus Piscichlamydia trichopodus" (CPT) based on 16S rRNA phylogenetic analysis. Systemic intracellular CPT infection was confirmed by histological examination, in situ hybridization, PCR assay, and sequencing of 16S rRNA. This novel pathogen belongs to the order Chlamydiales but differs in certain aspects from other species. The histopathological changes associated with CPT infection were different from the typical pathological lesions of epitheliocystis caused by previously known CLO. Unlike other CLO, CPT localized in the connective tissue rather than in the epithelial cells and formed smaller clumps of intracellular bacteria that stained dark blue with hematoxylin. On the other hand, typical myxospores of the genus Henneguya with tails were observed in the gill sections. Infection with Henneguya sp. resulted in extensive destruction of the gill filaments, most likely leading to respiratory distress. Due to the frequency of co-infections and the unavailability of culture methods for CLO and Henneguya sp, it was difficult to determine which pathogens were directly responsible for the associated mortality. However, co-infections may increase the negative impact on the host and the severity of the disease. Given the commercial importance of the snakeskin gourami and its significant aquaculture potential, the findings of this study are important for further studies on disease prevention. Rapid global phaseout of animal agriculture has the potential to stabilize greenhouse gas levels for 30 years and offset 68 percent of CO2 emissions this century Animal agriculture contributes significantly to global warming through ongoing emissions of the potent greenhouse gases methane and nitrous oxide, and displacement of biomass carbon on the land used to support livestock. However, because estimates of the magnitude of the effect of ending animal agriculture often focus on only one factor, the full potential benefit of a more radical change remains underappreciated. Here we quantify the full "climate opportunity cost" of current global livestock production, by modeling the combined, long-term effects of emission reductions and biomass recovery that would be unlocked by a phaseout of animal agriculture. We show that, even in the absence of any other emission reductions, persistent drops in atmospheric methane and nitrous oxide levels, and slower carbon dioxide accumulation, following a phaseout of livestock production would, through the end of the century, have the same cumulative effect on the warming potential of the atmosphere as a 25 gigaton per year reduction in anthropogenic CO2 emissions, providing half of the net emission reductions necessary to limit warming to 2{degrees}C. The magnitude and rapidity of these potential effects should place the reduction or elimination of animal agriculture at the forefront of strategies for averting disastrous climate change.

Significance StatementThe use of animals to produce food has a negative impact on the climate, but the benefits of a global switch to a plant based diet are underappreciated. We show that the global warming impact, through the rest of this century, of eliminating greenhouse gas emissions from livestock and allowing native ecosystems to regrow on the land currently used to house and feed livestock, would be equivalent to a 68% reduction in carbon dioxide emissions. We hope putting clearer numbers on the "climate opportunity cost" of our continued use of animals as food technology will help policymakers and the public properly prioritize dietary change as a climate defense strategy.

Declaration of Conflict of InterestPatrick Brown is the founder and CEO of Impossible Foods, a company developing alternatives to animals in food-production. Michael Eisen is an advisor to Impossible Foods. Both are shareholders in the company and thus stand to benefit financially from reduction of animal agriculture. Structural predictions of the SNX-RGS proteins suggest they belong to a new class of lipid transfer proteins Recent advances in protein structure prediction using machine learning such as AlphaFold2 and RosettaFold presage a revolution in structural biology. Genome-wide predictions of protein structures are providing unprecedented insights into their architecture and intradomain interactions, and applications have already progressed towards assessing protein complex formation. Here we present detailed analyses of the sorting nexin proteins that contain regulator of G-protein signalling domains (SNX-RGS proteins), providing a key example of the ability of AlphaFold2 to reveal novel structures with previously unsuspected biological functions. These large proteins are conserved in most eukaryotes and are known to associate with lipid droplets (LDs) and sites of LD-membrane contacts, with key roles in regulating lipid metabolism. They possess five domains, including an N-terminal transmembrane domain that anchors them to the endoplasmic reticulum, an RGS domain, a lipid interacting phox homology (PX) domain and two additional domains named the PXA and PXC domains of unknown structure and function. Here we report the crystal structure of the RGS domain of sorting nexin 25 (SNX25) and show that the AlphaFold2 prediction closely matches the experimental structure. Analysing the full-length SNX-RGS proteins across multiple homologues and species we find that the distant PXA and PXC domains in fact fold into a single unique structure that notably features a large and conserved hydrophobic pocket. The nature of this pocket strongly suggests a role in lipid or fatty acid binding, and we propose that these molecules represent a new class of conserved lipid transfer proteins. First detection of herpesvirus and prevalence of mycoplasma infection in free-ranging Hermann s tortoises (Testudo hermanni), and in potential pet vectors AO_SCPLOWBSTRACTC_SCPLOWTwo types of pathogens cause highly contagious upper respiratory tract diseases (URTD) in Chelonians: testudinid herpesviruses (TeHV) and a mycoplasma (Mycoplasma agassizii). In captivity, these infections are frequent and can provoke outbreaks. Pet trade generates international flow of tortoises, often without sanitary checking; individuals intentionally or accidentally released in the wild may spread pathogens. A better understanding of the transmission of infectious agents from captivity to wild tortoises is needed. Many exotic individuals have been introduced in populations of the endangered western Hermanns tortoise (Testudo hermanni hermanni), notably spur-thighed tortoises (Testudo graeca). We assessed the presence of TeHV and mycoplasma in native western Hermanns tortoises and in potential pet vectors in south-eastern France. Using a large sample (N=572 tortoises), this study revealed, by PCR, the worrying presence of herpesvirus in 7 free-ranging individuals (3 sub-populations). Additionally, Mycoplasma agassizii was detected, by PCR, in 15 of the 18 populations sampled with a frequency ranging from 3.4% (1 of 29 tortoises) to 25% (3 of 12 tortoises). Exotic spur-thighed tortoises showed high frequency of mycoplasma infection in captivity (18.2%) and in individuals (50%) found in native Hermanns tortoise sub-populations, suggesting that this species could be a significant vector. The paucity of information of TeHV on European tortoise URTD in natural settings, especially in combination with mycoplasma, prompts for further studies. Indeed, sick tortoises remain concealed and may not be easily detected in the field. Our results indicate a good health for most infected tortoise but it should be screened in the field as well as in captivity. The C. elegans gonadal sheath Sh1 cells extend asymmetrically over a differentiating germ cell population in the proliferative zone The C. elegans adult hermaphrodite germ line is surrounded by a thin tube formed by somatic sheath cells that support germ cells as they mature from the stem-like mitotic state through meiosis, gametogenesis and ovulation. Recently, we discovered that the distal-most Sh1 sheath cells associate with mitotic germ cells as they exit the niche. Here we report that these distal sheath-associated germ cells differentiate first in animals with temperature-sensitive mutations affecting germ cell state, and stem-like germ cells are maintained distal to the Sh1 boundary. We analyze several markers of the distal sheath, which is best visualized with endogenously tagged membrane proteins, as overexpressed fluorescent proteins fail to localize to distal membrane processes and can cause gonad morphology defects. However, such reagents with highly variable expression can be used to determine the relative positions of the two Sh1 cells, one of which often extends further distal than the other. 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.