Insertions in SARS-CoV-2 genome caused by template switch and duplications give rise to new variants that merit monitoring The appearance of multiple new SARS-CoV-2 variants during the winter of 2020-2021 is a matter of grave concern. Some of these new variants, such as B.1.617.2, B.1.1.7, and B.1.351, manifest higher infectivity and virulence than the earlier SARS-CoV-2 variants, with potential dramatic effects on the course of the COVID-19 pandemic. So far, analysis of new SARS-CoV-2 variants focused primarily on point nucleotide substitutions and short deletions that are readily identifiable by comparison to consensus genome sequences. In contrast, insertions have largely escaped the attention of researchers although the furin site insert in the spike protein is thought to be a determinant of SARS-CoV-2 virulence and other inserts might have contributed to coronavirus pathogenicity as well. Here, we investigate insertions in SARS-CoV-2 genomes and identify 347 unique inserts of different lengths. We present evidence that these inserts reflect actual virus variance rather than sequencing errors. Two principal mechanisms appear to account for the inserts in the SARS-CoV-2 genomes, polymerase slippage and template switch that might be associated with the synthesis of subgenomic RNAs. We show that inserts in the Spike glycoprotein can affect its antigenic properties and thus merit monitoring. At least, three inserts in the N-terminal domain of the Spike (ins245IME, ins246DSWG, and ins248SSLT) that were first detected in 2021 are predicted to lead to escape from neutralizing antibodies, whereas other inserts might result in escape from T-cell immunity.
Genetic models of latent tuberculosis in mice reveal differential control by adaptive immunity Studying latent Mycobacterium tuberculosis (Mtb) infection has been limited by the lack of a suitable mouse model. We discovered that transient depletion of protein biotin ligase (BPL) and thioredoxin reductase (TrxB2) results in latent infections during which Mtb cannot be detected but that relapse in a subset of mice. The immune requirements for Mtb control during latency, and the frequency of relapse, were strikingly different depending on how latency was established. TrxB2 depletion resulted in a latent infection that required adaptive immunity for control and reactivated with high frequency, whereas latent infection after BPL depletion was independent of adaptive immunity and rarely reactivated. We identified immune signatures of T cells indicative of relapse and demonstrated that BCG vaccination failed to protect mice from TB relapse. These reproducible genetic latency models allow investigating the host immunological determinants that control the latent state and offer opportunities to evaluate therapeutic strategies in settings that mimic aspects of latency and TB relapse in humans.
Autotaxin is a Potential Link between Genetic Risk Factors and Pathogenesis of Systematic Lupus Erythematosus in Plasmacytoid Dendritic Cells BackgroundThe importance of autotaxin, an enzyme that catalyzes lysophospholipid production, has recently been recognized in various diseases, including cancer and autoimmune diseases. Herein, we examined the role of autotaxin in systemic lupus erythematosus (SLE), utilizing data from ImmuNexUT, a comprehensive database consisting of transcriptome data and expression quantitative trait locus (eQTL) data of immune cells from patients with immune-mediated disorders.

MethodsSerum autotaxin concentrations in patients with SLE and healthy controls (HCs) were compared. The transcriptome data of patients with SLE and age-and sex-matched HCs were obtained from ImmuNexUT. The expression of ENPP2, the gene encoding autotaxin, was examined in peripheral blood immune cells. Next, weighted gene correlation network analysis (WGCNA) was performed to identify genes with expression patterns similar to ENPP2. The ImmuNexUT eQTL database and public epigenomic databases were used to infer the relationship between autotaxin and pathogenesis of SLE.

ResultsAutotaxin levels were elevated in the serum of patients with SLE compared to HCs. Furthermore, the expression of ENPP2 was higher in plasmacytoid dendritic cells (pDCs) than in other immune cell subsets, and its expression was elevated in pDCs of patients with SLE compared to HCs. In WGCNA, ENPP2 belonged to a module that correlated with disease activity. This module was enriched in interferon-associated genes and included genes whose expression was influenced by single-nucleotide polymorphisms associated with SLE, suggesting that it is a key module connecting genetic risk factors of SLE with disease pathogenesis. Analysis utilizing the ImmuNexUT eQTL database and public epigenomic databases suggested that the increased expression of ENPP2 in pDCs from patients with SLE may be caused by increased expression of interferon-associated genes and increased binding of STAT3 complexes to the regulatory region of ENPP2.

ConclusionsAutotaxin may play a critical role in connecting genetic risk factors of SLE to disease pathogenesis in pDCs. 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. Honeybee cognition as a tool for scientific engagement In addition to the crowdsourcing of experimental data, citizen science, and scientific engagement more broadly, serve as a bridge between researchers and the wider community. This serves to foster a greater understanding of the scientific method and science-based solutions generally. Apis mellifera (honeybees) are a well-established model for the study of learning and cognition and can serve as an engaging outreach system for this wider community. Here, we developed and implemented a protocol using well established honeybee conditioning protocols to safely study the effects of caffeine and dopamine on learning performance. Using this protocol, a group of high-school aged students as part of the Ryukyu Girls program demonstrated that caffeine, but not dopamine, significantly reduced the number of trials required for a successful conditioning response. This allowed these students to explore the scientific method in a relatable and engaging way.

Simple SummaryGlobal scientific literacy can be improved through widespread and effective community engagement by researchers. We propose Apis mellifera (honeybee) as an public engagement tool due to widespread awareness of colony collapse and the bees importance in food production. Moreover, their cognitive abilities make for engaging experiments. Their relative ease of cultivation means that studies can be performed cost-effectively, especially when partnering with local aperists. Using a proxy for honeybee learning, a group of non-specialist high-school-aged participants obtained data suggesting that caffeine, but not dopamine, improved learning. This hands-on experience facilitated student understanding of the scientific method, factors that shape learning and the importance of learning for hive health. A novel two-generation approach to the population dynamics of gregarious parasitoids Parasitoids, as important natural enemies, help maintain balance in natural ecosystems. Their population dynamics is generally predicted from the number of individuals. Here, using gregarious parasitoids as models, we show that this traditional approach omits one important parameter: the mothers manipulation of offspring fertility due to the clutch size-body size- fertility correlation. As a result of this correlation, when females deliberately adjust the number of offspring laid in a host, they determine not only the number but also the body sizes and reproductive potentials of those offspring. For the first time in the model species Anaphes flavipes, we determined the parasitoids offspring fertility from clutch size. Using this, we experimentally clarified the advantage of specific clutch size combinations and we show that identical fertility in the F1 generation can lead to distinctly different fertility values in the F2 generation. Even with the same number of hosts, lower fertility in the F1 generation can cause higher fertility in the F2 generation. Based on these results, we propose a novel two-generation approach which includes the clutch size-body size-fertility correlation. Our novel approach provides a new perspective for determining the individual fitness levels of gregarious parasitoids with new options for the modelling of parasitoid population dynamics. Weighted transitivity scores that account for triadic edge similarity in undirected graphs The graph transitivity measures the probability that adjacent vertices in a network are interconnected, thus revealing the existence of tightly connected neighborhoods playing a role in information and pathogen circulation. When the connections vary in strength, focusing on whether connections exist or not can be reductive. I score the weighted transitivity according to the similarity between the weights of the three possible links in each triad. I illustrate the biological relevance of that information with two reanalyses of animal contact networks. In the rhesus macaque Macaca mulatta, a species in which kin relationships strongly predict social relationships, the new metrics revealed striking similarities in the configuration of grooming networks in captive and free-ranging groups, but only as long as the matrilines were preserved. In the barnacle goose Branta leucopsis, in an experiment designed to test the long-term effect of the goslings social environment, the new metrics uncovered an excess of weak triplets closed by strong links in males compared to females, and consistent with the triadic process underlying goose dominance relationships. Rapid genotyping of tilapia lake virus (TiLV) using Nanopore sequencing Infectious diseases represent one of the major challenges to sustainable aquaculture production. Rapid, accurate diagnosis and genotyping of emerging pathogens during early-suspected disease cases is critical to facilitate timely response to deploy adequate control measures and prevent or reduce spread. Currently, most laboratories use PCR to amplify partial pathogen genomic regions, occasionally combined with sequencing of PCR amplicon(s) using conventional Sanger sequencing services for confirmatory diagnosis. The main limitation of this approach is the lengthy turnaround time. Here, we report an innovative approach using a previously developed specific PCR assay for pathogen diagnosis combined with a new Oxford Nanopore Technologies (ONT)-based amplicon sequencing method for pathogen genotyping. Using fish clinical samples, we applied this approach for the rapid confirmation of PCR amplicon sequences identity and genotyping of tilapia lake virus (TiLV), a disease-causing virus affecting tilapia aquaculture globally. The consensus sequences obtained after polishing exhibit strikingly high identity to references derived by Illumina and Sanger methods (99.83-100%). This study suggests that ONT-based amplicon sequencing is a promising platform to deploy in regional aquatic animal health diagnostic laboratories in low and medium income countries, for fast identification and genotyping of emerging infectious pathogens from field samples within a single day. Naturally occurring combinations of receptors from single cell transcriptomics in endothelial cells VEGF inhibitor drugs have been successful, especially in ophthalmology, but not all patients respond to them. Combinations of drugs are likely to be needed for more effective therapies of angiogenesis-related diseases. In this paper we describe naturally occurring combinations of receptors in endothelial cells that might help to understand how cells communicate and to identify targets for drug combinations. We also develop and share a new software tool called DECNEO to identify them.

Single-cell gene expression data are used to identify a set of co-expressed endothelial cell receptors, conserved among species (mice and humans) and enriched, within a network, of connections to up-regulated genes. This set includes several receptors previously shown to play a role in angiogenesis. Multiple statistical tests from large datasets, including an independent validation set, support the reproducibility, evolutionary conservation and role in angiogenesis of these naturally occurring combinations of receptors. We also show tissue-specific combinations and, in the case of choroid endothelial cells, consistency with both well-established and recent experimental findings, presented in a separate paper.

The results and methods presented here advance the understanding of signaling to endothelial cells. The methods are generally applicable to the decoding of intercellular combinations of signals. Maternal microbiota Bifidobacterium promotes placental morphogenesis, nutrient transport and fetal growth in mice The gut microbiota plays a central role in regulating host metabolism. However, while substantial progress has been made in discerning how the microbiota influences host functions post birth and beyond, little is known about how key members of the maternal gut microbiota can influence feto-placental growth. Here, using germ-free and specific-pathogen-free mice, we demonstrate that the bacterium Bifidobacterium breve UCC2003 modulates maternal body adaptations, placental structure and nutrient transporter capacity, with implications for fetal metabolism and growth. The effects of B. breve UCC2003 on feto-placental growth are mediated, in part, by changes in the maternal and placental metabolome (i.e. acetate and carnitine). Histological analysis of the placenta confirmed that Bifidobacterium modifies placental structure via changes in Igf2P0, Dlk1, Mapk1 and Mapk14 expression. Additionally, B. breve UCC2003, acting through Slc2a1 and Fatp1-4 transporters, was shown to restore fetal glycaemia and fetal growth in association with changes in the fetal hepatic transcriptome. This study provides knowledge towards a novel and safe therapeutic strategy for treating pregnancy disorders via modulation of the maternal gut microbiota. 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

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/464182v3_ufig1.gif" ALT="Figure 1">
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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 Chromosomal rearrangements with stable repertoires of genes and transposable elements in an invasive forest-pathogenic fungus AO_SCPLOWBSTRACTC_SCPLOWChromosomal rearrangements have been largely described among eukaryotes, and may have important consequences on evolution of species. High genome plasticity has been often reported in Fungi, which may explain their apparent ability to quickly adapt to new environments. Cryphonectria parasitica, causing the Chestnut blight disease, is an invasive fungal pathogen species associated with several recent host shifts during its successive introductions from Asia to North America and Europe. Previous cytological karyotyping and genomic studies suggested several chromosomal rearrangements which remains to be described in detail for this species. A serious limitation for valid genome comparisons is the access to robust genome assemblies that usually contain genomic regions of low complexity. We present a new de novo whole-genome assembly obtained from a new method of DNA extraction and long-reads sequencing Nanopore technology obtained from a Japanese isolate sampled in the native area of the species. The comparison with a recently published reference genome revealed stable gene and transposable elements (TEs) repertoires. We also showed that the C. parasitica genome is lowly compartmentalized, with a poor association between TEs and genes, such as those potentially involved in host interactions (i.e., genes coding for small secreted proteins or for secondary metabolites). This genome comparison, however, detected several large chromosomal rearrangements that may have important consequences in gene regulations and sexual mating in this invasive species. This study opens the way for more comparisons of high-quality assembled genomes, and questions the role of structural variations in the invasive success of this fungal pathogen species. 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. 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. Stimulus manipulations permit activation of fiber subpopulations in the mouse and rat vagus Afferent and efferent vagal fibers mediate bidirectional communication between the brain and visceral organs. Small, unmyelinated C-afferents constitute the majority of vagal fibers, play critical roles in numerous interoceptive circuits and autonomic reflexes in health and disease and may contribute to the efficacy and safety of vagus nerve stimulation (VNS). Selective engagement of C-afferents with electrical stimuli has not been feasible, due to the default fiber recruitment order: larger fibers first, smaller fibers last. Here, we determine and optimize an electrical stimulus that selectively engages vagal C-afferents. Intermittent KHz-frequency electrical stimulation (KES) activates motor and, preferentially, sensory vagal neurons in the brainstem. During KES, asynchronous activity of C-afferents increases, while that of larger fibers remains largely unchanged. In parallel, KES effectively blocks excitability of larger fibers while moderately suppressing excitability of C-afferents. By compiling selectivity indices in individual animals, we find that optimal KES parameters for C-afferents are >5KHz frequency and 7-10 times engagement threshold (xT) intensity in rats, 15-25xT in mice. These effects can be explained in computational models by how sodium channel responses to KES are shaped by axonal size and myelin. Our results indicate that selective engagement of vagal C-afferents is attainable by intermittent KES. High-Speed, Cortex-Wide Volumetric Recording of Neuroactivity at Cellular Resolution using Light Beads Microscopy Two-photon microscopy together with genetically encodable calcium indicators has emerged as a standard tool for high-resolution imaging of neuroactivity in scattering brain tissue. However, its various realizations have not overcome the inherent tradeoffs between speed and spatiotemporal sampling in a principled manner which would be necessary to enable, amongst other applications, mesoscale volumetric recording of neuroactivity at cellular resolution and speed compatible with resolving calcium transients. Here, we introduce Light Beads Microscopy (LBM), a scalable and spatiotemporally optimal acquisition approach limited only by fluorescence life-time, where a set of axially-separated and temporally-distinct foci record the entire axial imaging range near-simultaneously, enabling volumetric recording at 1.41 x 108 voxels per second. Using LBM, we demonstrate mesoscopic and volumetric imaging at multiple scales in the mouse cortex, including cellular resolution recordings within ~3x5x0.5 mm3 volumes containing >200,000 neurons at ~5 Hz, recording of populations of ~1 million neurons within ~5.4x6x0.5 mm3 volumes at ~2Hz as well as higher-speed (9.6 Hz) sub-cellular resolution volumetric recordings. LBM provides an unprecedented opportunity for discovering the neurocomputations underlying cortex-wide encoding and processing of information in the mammalian brain. Leaf Aqueous Extract of Manilkara hexandra influenced Glucose Metabolism in fish: An observation with Labeo rohita Fingerlings as model organism Manilkara hexandra (Roxb.) Dubard (common name Khirni, family: Sapotaceae) is an evergreen traditionally used medicinal plant. The present study aimed to see the effects of leaf aqueous extract of M. hexandra (LAEMH) on digestive and glucose metabolic enzyme action and serum glucose level in rohu, Labeo rohita, fingerlings. Experimental fish were fed a basal diet (Group I), and diets supplemented with LAEMH at 300 mg (Group II) and 600 mg (Group III) kg-1body weight. A significant reduction in serum glucose was noticed in the treated groups when compared with the control group. The reduced amylase activity was noticed in the treated fingerlings in a dose-dependent manner. However, lipase and protease activities didnt differ significantly among the experimental groups. Reduced serum glucose level might be correlated with the decline in the activity of digestive amylase in fish. Group II (3.35{+/-}0.19 U) and Group III (3.49{+/-}0.13 U) were recorded with reduced glucose-6-phosphatase activities than the control group (4.4{+/-}0.39 U). Moreover, the study revealed a decline in fructose-1,6-phosphatase activities in the treated groups in comparison to the control group. The decline in the activities of the metabolic enzymes might be associated with the non-availability of glucose owing to reduced activity of the digestive amylase in the treated groups. In conclusion, the present study established the hypoglycaemic effect of the leaf aqueous extract of M. hexandra in a fish model. Dose-dependent thresholds of dexamethasone destabilize CAR T-cell treatment efficacy Chimeric antigen receptor (CAR) T-cell therapy is potentially an effective targeted immunotherapy for glioblastoma, yet there is presently little known about the efficacy of CAR T-cell treatment when combined with the widely used anti-inflammatory and immunosuppressant glucocorticoid, dexamethasone. Here we present a mathematical model-based analysis of three patient-derived glioblastoma cell lines treated in vitro with CAR T-cells and dexamethasone. Advanced in vitro experimental cell killing assay technologies allow for highly resolved temporal dynamics of tumor cells treated with CAR T-cells and dexamethasone, making this a valuable model system for studying the rich dynamics of nonlinear biological processes with translational applications. We model the system as a nonautonomous, two-species predator-prey interaction of tumor cells and CAR T-cells, with explicit time-dependence in the clearance rate of dexamethasone. Using time as a bifurcation parameter, we show that (1) dexamethasone destabilizes coexistence equilibria between CAR T-cells and tumor cells in a dose-dependent manner and (2) as dexamethasone is cleared from the system, a stable coexistence equilibrium returns in the form of a Hopf bifurcation. With the model fit to experimental data, we demonstrate that high concentrations of dexamethasone antagonizes CAR T-cell efficacy by exhausting, or reducing the activity of CAR T-cells, and by promoting tumor cell growth. Finally, we identify a critical threshold in the ratio of CAR T-cell death to CAR T-cell proliferation rates that predicts eventual treatment success or failure that may be used to guide the dose and timing of CAR T-cell therapy in the presence of dexamethasone in patients.

Author summaryBioengineering and gene-editing technologies have paved the way for advance immunotherapies that can target patient-specific tumor cells. One of these therapies, chimeric antigen receptor (CAR) T-cell therapy has recently shown promise in treating glioblastoma, an aggressive brain cancer often with poor patient prognosis. Dexamethasone is a commonly prescribed anti-inflammatory medication due to the health complications of tumor associated swelling in the brain. However, the immunosuppressant effects of dexamethasone on the immunotherapeutic CAR T-cells are not well understood. To address this issue, we use mathematical modeling to study in vitro dynamics of dexamethasone and CAR T-cells in three patient-derived glioblastoma cell lines. We find that in each cell line studied there is a threshold of tolerable dexamethasone concentration. Below this threshold, CAR T-cells are successful at eliminating the cancer cells, while above this threshold, dexamethasone critically inhibits CAR T-cell efficacy. Our modeling suggests that in the presence of high dexamethasone reduced CAR T-cell efficacy, or increased exhaustion, can occur and result in CAR T-cell treatment failure. 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. Phenomics data processing: Extracting dose-response curve parameters from high-resolution temperature courses and repeated field-based wheat height measurements Temperature is a main driver of plant growth and development. New phenotyping tools enable quantifying the temperature response of hundreds of genotypes. Yet, for field-derived data, temperature response modeling bears flaws and pitfalls concerning the interpretation of derived parameters. In this study, climate data from five growing seasons with differing temperature distributions served as starting point for a growth simulation of wheat stem elongation, based on a four-parametric temperature response function (Wang-Engel) including all cardinal temperatures. In a novel approach, we re-extracted dose-responses from the simulation by combining high-resolution (hours) temperature courses with low-resolution (days) height data. The collection of such data is common in field phenotyping platforms. To take advantage of the lack of supra-optimal temperatures during the stem elongation, simpler (linear and asymptotic) models to predict temperature-response parameters were investigated. The asymptotic model extracted the base temperature of growth and the maximum absolute growth rate with high precision, whereas simpler, linear models failed to do so. Additionally, the asymptotic model provided a proxy estimate for the optimum temperature. However, when including seasonally changing cardinal temperatures, the prediction accuracy of the asymptotic model was strongly reduced. In a field study with three winter wheat varieties, significant differences were found for all three asymptotic dose-response curve parameters. We conclude that the asymptotic model based on high-resolution temperature courses is suitable to extract meaningful parameters from field-based data. Spatial organization of nuclear pores in Xenopus laevis oocytes Nuclear pores are protein assemblies inserted in the nuclear envelope of eukaryotic cells, acting as main gates for communication between nucleus and cytoplasm. So far, nuclear pores have been extensively studied to determine their structure and composition, yet their spatial organization and geometric arrangement on the nuclear surface are still poorly understood. Here, we analyze super-resolution images of the surface of Xenopus laevis oocyte nuclei during development, and characterize the arrangement of nuclear pores using tools commonly employed to study the atomic structural and topological features of soft matter. To interpret the experimental results, we hypothesize an effective interaction among nuclear pores and implemented it in extensive numerical simulations of octagonal clusters mimicking typical pore shapes. Thanks to our simple model, we find simulated spatial distributions of nuclear pores that are in excellent agreement with experiments, suggesting that an effective interaction among nuclear pores exists and could explain their geometrical arrangement. Furthermore, our results show that the statistical features of the geometric arrangement of nuclear pores do not depend on the type of pore-pore interaction, attractive or repulsive, but are mainly determined by the octagonal symmetry of each single pore. These results pave the way to further studies needed to determine the biological nature of pore-pore interactions. New phylogenetic Markov models for inapplicable morphological characters This paper describes new Markov models for phylogenetic inference with inapplicable (anatomically dependent) morphological characters. It shows that the new solutions fall into two categories depending on the type of character hierarchy and require use of different Markov models. A combination of the new models and previous solutions is capable to model all general types of dependencies that may exists between morphological traits. The performance of the new models is assessed using theory and simulations. Bayesian phylogenetic inference with the new models and their construction for a hierarchy of custom complexity is demonstrated using RevBayes and the R package rphenoscate respectively. A signalling axis involving CNOT3, Aurora B and ERK promotes differentiation and survival of mesendodermal progenitor cells Mesendoderm cells are key intermediate progenitors that form at the early primitive streak (PrS) and give rise to mesoderm and endoderm in the gastrulating embryo. We have identified an interaction between CNOT3 and the cell cycle kinase Aurora B, which requires sequences in the NOT box domain of CNOT3, and regulates MAPK/ERK signalling during mesendoderm differentiation. Aurora B phosphorylates CNOT3 at two sites located close to a nuclear localization signal and promotes localization of CNOT3 to the nuclei of mouse ES cells (ESCs) and metastatic lung cancer cells. ESCs that have both sites mutated give rise to embryoid bodies that are largely devoid of mesoderm and endoderm and are composed mainly of ectoderm. The mutant ESCs are also compromised in their ability to differentiate into mesendoderm in response to FGF2, BMP4 and Wnt3. The double mutation affects interaction of CNOT3 with Aurora B and with ERK and reduces phosphorylation of ERK in response to FGF2, impacting on survival of the differentiated ME cells. Our results identify an adaptor function for CNOT3 that regulates a key pathway in embryogenesis and cancer. Inferring mammalian tissue-specific regulatory conservation by predicting tissue-specific differences in open chromatin BackgroundEvolutionary conservation is an invaluable tool for inferring functional significance in the genome, including regions that are crucial across many species and those that have undergone convergent evolution. Computational methods to test for sequence conservation are dominated by algorithms that examine the ability of one or more nucleotides to align across large evolutionary distances. While these nucleotide alignment-based approaches have proven powerful for protein-coding genes and some non-coding elements, they fail to capture conservation at many enhancers, distal regulatory elements that control spatio-temporal patterns of gene expression. The function of enhancers is governed by a complex, often tissue- and cell type-specific, code that links combinations of transcription factor binding sites and other regulation-related sequence patterns to regulatory activity. Thus, function of orthologous enhancer regions can be conserved across large evolutionary distances, even when nucleotide turnover is high.

ResultsWe present a new machine learning-based approach for evaluating enhancer conservation that leverages the combinatorial sequence code of enhancer activity rather than relying on the alignment of individual nucleotides. We first train a convolutional neural network model that is able to predict tissue-specific open chromatin, a proxy for enhancer activity, across mammals. Then, we apply that model to distinguish instances where the genome sequence would predict conserved function versus a loss regulatory activity in that tissue. We present criteria for systematically evaluating model performance for this task and use them to demonstrate that our models accurately predict tissue-specific conservation and divergence in open chromatin between primate and rodent species, vastly out-performing leading nucleotide alignment-based approaches. We then apply our models to predict open chromatin at orthologs of brain and liver open chromatin regions across hundreds of mammals and find that brain enhancers associated with neuron activity and liver enhancers associated with liver regeneration have a stronger tendency than the general population to have predicted lineage-specific open chromatin.

ConclusionThe framework presented here provides a mechanism to annotate tissue-specific regulatory function across hundreds of genomes and to study enhancer evolution using predicted regulatory differences rather than nucleotide-level conservation measurements. Affordable Caenorhabditis elegans tracking system for classroom use For decades the nematode C. elegans has served as an outstanding research organism owing to its unsurpassed experimental amenability. This advantage has also made this tiny worm an attractive vehicle for science instruction across higher learning institutions. However, the prohibitive cost associated with the automated behavioral assessment of these animals remains an obstacle preventing their full adoption in undergraduate and high school settings. To improve this situation, we developed an inexpensive worm tracking system for use by high school interns and undergraduate students. Over the past two years this tracker has been successfully used by undergraduate students in our introductory Cell and Molecular lab (BSC220) at Illinois State University. Here we describe and demonstrate the use of our inexpensive worm tracking system. Getting more out of FLAG-Tag co-immunoprecipitation mass spectrometry experiments using FAIMS. Co-immunoprecipitation of proteins coupled to mass spectrometry is critical for the understanding of protein interaction networks. In instances where a suitable antibody is not available, it is common to graft synthetic tags onto target protein sequences and allowing the use of commercially available antibodies for affinity purification. A common approach is through FLAG-Tag co-immunoprecipitation. To allow the selective elution of protein complexes, competitive displacement using a large molar excess of the tag peptides is often carried out. Yet, this creates downstream challenges for the mass spectrometry analysis due to the presence of large quantities of these peptides. Here, we demonstrate that Field Asymmetric Ion Mobility Spectrometry (FAIMS), a gas phase ion separation device prior to mass spectrometry analysis can be applied to FLAG-Tag co-immunoprecipitation experiment to increase the depth of protein coverage. By excluding these abundant tag peptides, we were able to observe deeper coverage of interacting proteins and as a result, deeper biological insights, without the need for additional sample handling or altering sample preparation protocols.