The Digital Brain Bank, an open access platform for post-mortem datasets Post-mortem MRI provides the opportunity to acquire high-resolution datasets to investigate neuroanatomy, and validate the origins of image contrast through microscopy comparisons. We introduce the Digital Brain Bank (open.win.ox.ac.uk/DigitalBrainBank), a data release platform providing open access to curated, multimodal post-mortem neuroimaging datasets. Datasets span three themes - Digital Neuroanatomist: datasets for detailed neuroanatomical investigations; Digital Brain Zoo: datasets for comparative neuroanatomy; Digital Pathologist: datasets for neuropathology investigations. The first Digital Brain Bank release includes twenty one distinctive whole-brain diffusion MRI datasets for structural connectivity investigations, alongside microscopy and complementary MRI modalities. This includes one of the highest-resolution whole-brain human diffusion MRI datasets ever acquired, whole-brain diffusion MRI in fourteen non-human primate species, and one of the largest post-mortem whole-brain cohort imaging studies in neurodegeneration. The Digital Brain Bank is the culmination of our labs investment into post-mortem MRI methodology and MRI-microscopy analysis techniques. This manuscript provides a detailed overview of our work with post-mortem imaging to date, including the development of diffusion MRI methods to image large post-mortem samples, including whole, human brains. Taken together, the Digital Brain Bank provides crossscale, cross-species datasets facilitating the incorporation of post-mortem data into neuroimaging studies.
Fast event-related mapping of population fingertip tuning properties in human sensorimotor cortex at 7T fMRI studies that investigate somatotopic tactile representations in the human cortex typically use either block or phase-encoded stimulation designs. Event-related (ER) designs allow for more flexible and unpredictable stimulation sequences than the other methods, but they are less efficient. Here we compared an efficiency-optimized fast ER design (2.8s average intertrial interval, ITI) to a conventional slow ER design (8s average ITI) for mapping voxelwise fingertip tactile tuning properties in the sensorimotor cortex of 6 participants at 7 Tesla. The fast ER design yielded more reliable responses compared to the slow ER design, but with otherwise similar tuning properties. Concatenating the fast and slow ER data, we demonstrate in each individual brain the existence of two separate somatotopically-organized representations of the fingertips, one in the primary somatosensory cortex (S1) on the post-central gyrus, and the other shared across the motor and pre-motor cortices on the pre-central gyrus. In both S1 and motor representations, fingertip selectivity decreased progressively, from narrowly-tuned Brodmann areas 3b and 4a respectively, towards associative parietal and frontal regions that responded equally to all fingertips, suggesting increasing information integration along these two pathways. In addition, fingertip selectivity in S1 decreased from the cortical representation of the thumb to that of the pinky.

Significance StatementSensory and motor cortices in the human brain contain map-like representations of the body in which adjacent brain regions respond to adjacent body parts. The properties of these somatotopic maps provide important insight into how tactile and motor information is processed by the brain. Here, we describe an efficient mapping method using functional magnetic resonance imaging (fMRI) to measure somatotopic maps and their tuning properties. We used a fast event-related sequence to map the five fingers of the left hand in six human participants, and show that this method is superior to a conventional, slower event-related design. Furthermore, we confirm previously-identified tuning properties of fingertip representations in somatosensory cortex, and reveal a hitherto unknown fingertip map in the motor cortex.
Two Types of Cinnamoyl-CoA Reductase Function Divergently in Tissue Lignification, Phenylpropanoids Flux Control, and Inter-pathway Cross-talk with Glucosinolates as Revealed in Brassica napus Cinnamoyl-CoA reductase (CCR) is the entry point of lignin pathway and a crucial locus in dissection and manipulation of associated traits, but its functional dissection in Brassicaceae plants is largely lagged behind though Arabidopsis thaliana CCR1 has been characterized to certain extent. Here, 16 CCR genes are identified from Brassica napus and its parental species B. rapa and B. oleracea. Brassicaceae CCR genes are divided into CCR1 subfamily and CCR2 subfamily with divergent organ-specificity, yellow-seed trait participation and stresses responsiveness. CCR1 is preferential in G- and H-lignins biosynthesis and vascular development, while CCR2 has a deviation to S-lignin biosynthesis and interfascicular fiber development. CCR1 has stronger effects on lignification-related development, lodging resistance, phenylpropanoid flux control and seed coat pigmentation, whereas CCR2 controls sinapates levels. CCR1 upregulation could delay bolting and flowering time, while CCR2 upregulation weakens vascular system in leaf due to suppressed G lignin accumulation. Besides, CCR1 and CCR2 are deeply but almost oppositely linked with glucosinolates metabolism through inter-pathway crosstalk. Strangely, upregulation of both CCR1 and CCR2 could not enhance resistance to UV-B and S. sclerotiorum though CCR2 is sharply induced by them. These results provide systemic dissection on Brassica CCRs and CCR1-CCR2 divergence in Brassicaceae.

HighlightBrassicaceae contains two types of Cinnamoyl-CoA reductase. As revealed in Brassica napus, they are divergently involved in lignin monomer biosynthesis, tissue lignification, phenylpropanoid flux control, and inter-pathway crosstalk with glucosinolates. Identification of a conserved neutralizing epitope present on spike proteins from all highly pathogenic coronaviruses Three pathogenic human coronaviruses have emerged within the last 20 years, with SARS-CoV-2 causing a global pandemic. Although therapeutic antibodies targeting the SARS-CoV-2 spike currently focus on the poorly conserved receptor-binding domain, targeting essential neutralizing epitopes on the more conserved S2 domain may provide broader protection. We report an antibody binding an epitope conserved in the pre-fusion core of MERS-CoV, SARS-CoV and SARS-CoV-2 spike S2 domains. Antibody 3A3 binds a conformational epitope with ~2.5 nM affinity and neutralizes spike from SARS-CoV, SARS-CoV-2 and variants of concern in in vitro pseudovirus assays. Hydrogen-deuterium exchange mass spectrometry identified residues 980-1006 in the flexible hinge region at the S2 apex as the 3A3 epitope, suggesting 3A3 prevents the S2 conformational rearrangements required for conversion to the spike post-fusion state and virus-host cell fusion. This work defines a conserved vulnerable site on the SARS-CoV-2 S2 domain and guides the design of pan-protective spike immunogens. Ineffectual AEC1 differentiation from KRT8hi transitional state without fibrosis is associated with fatal COVID-19 ARDS ARDS due to COVID-19 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; clinical recovery requires epithelial regeneration. During physiologic regeneration in mice, AEC2s proliferate, exit the cell cycle, and transiently assume a transitional state before differentiating into AEC1s; persistence of the transitional state is associated with pulmonary fibrosis in humans. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiologic regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and fatal acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells in organized monolayers on alveolar septa without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or interrogation of scRNAseq datasets revealed that transitional cells in mouse models of physiologic regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure, but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis. ITS-based assessment of Madagascar's fungal diversity and arrival of ectomycorrhizal fungi to the island Madagascar is known for its high diversity and endemism of Fauna and Flora, which makes it particularly interesting for research on diversity and evolution. Fungi, however, have been largely overlooked, and whether fungi exhibit the same patterns as animals and plants has yet to be further examined.

We collected fungal sporocarps and ectomycorrhizal (EcM) roots during opportunistic surveys in five forests in Madagascar and generated a dataset of fungal Internal Transcribed Spacer (ITS) DNA sequences. We analysed them together with all publicly available fungal ITS DNA sequences. We identified 620 Operational Taxonomic Units (OTUs) from Madagascar, 10% of which contained only sequences from our surveys. We found 292 OTUs belonging to EcM species with /russula-lactarius, /boletus, /tomentella-telephora, /cortinarius and /amanita as the most abundant EcM lineages. Overall, sixty percent of all the fungi and 81% of the EcM species found are endemic to Madagascar. Lastly, we conducted a phylogenetic analysis using all the OTUs in Amanitaceae, Boletaceae and Russulaceae families to elucidate their relative timing of arrival in Madagascar. We found that the EcM species from Madagascar in the three families diverged recently (less than 34 mya), long after the separation of India and Madagascar (88 mya), which is consistent with a dispersal mediated process of arrival on the island. Our study provides the first comprehensive view of the current state of knowledge of EcM fungi in Madagascar represented by molecular data useful for ecological and evolutionary studies. Define protein variant functions with high-complexity mutagenesis libraries and enhanced mutation detection software ASMv1.0 Open reading frame (ORF) variant libraries have advanced our ability to query the functions of a large number of variants of a protein simultaneously in a single experiment. Variant libraries targeting full-length ORFs typically consists of all possible single-amino-acid substitutions and a stop codon at each amino-acid position. Because a variant differs from the template ORF by a single codon variation, variant quantification presents the most profound challenge to this technology. Efforts such as dividing a library into sub-libraries for direct sequencing, or tag-directed subassembly are practical only for small ORFs. Our approach, however, features an enhanced variant-calling tool that processes massive shotgun sequencing data, allowing one to screen a single variant library for ORFs sized up to [~]3600 bases. The variant-calling tool detects variants reliably, and also presents variant annotations in datafiles enabling analyses that have reshaped our strategies governing library design, screen deconvolution, sequencing and sequencing data analysis. Early postnatal activation of the hypoxia pathway disrupts β-cell function Hypoxic insults in the perinatal period can lead to persistent hyperinsulinism and profound hypoglycemia in newborns. We studied the impact of the hypoxia-inducible factor 1A (HIF1A) pathway on postnatal {beta}-cell function. Rat pups were treated daily between postnatal day (P)7 to P10 with adaptaquin (AQ), an inhibitor of prolyl hydroxylases, which stabilizes HIF1A. AQ-treated pups were hypoglycemic and had higher plasma insulin concentrations. Their islets had a decreased glucose threshold for insulin secretion, indicative of a delay in {beta}-cell postnatal functional maturation. Histology analyses revealed that AQ-treated pups had increased pancreatic insulin-positive area but no changes in the number of islets or number of {beta}-cells per islet, suggesting larger average {beta}-cell size. AQ-treated rat pups had decreased expression of cell cycle genes and decreased numbers of proliferating {beta}-cells. In conclusion, pharmacologic activation of the HIF1A pathway in the early postnatal period leads to hyperinsulinism, due to the persistence of a low glucose threshold for insulin secretion, and to decreased early postnatal {beta}-cell proliferation, suggesting it can impact adult {beta}-cell mass and diabetes risk. Pak1 and PP2A antagonize aPKC function to support cortical tension induced by the Crumbs-Yurt complex The Drosophila polarity protein Crumbs is essential for the establishment and growth of the apical domain in epithelial cells. The protein Yurt limits the ability of Crumbs to promote apical membrane growth, thereby defining proper apical/lateral membrane ratio that is crucial for forming and maintaining complex epithelial structures such as tubes or acini. Here, we show that Yurt also increases Myosin-dependent cortical tension downstream of Crumbs. Yurt overexpression thus induces apical constriction in epithelial cells. The kinase aPKC phosphorylates Yurt, thereby dislodging the latter from the apical domain and releasing apical tension. In contrast, the kinase Pak1 promotes Yurt dephosphorylation through activation of the phosphatase PP2A. The Pak1- PP2A module thus opposes aPKC function and supports Yurt-induced apical constriction. Hence, the complex interplay between Yurt, aPKC, Pak1 and PP2A contributes to the functional plasticity of Crumbs. Overall, our data increase our understanding of how proteins sustaining epithelial cell polarization and Myosin-dependent cell contractility interact with one another to control epithelial tissue architecture. Reliance of neuronal gene expression on cohesin scales with chromatin loop length Cohesin and CTCF are major drivers of 3D genome organization, but their role in neurons is still emerging. Here we show a prominent role for cohesin in the expression of genes that facilitate neuronal maturation and homeostasis. Unexpectedly, we observed two major classes of activity-regulated genes with distinct reliance on cohesin in primary cortical neurons. Immediate early genes remained fully inducible by KCl and BDNF, and short-range enhancer-promoter contacts at the Immediate early gene Fos formed robustly in the absence of cohesin. In contrast, cohesin was required for full expression of a subset of secondary response genes characterised by long-range chromatin contacts. Cohesin-dependence of constitutive neuronal genes with key functions in synaptic transmission and neurotransmitter signaling also scaled with chromatin loop length. Our data demonstrate that key genes required for the maturation and activation of primary cortical neurons depend cohesin for their full expression, and that the degree to which these genes rely on cohesin scales with the genomic distance traversed by their chromatin contacts. Propionate ameliorates diabetes-induced neurological dysfunction through regulating the PI3K/Akt/eNOS signaling pathway A large body of research has established diabetes-related cognitive deterioration, sometimes known as "diabetic encephalopathy." Current evidence supports that oxidative stress, neuronal apoptosis, and cerebral microcirculation weakness are associated with cognition deficits induced by diabetes. The present study explores the effect of propionate on neurological deficits, cerebral blood flow, and oxidative stress in diabetic mice. Propionate can markedly improve neurological function, which is correlated with its capabilities of stimulating nitric oxide (NO) production, increasing cerebral microcirculation, suppressing oxidative stress and reducing neuron loss in the hippocampus. In addition, the results of Western Blotting indicated that the brain-protective function of propionate in streptozocin (STZ)-induced insulin-dependent diabetes mellitus (IDDM) mice is related to phosphoinositide 3-kinase (PI3K)/serine-threonine protein kinase (Akt)/endothelial nitric oxide synthase(eNOS) signaling pathway. In a diabetic mouse model, propionate reduces cerebral microcirculation, hippocampus apoptosis, and neurological impairment. Thus, propionate, now employed as a food preservative, may also help slow diabetes-induced cognitive loss.

Graphical Abstract

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org.highwire.dtl.DTLVardef@81fa09org.highwire.dtl.DTLVardef@1469451org.highwire.dtl.DTLVardef@b37fa1org.highwire.dtl.DTLVardef@8037e7_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIPropionate ameliorates diabetic-induced depression-like behavior, spatial learning and memory deficits.
C_LIO_LIPropionate increases cerebral microcirculation by enhancing NO production in diabetic mice.
C_LIO_LIPropionate suppresses oxidative stress and reduces neuron loss in the hippocampus.
C_LIO_LIThe brain-protective function of propionate is related to PI3K/Akt/eNOS signaling pathway.
C_LI Extending Bayesian Elo-rating to quantify the steepness of dominance hierarchies The steepness of dominance hierarchies provides information about the degree of competition within animal social groups and is thus an important concept in socioecology. The currently most widely-used metrics to quantify steepness are based on Davids scores (DS) derived from dominance interaction networks. One serious drawback of these DS-based metrics is that they are biased, i.e., network density systematically decreases steepness values. Here, we provide a novel approach to estimate steepness based on Elo-ratings, implemented in a Bayesian framework (STEER: Steepness estimation with Elo-rating). Our new metric has two key advantages. First, STEER is unbiased, precise and more robust to data density than DS-based steepness. Second, it provides explicit probability distributions of the estimated steepness coefficient, which allows uncertainty assessment. In addition, it relies on the same underlying concept and is on the same scale as the original measure, and thus allows comparison to existing published results. We evaluate and validate performance of STEER by means of experimentation on empirical and artificial data sets and compare its performance to that of several other steepness estimators. Our results suggest that STEER provides a considerable improvement over existing methods to estimate dominance hierarchy steepness. We provide an R package EloSteepness to calculate the new steepness measure, and also show an example of using steepness in a comparative analysis. 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. Beta-cell specific Insr deletion promotes insulin hypersecretion and improves glucose tolerance prior to global insulin resistance Abstract

Insulin receptor (Insr) protein can be found at higher levels in pancreatic {beta}-cells than in most other tissues, but the consequences of {beta}-cell insulin resistance remain enigmatic. Ins1cre allele was used to delete Insr specifically in {beta}-cells of both female and male mice. Experimental mice were compared to Ins1cre-containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined {beta}-cells revealed transcriptomic consequences of Insr loss, which differed between female and male mice. Action potential and calcium oscillation frequencies were increased in Insr knockout {beta}- cells from female, but not male mice, whereas only male {beta}InsrKO mice had reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female {beta}InsrKO and {beta}InsrHET mice exhibited elevated insulin release in perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr did not alter {beta}-cell area up to 9 months of age, nor did it impair hyperglycemia-induced proliferation. Based on our data, we adapted a mathematical model to include {beta}-cell insulin resistance, which predicted that {beta}-cell Insr knockout would improve glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance was significantly improved in female {beta}InsrKO and {beta}InsrHET mice when compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We did not observe improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of {beta}-cell specific insulin resistance. The propensity for hyperinsulinemia was associated with mildly reduced fasting glucose and increased body weight. We further validated our main in vivo findings using the Ins1-CreERT transgenic line and found that male mice had improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that loss of {beta}-cell Insr contributes to glucose-induced hyperinsulinemia, thereby improving glucose homeostasis in otherwise insulin sensitive dietary and age contexts. 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. Deficiency of gluconeogenic enzyme PCK1 promotes murine NASH progression and fibrosis by activation PI3K/AKT/PDGF axis Background and AimsNonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by hepatic lipid accumulation, inflammation, and progressive fibrosis. However, the pathomechanisms underlying NASH are incompletely explored. Phosphoenolpyruvate carboxykinase 1 (PCK1) catalyzes the first rate-limiting step of gluconeogenesis. This study was designed to determine the role of PCK1 in regulating NASH progression.

MethodsLiver metabolism, hepatic steatosis, and fibrosis were evaluated at 24 weeks in liver-specific Pck1-knockout mice fed with NASH diet or chow diet. Gain- or loss-of-function approaches were used to explore the underlying mechanism in vitro. AKT and RhoA inhibitors were evaluated for NASH treatment in vivo.

ResultsHepatic PCK1 was downregulated in patients with NASH and mouse models of NASH. Mice with liver Pck1 deficiency displayed hepatic lipid disorder and liver injury fed with normal diet, while showed aggravated fibrosis and inflammation when fed NASH diet. Mechanistically, PCK1 deficiency upregulated genes involved in fatty acid transport and lipid droplet formation. Moreover, metabolomics analysis showed the accumulation of glycerol 3-phosphate, the substrate of triglyceride synthesis. Furthermore, the loss of hepatic PCK1 could activate the RhoA/PI3K/AKT pathway, which leads to increased secretion of PDGF-AA and promotes the activation of hepatic stellate cells. Accordingly, treatment with RhoA and AKT inhibitors alleviated NASH progression in the presence of Pck1 deletion in vivo.

ConclusionsPCK1 deficiency plays a key role in the development of hepatic steatosis and fibrosis by facilitating the RhoA/PI3K/AKT/PDGF-AA axis. These findings provide a novel insight into therapeutic approaches for the treatment of NASH. The glycocalyx affects the mechanotransductive perception of the topographical microenvironment The cell/microenvironment interface is the starting point of integrin-mediated mechanotransduction, but many details of mechanotransductive signal integration remain elusive due to the complexity of the involved (extra)cellular structures, such as the glycocalyx.

We used nano-bio-interfaces reproducing the complex nanotopographical features of the extracellular matrix to analyse the glycocalyx impact on PC12 cell mechanosensing at the nanoscale (e.g., by force spectroscopy with functionalised probes). Our data demonstrates that the glycocalyx configuration affects spatio-temporal nanotopography-sensitive mechanotransductive events at the cell/microenvironment interface. Opposing effects of glycocalyx removal were observed, when comparing flat and specific nanotopographical conditions. The excessive retrograde actin flow speed and force loading are strongly reduced on certain nanotopographies upon removal of the native glycocalyx, while on the flat substrate we observe the opposite trend.

Our results highlight the importance of the glycocalyx configuration in a molecular clutch force loading-dependent cellular mechanism for mechanosensing of microenvironmental nanotopographical features. Phenotypic plasticity is aligned with phenological adaptation on both micro- and macroevolutionary timescales In seasonally-variable environments, phenotypic plasticity in phenology may be critical for adaptation to fluctuating environmental (temperature) conditions. Using an 18-generation longitudinal dataset from natural insect (damselfly) populations, we show that phenology has strongly advanced. Individual fitness data suggest this is likely an adaptive response towards a thermally-dependent fitness optimum. A laboratory experiment revealed that developmental plasticity qualitatively matches environmental-dependence of selection, partially explaining observed phenological advance. Expanding our analysis to the macroevolutionary level, we use a database of over 1-million occurrence records and spatiotemporally-matched temperature data from 49 Swedish Odonate species to infer macroevolutionary dynamics of phenology. Phenological plasticity was more closely aligned with adaptation for species that have recently colonized northern latitudes, with more phenological mismatch at lower latitudes. Our results show that phenological plasticity plays a key role in microevolutionary dynamics within a single species, and such plasticity may have facilitated post-Pleistocene range expansion in this insect clade. Adaptive anti-tumor immunity is orchestrated by a population of CCL5-producing tissue-resident NK cells T cell-directed cancer immunotherapy often fails to generate lasting tumor control. Harnessing additional effectors of the immune response against tumors may strengthen the clinical benefit of immunotherapies. Here, we demonstrate that therapeutic targeting of the IFN{gamma}-IL-12 pathway relies on the ability of a population of tissue-resident NK (trNK) cells to orchestrate an anti-tumor microenvironment. Particularly, utilizing an engineered adenoviral platform, we show that paracrine IL-12 enhances functional DC-CD8 T cell interactions to generate adaptive anti-tumor immunity. This effect depends on the abundance of trNK cells and specifically their capacity to produce the cDC1-chemoattractant CCL5. Failure to respond to IL-12 and other IFN{gamma}-inducing therapies such as immune checkpoint blockade in tumors with low trNK cell infiltration could be overcome by intra-tumoral delivery of CCL5. Our findings reveal a novel barrier for T cell-focused therapies and offer mechanistic insights into how T cell-NK cell-DC crosstalk can be enhanced to promote anti-tumor immunity and overcome resistance.

SignificanceWe identified the lack of CCL5-producing, tissue-resident NK (trNK) cells as a barrier to T cell-focused therapies. While IL-12 induces anti-tumoral DC-T cell crosstalk in trNK cellrich tumors, resistance to IL-12 or anti-PD-1 in trNK cellpoor tumors can be overcome by the additional delivery of CCL5. Sablefish (Anoplopoma fimbria Pallas, 1814) plasma biochemistry and hematology reference intervals including blood cell morphology Plasma biochemistry and hematology reference intervals are integral health assessment tools in all medical fields, including aquatic animal health. As sablefish (Anoplopoma fimbria) are becoming aquaculturally and economically more important, this manuscript provides essential reference intervals (RI) for their plasma biochemistry and hematology along with reference photomicrographs of blood cells in healthy, fasted sablefish. Blood cell morphology can differ between fish species. In addition, blood cell counts and blood chemistry can vary between fish species, demographics, water conditions, seasons, diets, and culture systems, which precludes the use of RIs from other fish species. For this study, blood was collected for plasma biochemistry and hematology analysis between June 20 and July 18, 2019, from healthy, yearling sablefish, hatched and reared in captivity on a commercial diet. Overnight fast of 16-18 hours did not sufficiently reduce lipids in the blood, which led to visible lipemia and frequent rupture of blood cells during analysis. Therefore, sablefish should be fasted for 24 to 36 hours before blood is collected to reduce hematology artifacts or possible reagent interference in plasma biochemistry analysis. Lymphocytes were the most dominant leukocytes (98%), while eosinophils were rare, and basophils were not detected in sablefish. Neutrophils were very large cells with Dohle bodies. In mammals and avian species, Dohle bodies are usually signs of toxic change from inflammation, but no such association was found in these fish. In conclusion, lipemia can interfere with sablefish blood analysis, and available removal methods should be evaluated as fasting for up to 36 h might not always be feasible. Also, more studies are required to establish RI for different developmental stages and rearing conditions. Comparison of hybrid learning and remote education in the implementation of the 'Adopt a Microorganism' methodology The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook(R), represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, "Adopt a Microorganism", was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of Sao Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students discourse richness in microbiology even without face-to-face traditional classes. NOMA: a high-throughput microchip for robust, sequential measurements of secretions from the same single-cells Despite recent advances in single-cell analysis technologies, lacking simple methods to keep the live single-cells traceable for longitudinal detection reliably poses a significant obstacle in single-cell secretion analysis. Here we reported the high-density NOMA (narrow-opening microwell array) microchip that realized the retention of[&ge;] 97% of trapped single cells in dedicated spatial locations during repetitive detection procedures, verified with both adherent and suspension cells by two researchers independently. We applied it in monitoring single-cell protein secretions sequentially from the same single cells, and we found the digital protein secretion patterns dominate the protein secretion. We also demonstrated the microchip for longitudinally tracking IL-8 and the CD81+EV secretions from the same single-cells over days, which revealed the presence of "super secretors" within the cell population be more persistent to secrete protein or extracellular vesicle for an extended period. The NOMA platform reported here is simple, robust, and easy to operate for tracking sequential measurements from the same single cells, representing a novel and informative tool to inspire new observations in biomedical research.

Table of Contents Graphic

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org.highwire.dtl.DTLVardef@aa69c7org.highwire.dtl.DTLVardef@6bc79org.highwire.dtl.DTLVardef@3fcbbforg.highwire.dtl.DTLVardef@597f4b_HPS_FORMAT_FIGEXP  M_FIG C_FIG Development and Characterization of PAR-Trackers: New Tools for Detecting Poly(ADP-ribose) In Vitro and In Vivo AO_SCPLOWBSTRACTC_SCPLOWADP-ribosylation (ADPRylation) is a reversible post-translation modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally-occurring protein motifs and domains, including WWEs, PBZs, and macrodomains, act as "readers" for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe and characterize a set of poly(ADP-ribose) (PAR) Trackers (PAR-Ts) - optimized dimerization-dependent or split-protein reassembly PAR sensors in which a naturally occurring PAR binding domain, WWE, was fused to both halves of dimerization-dependent GFP (ddGFP) or split Nano Luciferase (NanoLuc), respectively. We demonstrate that these new tools allow the detection and quantification of PAR levels in extracts, living cells, and living tissues with greater sensitivity, as well as temporal and spatial precision. Importantly, these sensors detect changes in cellular ADPR levels in response to physiological cues (e.g., hormone-dependent induction of adipogenesis without DNA damage), as well as xenograft tumor tissues in living mice. Our results indicate that PAR Trackers have broad utility for detecting ADPR in many different experimental and biological systems. Using Hierarchical Similarity To Examine The Genetics of Behcet's disease Behcets disease (BD) is a multisystem inflammatory disease that affects patients along the historic silk road. Thus far, the pathogenesis of the disease has proved elusive due to the complex genetic interactions and unknown environmental or viral triggering factors of the disease. In this paper, we seek to clarify the genetic factors of the disease while also uncovering other diseases of interest that present with a similar genotype as BD. To do this, we employ a convergent functional genomics approach by leveraging the hierarchical similarity tool available in Geneweaver. Through our analysis, we were able to ascertain 7 BD consensus genes and 16 autoimmune diseases with genetic overlap with BD. The results of our study will inform further research into the pathogenesis of Behcets Disease. Generation of a novel SARS-CoV-2 sub-genomic RNA due to the R203K/G204R variant in nucleocapsid: homologous recombination has potential to change SARS-CoV-2 at both protein and RNA level BackgroundGenetic variations across the SARS-CoV-2 genome may influence transmissibility of the virus and the hosts anti-viral immune response, in turn affecting the frequency of variants over-time. In this study, we examined the adjacent amino acid polymorphisms in the nucleocapsid (R203K/G204R) of SARS-CoV-2 that arose on the background of the spike D614G change and describe how strains harboring these changes became dominant circulating strains globally.

MethodsDeep sequencing data of SARS-CoV-2 from public databases and from clinical samples were analyzed to identify and map genetic variants and sub-genomic RNA transcripts across the genome.

ResultsSequence analysis suggests that the three adjacent nucleotide changes that result in the K203/R204 variant have arisen by homologous recombination from the core sequence (CS) of the leader transcription-regulating sequence (TRS) rather than by stepwise mutation. The resulting sequence changes generate a novel sub-genomic RNA transcript for the C-terminal dimerization domain of nucleocapsid. Deep sequencing data from 981 clinical samples confirmed the presence of the novel TRS-CS-dimerization domain RNA in individuals with the K203/R204 variant. Quantification of sub-genomic RNA indicates that viruses with the K203/R204 variant may also have increased expression of sub-genomic RNA from other open reading frames.

ConclusionsThe finding that homologous recombination from the TRS may have occurred since the introduction of SARS-CoV-2 in humans resulting in both coding changes and novel sub-genomic RNA transcripts suggests this as a mechanism for diversification and adaptation within its new host. Temporal and Spatial Dynamics of Hepatic Stellate Cell Activation in Intrahepatic Cholangiocarcinoma Intrahepatic cholangiocarcinoma (iCCA) is an aggressive type of primary liver cancer characterized by its highly desmoplastic stroma. Compared to the ample knowledge on cancer-associated fibroblast residing within iCCA tumor mass, little is known about the fibrotic response from the tumor-surrounding liver and its role in iCCA development. In this study, we find a significant accumulation of peritumoral myofibroblasts (pMFs) in both patient iCCA and tumors from an iCCA orthotopic allograft mouse model. Using tumor and liver spheroid coculture we show that iCCA-liver interaction induces rapid pMF accumulation at the interface. We find pMFs placed around iCCA spheroids exert a strong suppressive effect on tumor cells growth, in contrast to the pro-proliferative effect of MFs mixed within tumor spheroids. However, prolonged iCCA-pMF interaction elicits tumor cell dissemination in vitro. We find an upregulation of Vcam1 in tumor cells in the early phase of iCCA-pMF interaction both in vitro and in vivo which is downregulated when tumor cells disseminate. Blocking Vcam1 activity in iCCA allograft mouse models slows primary tumor growth but lead to increased tumor metastasis. Our data suggest that pMFs are beyond simple pro- or anti-tumorigenic in iCCA, with the ability to suppress tumor growth but elicit tumor cell dissemination.