Population-level variation of enhancer expression identifies novel disease mechanisms in the human brain Identification of risk variants for neuropsychiatric diseases within enhancers underscores the importance of understanding the population-level variation of enhancers in the human brain. Besides regulating tissue- and cell-type-specific transcription of target genes, enhancers themselves can be transcribed. We expanded the catalog of known human brain transcribed enhancers by an order of magnitude by generating and jointly analyzing large-scale cell-type-specific transcriptome and regulome data. Examination of the transcriptome in 1,382 brain samples in two independent cohorts identified robust expression of transcribed enhancers. We explored gene-enhancer coordination and found that enhancer-linked genes are strongly implicated in neuropsychiatric disease. We identified significant expression quantitative trait loci (eQTL) for 25,958 enhancers which mediate 6.8% of schizophrenia heritability, mostly independent from standard gene eQTL. Inclusion of enhancer eQTL in transcriptome-wide association studies enhanced functional interpretation of disease loci. Overall, our study characterizes the enhancer-gene regulome and genetic mechanisms in the human cortex in both healthy and disease states.
Distinct signatures of loss of consciousness during Focal Impaired Awareness versus Focal to Bilateral Tonic-Clonic seizures Loss of consciousness (LOC) is a hallmark of many epileptic seizures and carries risks of serious injury and sudden death. While cortical sleep-like activities accompany LOC during focal impaired awareness (FIA) seizures, the mechanisms of LOC during focal to bilateral tonic-clonic (FBTC) seizures remain unclear. Quantifying differences in markers of cortical activation and ictal recruitment between FIA and FBTC seizures may also help to understand their different consequences for clinical outcomes and to optimize neuromodulation therapies.

We quantified clinical signs of LOC and intracranial EEG (iEEG) activity during 129 FIA and 50 FBTC from 41 patients. We characterized iEEG changes both in the seizure onset zone (SOZ) and in areas remote from SOZ with a total of 3386 electrodes distributed across brain areas. First, we compared the dynamics of iEEG sleep-like activities: slow-wave activity (SWA; 1-4 Hz) and beta/delta ratio (B/D; a validated marker of cortical activation) during FIA vs. FBTC. Second, we quantified differences between FBTC and FIA for a marker validated to detect ictal cross-frequency coupling: phase-locked high-gamma (PLHG; high gamma phased locked to low frequencies) and a marker of ictal recruitment: the epileptogenicity index (i.e. the number of channels crossing an energy ratio threshold for high vs. low frequency power). Third, we assessed changes in iEEG activity preceding and accompanying behavioral generalization onset and their correlation with electromyogram (EMG) channels. In addition, we analyzed human cortical multi-unit activity recorded with Utah arrays during three FBTC.

Compared to FIA, FBTC seizures were characterized by deeper LOC and by stronger increases in SWA in parieto-occipital cortex. FBTC also displayed more widespread increases in cortical activation (B/D), ictal cross-frequency coupling (PLHG) and ictal recruitment (epileptogenicity index). Even before generalization, FBTC displayed deeper LOC; this early LOC was accompanied by a paradoxical increase in B/D in fronto-parietal cortex. Behavioral generalization coincided with complete loss of responsiveness and a subsequent increase in high-gamma in the whole brain, which was especially synchronous in deep sources and could not be explained by EMG. Similarly, multi-unit activity analysis of FBTC revealed sustained increases in cortical firing rates during and after generalization onset in areas remote from the SOZ.

Unlike during FIA, LOC during FBTC is characterized by a paradoxical increase in cortical activation and neuronal firing. These findings suggest differences in the mechanisms of ictal LOC between FIA and FBTC and may account for the more negative prognostic consequences of FBTC.
The Tabula Sapiens: a multiple organ single cell transcriptomic atlas of humans Molecular characterization of cell types using single cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues and tissue specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, the tissue specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type specific RNA splicing was discovered and analyzed across tissues within an individual. Preprints in motion: tracking changes between preprint posting and journal publication during a pandemic Amidst the COVID-19 pandemic, preprints in the biomedical sciences are being posted and accessed at unprecedented rates, drawing widespread attention from the general public, press and policymakers for the first time. This phenomenon has sharpened longstanding questions about the reliability of information shared prior to journal peer review. Does the information shared in preprints typically withstand the scrutiny of peer review, or are conclusions likely to change in the version of record? We assessed preprints from bioRxiv and medRxiv that had been posted and subsequently published in a journal through 30th April 2020, representing the initial phase of the pandemic response. We utilised a combination of automatic and manual annotations to quantify how an article changed between the preprinted and published version. We found that the total number of figure panels and tables changed little between preprint and published articles. Moreover, the conclusions of 7.2% of non-COVID-19-related and 17.2% of COVID-19-related abstracts undergo a discrete change by the time of publication, but the majority of these changes do not qualitatively change the conclusions of the paper. 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. Reconstitution of minimal motility system based on Spiroplasma swimming by expressing two bacterial actins in synthetic minimal bacterium Motility is one of the most important features of life, but its evolutionary origin remains unknown. In this study, we focused on Spiroplasma, commensal, or parasitic bacteria. They swim by switching the helicity of a ribbon-like cytoskeleton that comprises six proteins, each of which evolved from a nucleosidase and bacterial actin called MreB. We expressed these proteins in a synthetic, non-motile minimal bacterium, JCVI-syn3.0B, whose reduced genome was computer-designed and chemically synthesized. The synthetic bacterium exhibited swimming motility with features characteristic of Spiroplasma swimming. Moreover, some combinations of the two proteins produced a helical cell shape and swimming, suggesting that the swimming originated from the differentiation and coupling of bacterial actins, and we also obtained a minimal system for motility of the synthetic bacterium.

One-Sentence SummaryThe minimal system comprised two bacterial actins that provided cell helicity and swimming to the synthetic minimal bacterium. GPI-anchored FGF directs cytoneme-mediated bidirectional signaling to self-regulate tissue-specific dispersion How signaling proteins generate a multitude of information to organize tissue patterns is critical to understanding morphogenesis. In Drosophila, FGF produced in wing-disc cells regulates the development of the disc-associated air-sac-primordium (ASP). Here, we show that FGF is Glycosylphosphatidylinositol-anchored to the producing cell surface and that this modification both inhibits free FGF secretion and activates target-specific bidirectional FGF-FGFR signaling through cytonemes. FGF-source and recipient ASP cells extend cytonemes that present FGF and FGFR on their surfaces and reciprocally recognize each other over distance by contacting through CAM-like FGF-FGFR binding. Contact-mediated FGF-FGFR binding induces bidirectional signaling, which, in turn, promotes ASP and source cells to polarize cytonemes toward each other and reinforce signaling contacts. Subsequent un-anchoring of FGFR-bound-FGF from the source cell membrane dissociates cytoneme contacts and delivers FGF target-specifically to ASP cytonemes for paracrine functions. Thus, GPI-anchored FGF organizes both source and recipient cells and self-regulates its cytoneme-mediated tissue-specific dispersion and signaling. Venomics and antivenomics of Indian spectacled cobra (Naja naja) from the Western Ghats Venom proteome profiling of Naja naja from the Western Ghats region in Kerala was achieved through SDS-PAGE and RP-HPLC followed by Q-TOF LC-MS/MS analysis, incorporating PEAKS and Novor assisted de novo sequencing methodologies. A total of 115 proteins distributed across 17 different enzymatic and non-enzymatic venom protein families were identified through conventional and 39 peptides through homology-driven proteomics approaches. Fourteen peptides derived through de novo complements the Mascot data indicating the importance of homology-driven approaches in improving protein sequence information. Among the protein families identified, glutathione peroxidase and endonuclease were reported for the first time in the Indian cobra venom. Immunological cross-reactivity assessed using Indian polyvalent antivenoms suggested that VINS showed better EC50 (2.48 g/mL) value than that of PSAV (6.04 g/mL) and Virchow (6.03 g/mL) antivenoms. Western blotting experiments indicated that all the antivenoms elicited poor binding specificities, especially towards low molecular mass proteins. Second-generation antivenomics studies revealed that VINS antivenom was less efficient to detect many low molecular mass proteins such as three-finger toxins and Kunitz-type serine protease Inhibitors. Taken together, the present study enabled a large-scale characterization of the venom proteome of Naja naja from the Western Ghats and emphasized the need for developing more efficient antivenoms.

HighlightsO_LIProteomics of cobra venom resulted in the identification of 115 proteins representing 17 snake venom protein families.
C_LIO_LIDe novo approaches exclusively yielded 39 peptides harbouring multiple amino acid mutations.
C_LIO_LIGlutathione peroxidase and endonuclease were identified for the first time in Indian cobra venom.
C_LIO_LIIndian polyvalent antivenoms showed varying cross-reactivity towards cobra venom.
C_LIO_LIVINS antivenom was less efficient to detect many low molecular mass proteins (< 20 kDa).
C_LI Nontypeable Haemophilus influenzae infection impedes Pseudomonas aeruginosa colonization and persistence in mouse respiratory tract Patients with cystic fibrosis (CF) experience lifelong respiratory infections which are a significant cause of morbidity and mortality. These infections are polymicrobial in nature, and the predominant bacterial species undergo a predictable series of changes as patients age. Young patients have populations dominated by opportunists that are typically found within the microbiome of the human nasopharynx, such as nontypeable Haemophilus influenzae (NTHi); these are eventually supplanted and the population within the CF lung is later dominated by pathogens such as Pseudomonas aeruginosa (Pa). In this study, we investigated how initial colonization with NTHi impacts colonization and persistence of Pa in the respiratory tract.

Analysis of polymicrobial biofilms in vitro by confocal microscopy revealed that NTHi promoted greater levels of Pa biofilm volume and diffusion. However, sequential respiratory infection of mice with NTHi followed by Pa resulted in significantly lower Pa as compared to infection with Pa alone. Coinfected mice also had reduced airway tissue damage and lower levels of inflammatory cytokines as compared with Pa infected mice. Similar results were observed after instillation of heat-inactivated NTHi bacteria or purified NTHi lipooligosaccharide (LOS) endotoxin prior to Pa introduction. Based on these results, we conclude that NTHi significantly reduces susceptibility to subsequent Pa infection, most likely due to priming of host innate immunity rather than a direct competitive interaction between species. These findings have potential significance with regard to therapeutic management of early life infections in patients with CF. Microbial colonization of tannin-rich tropical plants: interplay between degradability, methane production and tannin disappearance in the rumen AO_SCPLOWBSTRACTC_SCPLOWCondensed tannins in plants are found free and attached to protein and fibre but it is not known whether these fractions influence rumen degradation and microbial colonization. This study explored the rumen degradation of tropical tannins-rich plants and the relationship between condensed tannins fractions disappearance and microbial communities colonising plant particles. Leaves from Calliandra calothyrsus, Gliricidia sepium, and Leucaena leucocephala, pods from Acacia nilotica and the leaves of two agricultural by-products: Manihot esculenta and Musa spp. were incubated in situ in the rumen of dairy cows. An in vitro approach was also used to assess the effects of these plants on rumen fermentation parameters. All plants contained more than 100 g/kg of condensed tannins with a large proportion (32 to 61%) bound to proteins. Calliandra calothyrsus had the highest concentration of condensed tannins at 361 g/kg, whereas Acacia nilotica was particularly rich in hydrolysable tannins (350 g/kg). Hydrolysable and free condensed tannins from all plants completely disappeared after 24 h incubation in the rumen. Disappearance of protein-bound condensed tannins was variable with values ranging from 93% for Gliricidia sepium to 21% for Acacia nilotica. In contrast, fibre-bound condensed tannins disappearance averaged ~82% and did not vary between plants. Disappearance of bound fractions of condensed tannins was not associated with degradability of plant fractions. The presence of tannins interfered with the microbial colonisation of plants. Each plant had distinct bacterial and archaeal communities after 3 and 12 h of incubation in the rumen and distinct protozoal communities at 3 h. Adherent communities in tannin-rich plants had a lower relative abundance of fibrolytic microbes, notably Fibrobacter spp. Whereas, archaea diversity was reduced in high tannin-containing Calliandra calothyrsus and Acacia nilotica at 12 h of incubation. Concurrently, in vitro methane production was lower for Calliandra calothyrsus, Acacia nilotica and Leucaena leucocephala although for the latter total volatile fatty acids production was not affected and was similar to control. Here we show that the total amount of hydrolysable and condensed tannins contained in a plant govern the interaction with rumen microbes affecting degradability and fermentation. The effect of protein- and fibre-bound condensed tannins on degradability is less important. 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. Propelling and perturbing appendages together facilitate strenuous ground self-righting Terrestrial animals must self-right when overturned on the ground, but this locomotor task is strenuous. To do so, the discoid cockroach often pushes its wings against the ground to begin a somersault which rarely succeeds. As it repeatedly attempts this, the animal probabilistically rolls to the side to self- right. During winged self-righting, the animal flails its legs vigorously. Here, we studied whether wing opening and leg flailing together facilitate strenuous ground self-righting. Adding mass to increase hind leg flailing kinetic energy increased the animals self-righting probability. We then developed a robot with similar strenuous self-righting behavior and used it as a physical model for systematic experiments. The robots self-righting probability increased with wing opening and leg flailing amplitudes. A potential energy landscape model revealed that, although wing opening did not generate sufficient kinetic energy to overcome the high pitch potential energy barrier to somersault, it reduced the barrier for rolling, facilitating the small kinetic energy from leg flailing to probabilistically overcome it to self-right. The model also revealed that the stereotyped body motion during self-righting emerged from physical interaction of the body and appendages with the ground. Our work demonstrated the usefulness of potential energy landscape for modeling self-righting transitions.

Impact statementAnimals can, and robots should, use different types of appendages together to propel and perturb themselves to self-right when overturned, a strenuous yet crucial locomotor task. Atg11 promotes Kar9-mediated alignment of the mitotic spindle Asymmetric spindle pole body (SPB) inheritance requires a cascade of events that involve kinases, phosphatases and structural scaffold proteins including molecular motors and microtubule-associated proteins. Higher levels of an SPB component Spc72 and the spindle positioning factor Kar9 at the old SPB, which migrates to the daughter cell, ensure asymmetric SPB inheritance. In this study, by analyzing the protein interaction network of autophagy (Atg)-related proteins with those that constitute the chromosome segregation machinery, and growth inhibition kinetics in the presence of a microtubule poison, we identified Atg11 as a potential regulator of chromosome transmission from a cohort of 35 autophagy mutants in Saccharomyces cerevisiae. Cells lacking Atg11 did not show kinetochore defects but displayed a high rate of chromosome loss and delayed anaphase onset. Atg11 positively interacted with Kar9 and Kip2 and negatively with Dyn1 and Kar3 in mediating proper positioning and alignment of the mitotic spindle that was found to be under the surveillance of the spindle positioning checkpoint. Indeed, atg11{Delta} cells displayed an inverted SPB inheritance. Our experiments further revealed that via the Kar9 pathway, Atg11 promotes asymmetric localization of Spc72 and Kar9 on the old SPB. Atg11 physically interacted with Spc72 and transiently localized close to the old SPB during metaphase-to-anaphase progression. Thus, we uncover an autophagy-independent role of Atg11 in spindle alignment. This study emphasizes the importance of screens to identify factors mediating the complex and intricate crosstalk between biological processes fundamental to genomic integrity. Multiple lines of evidence combine to understand tree species coexistence Understanding drivers of species coexistence is a central challenge in ecology (1, 2). Coexistence cannot be observed directly, and while species co-occurrence in time and space is necessary for coexistence, it is not sufficient to prove coexistence (3, 4). Species exclusion from a region is potentially observable, but can take decades to occur, and still might just occur stochastically (5, 6). Thus, ecologists generally use theory to identify indirect observations that are indicative of mechanisms driving coexistence or exclusion (7-9). Various methods have been developed to indirectly infer coexistence, each of which requires different data, none of which are usually conclusive on their own, and are not typically combined (e.g. (9-12)). Here, we combine three different approaches which are normally used in isolation to build a case about a lack of coexistence of multiple hardwood species. Importantly, though each method has its flaws, we find that all three point to the same conclusion which strengthens the case for a lack of coexistence. First, in an experimental planting of three mature tree species we found no relationship between productivity and species diversity, which has been hypothesized to occur due to a lack of niche differences among species. Second, we used modern coexistence theory to calculate niche and fitness differences for each pair of species, which confirmed the high niche overlap among species and competitive dominance of one species. Third, we used the United States Department of Agriculture Forest Inventory and Analysis data to examine co-occurrence patterns of our species across thousands of natural forest stands and found that, these three species were distributed randomly throughout the USA, and do not significantly co-occur as would be expected if coexistence occurred naturally. Given that these independent methods agree; we take this as strong evidence about a lack of coexistence. Soluble ECM promotes organotypic formation in lung alveolar model Micropatterned suspension culture creates consistently sized and shaped cell aggregates but has not produced organotypic structures from stable cells, thus restricting its use in accurate disease modeling. Here, we show that organotypic structure is achieved in hybrid suspension culture via supplementation of soluble extracellular matrix (ECM). We created a viable lung organoid from epithelial, endothelial, and fibroblast human stable cell lines in suspension culture. We demonstrate the importance of soluble ECM in organotypic patterning with the emergence of lumen-like structures with airspace showing feasible gas exchange units, formation of branching, perfusable vasculature, and long-term 70-day maintenance of lumen structure. Our results show a dependent relationship between enhanced fibronectin fibril assembly and the incorporation of ECM in the organoid. We successfully applied this technology in modeling lung fibrosis via bleomycin induction and test a potential antifibrotic drug in vitro while maintaining fundamental cell-cell interactions in lung tissue. Our human fluorescent lung organoid (hFLO) model represents features of pulmonary fibrosis which were ameliorated by fasudil treatment. We also demonstrate a 3D culture method with potential of creating organoids from mature cells, thus opening avenues for disease modeling and regenerative medicine, enhancing understanding of lung cell biology in health and lung disease. Spatial phylogenetics of Japanese ferns: Patterns, processes, and implications for conservation PremiseBiodiversity is often only measured with species richness. However, this ignores evolutionary history and is not sufficient for making conservation decisions. Here, we characterize multiple facets and drivers of biodiversity to understand how these relate to bioregions and conservation status in the ferns of Japan.

MethodsWe compiled a community dataset of 1,239 20 km x 20 km grid-cells including 672 taxa based on > 300,000 specimen records. We combined this with a phylogeny and functional traits to analyze taxonomic, phylogenetic, and functional diversity, and modeled biodiversity metrics in response to environmental factors and reproductive mode. Hierarchical clustering was used to delimit bioregions. Conservation status and threats were assessed by comparing the overlap of significantly diverse grid-cells with conservation zones and range maps of native Japanese deer.

ResultsTaxonomic richness was highest at mid-latitudes. Phylogenetic and functional diversity and phylogenetic endemism were highest in small southern islands. Relative phylogenetic and functional diversity were high at high and low latitudes, and low at mid-latitudes. Grid-cells were grouped into three (phylogenetic) or four (taxonomic) major bioregions. Temperature and apomixis were identified as drivers of biodiversity patterns. Conservation status was generally high for grid-cells with significantly high biodiversity, but the threat due to herbivory by deer was greater for taxonomic richness than other metrics.

ConclusionsOur integrative approach reveals previously undetected patterns and drivers of biodiversity in the ferns of Japan. Future conservation efforts should recognize that threats can vary by biodiversity metric and consider multiple multiple metrics when establishing conservation priorities. Comparison study of sixteen differential abundance testing methods using two large Parkinson disease gut microbiome datasets BackgroundWhen studying the relationship between the microbiome and a disease, a common question asked is what individual microbes are differentially abundant between a disease and healthy state. Numerous differential abundance (DA) testing methods exist and range from standard statistical tests to methods specifically designed for microbiome data. Comparison studies of DA testing methods have been performed, but none were performed on microbiome datasets collected for the study of real, complex disease. Due to this, we performed DA testing of microbial genera using 16 DA methods in two large, uniformly collected gut microbiome datasets on Parkinson disease (PD), and compared their results.

ResultsPairwise concordances between methods ranged from 46%-99% similarity. Average pairwise concordance per dataset was 76%, and dropped to 62% when taking replication of signals across datasets into account. Certain methods consistently resulted in above average concordances (e.g. Kruskal-Wallis, ALDEx2, GLM with centered-log-ratio transform), while others consistently resulted in lower than average concordances (e.g. edgeR, fitZIG). Overall, [~]80% of genera tested were detected as differentially abundant by at least one method in each dataset. Requiring associations to replicate across datasets reduced significant signals by almost half. Further requirement of signals to be replicated by the majority of methods ([&ge;]8) yielded 19 associations. Only one genus (Agathobacter) was replicated by all methods. Use of hierarchical clustering revealed three groups of DA signatures that were (1) replicated by the majority of methods and included genera previously associated with PD, (2) replicated by few or no methods, and (3) replicated by a subset of methods and included rarer genera, all enriched in PD.

ConclusionsDifferential abundance tests yielded varied results. Using one method on one dataset may find true associations, but may also detect non-reproducible signals, adding to inconsistency in the literature. To help lower false positives, one might analyze data with two or more DA methods to gauge concordance, and use a built-in replication dataset to show reproducibility. This study corroborated previously reported microorganism associations in PD, and revealed a potential new group of microorganisms whose abundance is significantly elevated in PD, and might be worth pursuing in future investigations. Fractal polymer with loops recapitulates key features of chromosome organization Chromosomes are exceedingly long polymers compacted in a cell nucleus. While it has long been suggested that mammalian chromosomes during interphase are folded into loops, experimental detection of such loops has remained a daunting task. The most comprehensive experimental information about chromosome spatial organization is provided by Hi-C experiments that measure the frequency of contacts between all chromosomal positions. The lack of a tractable physical model of a polymer folded into loops limits our ability to interpret experimental data. It thus remains unknown how to obtain accurate and quantitative information about the nature of chromosomal looping from Hi-C. Here, we introduce a model of a polymer with random loops, solve it analytically and extend it by simulations for real chains. Remarkably, our model faithfully reproduces complex shapes of experimental contact probability curves universal among mammalian cells. Furthermore, our model allows one to estimate sizes of randomly positioned chromosomal loops from experimental data. We also show that excluded volume in real chains can induce osmotic and topological repulsion between loops, stiffening interphase chromosomes. Thus, our new framework allows interpretation of experimental data and suggests that interphase chromosomes are crumpled polymers further folded into a sequence of randomly positioned loops. Tuft cell-produced cysteinyl leukotrienes and IL-25 synergistically initiate lung type 2 inflammation Aeroallergen sensing by airway epithelial cells can trigger pathogenic immune responses leading to chronic type 2 inflammation, the hallmark of airway diseases such as asthma. Airway tuft cells are specialized chemosensory epithelial cells and the dominant source of the epithelial cytokine IL-25 in the trachea and of cysteinyl leukotrienes (CysLTs) in the naive murine nasal mucosa. The interaction of IL-25 and CysLTs and the contribution of tuft cell-derived CysLTs to the development of allergen-triggered inflammation in the airways has not been clarified. Here we show that inhalation of LTC4 in combination with a subthreshold dose of IL-25 leads to dramatic synergistic induction of type 2 inflammation throughout the lungs, causing rapid eosinophilia, dendritic cell (DC) and inflammatory type 2 innate lymphoid cell (ILC2) expansion, and goblet cell metaplasia. While lung eosinophilia is dominantly mediated through the classical CysLT receptor CysLT1R, type 2 cytokines and activation of innate immune cells require signaling through both CysLT1R and CysLT2R. Tuft cell-specific deletion of the terminal enzyme requisite for CysLT production, Ltc4s, was sufficient to reduce both the innate immune response in the lung - eosinophilia, KLRG1+ ILC2 activation and DC recruitment - and the systemic immune response in the draining lymph nodes after inhalation of the mold aeroallergen Alternaria. Our findings identify surprisingly potent synergy of CysLTs and IL-25 downstream of aeroallergen-trigged activation of airway tuft cells leading to a highly polarized type 2 immune response and further implicate airway tuft cells as powerful modulators of type 2 immunity in the lungs.

One Sentence SummaryTuft cells produce two highly synergistic mediators: LTC4 and IL-25 to cooperatively induce allergen-driven airway inflammation. Optogenetic actuator/ERK biosensor circuits identify MAPK network nodes that shape ERK dynamics Combining single-cell measurements of ERK activity dynamics with perturbations provides insights into the MAPK network topology. We built circuits consisting of an optogenetic actuator to activate MAPK signaling and an ERK biosensor to measure single-cell ERK dynamics. This allowed us to conduct RNAi screens to investigate the role of 50 MAPK proteins in ERK dynamics. We found that the MAPK network is robust against most node perturbations. We observed that the ERK-RAF and the ERK-RSK2-SOS negative feedbacks operate simultaneously to regulate ERK dynamics. Bypassing the RSK2-mediated feedback, either by direct optogenetic activation of RAS, or by RSK2 perturbation, sensitized ERK dynamics to further perturbations. Similarly, targeting this feedback in a human ErbB2-dependent oncogenic signaling model increased the efficiency of a MEK inhibitor. The RSK2-mediated feedback is thus important for the ability of the MAPK network to produce consistent ERK outputs and its perturbation can enhance the efficiency of MAPK inhibitors. A Novel Proposal for an Index for Regional Cerebral Perfusion Pressure - A Theoretical Approach Using Fluid Dynamics Cerebral blood flow (CBF) / cerebral blood volume (CBV) ratio derived by [15O] H2O/ CO2 and CO positron emission tomography (PET) examination has been used empirically as an index for cerebral perfusion pressure (CPP). However, this equation lacks a theoretical basis and furthermore, has not been confirmed to be proportionate to CPP, as direct measurement of local CPP is not practical.

We have developed a new index for CPP using the Poiseuille equation based on a simple model. Our model suggests that CBF/CBV2 is proportionate to CPP and that it is mathematically a more accurate index than CBF/CBV. SARS-CoV-2 spike-glycoprotein processing at S1/S2 and S2' and shedding of the ACE2 viral receptor: roles of Furin and TMPRSS2 and implications for viral infectivity and cell-to-cell fusion The spike (S)-protein of SARS-CoV-2 binds ACE2 and requires proteolytic "priming" at PRRAR685{downarrow} into S1 and S2 (cleavage at S1/S2), and "fusion-activation" at a S2 site for viral entry. In vitro, Furin cleaved peptides mimicking the S1/S2 cleavage site more efficiently than at the putative S2, whereas TMPRSS2 inefficiently cleaved both sites. In HeLa cells Furin-like enzymes mainly cleaved at S1/S2 during intracellular protein trafficking, and S2 processing by Furin at KPSKR815{downarrow} was strongly enhanced by ACE2, but not for the optimized S2 KRRKR815{downarrow} mutant (S2), whereas individual/double KR815AA mutants were retained in the endoplasmic reticulum. Pharmacological Furin-inhibitors (Boston Pharmaceuticals, BOS-inhibitors) effectively blocked endogenous S-protein processing in HeLa cells. Furthermore, we show using pseudotyped viruses that while entry by a "pH-dependent" endocytosis pathway in HEK293 cells did not require Furin processing at S1/S2, a "pH-independent" viral entry in lung-derived Calu-3 cells was sensitive to inhibitors of Furin (BOS) and TMPRSS2 (Camostat). Consistently, these inhibitors potently reduce infectious viral titer and cytopathic effects, an outcome enhanced when both compounds were combined. Quantitative analyses of cell-to-cell fusion and spike processing revealed the key importance of the Furin sites for syncytia formation. Our assays showed that TMPRSS2 enhances fusion and proteolysis at S2 in the absence of cleavage at S1/S2, an effect that is linked to ACE2 shedding by TMPRSS2. Overall, our results indicate that Furin and TMPRSS2 play synergistic roles in generating fusion-competent S-protein, and in promoting viral entry, supporting the combination of Furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2.

IMPORTANCESARS-CoV-2 is the etiological agent of COVID-19 that resulted in >5 million deaths. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S-protein is cleaved at two sites: S1/S2 and S2. Cleavage at S1/S2, induces a conformational change favoring the recognition of ACE2. The S2 cleavage is critical for cell-to-cell fusion and virus entry into host cells. Our study contributes to a better understanding of the dynamics of interaction between Furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a non-toxic Furin inhibitor with a TMPRSS2 inhibitor could significantly reduce viral entry in lung cells, as evidenced by an average synergistic [~]95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses. Water levels primarily drive variation in photosynthesis and nutrient use of scrub Red Mangroves in the southeastern Florida Everglades We investigated how mangrove-island micro-elevation (i.e., habitat: center vs. edge) affects tree physiology in a scrub mangrove forest of the southeastern Everglades. We measured leaf gas exchange rates of scrub Rhizophora mangle L. trees monthly during 2019, hypothesizing that CO2 assimilation (Anet) and stomatal conductance (gsw) would decline with increasing water levels and salinity, expecting more-considerable differences at mangrove-island edges than centers, where physiological stress is greatest. Water levels varied between 0 and 60 cm from the soil surface, rising during the wet season (May-October) relative to the dry season (November-April). Porewater salinity ranged from 15 to 30 ppt, being higher at mangrove-island edges than centers. Anet maximized at 15.1 {micro}mol m-2 s-1, and gsw was typically <0.2 mol m-2 s-1, both of which were greater in the dry than the wet season and greater at island centers than edges, with seasonal variability being roughly equal to variation between habitats. After accounting for season and habitat, water level positively affected Anet in both seasons but did not affect gsw. Our findings suggest that inundation stress (i.e., water level) is the primary driver of variation in leaf gas exchange rates of scrub mangroves in the Florida Everglades, while also constraining Anet more than gsw. The interaction between inundation stress due to permanent flooding and habitat varies with season as physiological stress is alleviated at higher-elevation mangrove-island center habitats during the dry season. Freshwater inflows during the wet season, increase water levels and inundation stress at higher-elevation mangrove-island centers, but also potentially alleviate salt and sulfide stress in soils. Thus, habitat heterogeneity leads to differences in nutrient and water acquisition and use between trees growing in island centers versus edges, creating distinct physiological controls on photosynthesis, which likely affect carbon flux dynamics of scrub mangroves in the Everglades. Chromatin-contact atlas reveals disorder-mediated protein interactions and moonlighting chromatin-associated RBPs RNA-binding proteins (RBPs) play diverse roles in regulating co-transcriptional RNA-processing and chromatin functions, but our knowledge of the repertoire of chromatin-associated RBPs (caRBPs) and their interactions with chromatin remains limited. Here, we developed SPACE (Silica Particle Assisted Chromatin Enrichment) to isolate global and regional chromatin components with high specificity and sensitivity, and SPACEmap to identify the chromatin-contact regions in proteins. Applied to mouse embryonic stem cells, SPACE identified 1,459 chromatin-associated proteins, [~]48% of which are annotated as RBPs, indicating their dual roles in chromatin and RNA-binding. Additionally, SPACEmap stringently verified chromatin-binding of 404 RBPs and identified their chromatin-contact regions. Notably, SPACEmap showed that about half of the caRBPs bind to chromatin by intrinsically disordered regions (IDRs). Studying SPACE and total proteome dynamics from mES cells grown in 2iL and serum medium indicates significant correlation (R = 0.62). One of the most dynamic caRBPs is Dazl, which we find co-localized with PRC2 at transcription start sites of genes that are distinct from Dazl mRNA binding. Dazl and other PRC2-colocalised caRBPs are rich in intrinsically disordered regions (IDRs), which could contribute to the formation and regulation of phase-separated PRC condensates. Together, our approach provides an unprecedented insight into IDR-mediated interactions and caRBPs with moonlighting functions in native chromatin. NPEPPS regulates intracellular import and sensitivity to cisplatin by interaction with volume regulated anion channels Despite routine use of platinum-based chemotherapeutics in cancer treatment, there remains a need to improve efficacy and patient selection. Multi-omic assessment of human bladder cancer cell lines and their cisplatin resistant derivatives and whole-genome CRISPR screens identified NPEPPS, the puromycin-sensitive aminopeptidase as a novel driver of cisplatin resistance. NPEPPS depletion increased cisplatin import and sensitization of resistant cells in vitro and in vivo. Pharmacologic inhibition of NPEPPS in cells and chemoresistant, patient-derived tumor organoids improved response to cisplatin. NPEPPS was found in complex with volume regulated anion channel (VRAC) subunits LRRC8A and LRRC8D, whose loss is known to enhance resistance to cisplatin. Depletion of LRRC8A the only obligate subunit for normal VRAC function abrogated the effect of NPEPPS-mediated cisplatin import. Our findings describe the first mechanism by which VRACs can be targeted for therapeutic benefit.

HIGHLIGHTSO_LIMulti-omic screening found NPEPPS is a driver of cisplatin resistance in cancer
C_LIO_LINPEPPS is in protein complex with LRRC8A and D, volume regulated anion channel (VRAC) subunits
C_LIO_LILRRC8A and D loss increases cisplatin resistance and reduce its intracellular levels
C_LIO_LINPEPPS regulates cisplatin import and sensitivity through VRACs
C_LIO_LINPEPPS function is inhibited by small molecules offering potential for clinical translation
C_LI Glucose inhibits haemostasis and accelerates diet-induced hyperlipidaemia in zebrafish larvae Hyperglycaemia damages the microvasculature in part through the reduced recruitment of immune cells and interference with platelet signalling, leading to poor wound healing and accelerated lipid deposition in mammals. We investigated the utility of zebrafish larvae to model the effect of glucose on neutrophil and macrophage recruitment to a tail wound, wound-induced haemostasis, and chicken egg yolk feed challenge-induced hyperlipidaemia by supplementing larvae with exogenous glucose by immersion or injection. Neither method of glucose supplementation affected the recruitment of neutrophils and macrophages following tail transection. Glucose injection reduced thrombocyte retention and fibrin plug formation while only thrombocyte retention was reduced by glucose immersion following tail transection. We observed accelerated lipid accumulation in glucose-injected larvae challenged with high fat chicken egg yolk feeding. Our study identifies conserved and divergent effects of high glucose on inflammation, haemostasis, and hyperlipidaemia in zebrafish larvae compared to mammals.