CRCM5484: A BET- BDII Selective Compound With Differential Anti-Leukemic Drug Modulation Differentially screening the Fr-PPIChem chemical library on the BET BRD4-BDII versus -BDI bromodomains led to the discovery of a BDII selective tetrahydropyridothienopyrimidinone (THPTP)-based compound. Structure-activity relationship (SAR) and hit-to-lead approaches allowed us to develop CRCM5484, a potent inhibitor of BET proteins with a preferential and 475-fold selectivity for the second bromodomain of the BRD3 protein (BRD3-BDII) over its first bromodomain (BRD3-BDI). Its very low activity was demonstrated in various cell-based assays, corresponding with recent data describing other selective BDII compounds. However, screening on a drug sensitivity and resistance-profiling platform revealed its ability to modulate the anti-leukemic activity in combination with various FDA-approved and/or in-development drugs in a cell- and context-dependent differential manner. Altogether, the results confirm the originality of the THPTP molecular mode of action in the BD cavity and its potential as starting scaffold for the development of potent and selective bromodomain inhibitors.
Redefining colorectal cancer classification and clinical stratification through a single-cell atlas Colorectal cancer (CRC), a disease of high incidence and mortality, has had few treatment advances owing to a large degree of inter- and intratumoral heterogeneity. Attempts to classify subtypes of colorectal cancer to develop treatment strategies has been attempted by Consensus Molecular Subtypes (CMS) classification. However, the cellular etiology of CMS classification is incompletely understood and controversial. Here, we generated and analyzed a single-cell transcriptome atlas of 49,859 CRC cells from 16 patients, validated with an additional 31,383 cells from an independent CRC patient cohort. We describe subclonal transcriptomic heterogeneity of CRC tumor epithelial cells, as well as discrete stromal populations of cancer-associated fibroblasts (CAFs). Within CRC CAFs, we identify the transcriptional signature of specific subtypes (CAF-S1 and CAF-S4) in more than 1,500 CRC patients using bulk transcriptomic data that significantly stratifies overall survival in multiple independent cohorts. We also uncovered two CAF-S1 subpopulations, ecm-myCAF and TGF{beta}-myCAF, known to be associated with primary resistance to immunotherapies. We demonstrate that scRNA analysis of malignant, stromal, and immune cells exhibit a more complex picture than portrayed by bulk transcriptomic-based Consensus Molecular Subtypes (CMS) classification. By demonstrating an abundant degree of heterogeneity amongst these cell types, our work shows that CRC is best represented in a transcriptomic continuum crossing traditional classification systems boundaries. Overall, this CRC cell map provides a framework to re-evaluate CRC tumor biology with implications for clinical trial design and therapeutic development.
Feed efficiency of lactating Holstein cows was not as repeatable across diets as within diet over subsequent lactation stages BackgroundImproving feed efficiency has become a common target for dairy farmers to meet the requirement of producing more milk with fewer resources. To improve feed efficiency, a prerequisite is to ensure that the cows identified as most or least efficient will remain as such, independently of diet composition. Therefore, the current research analysed the ability of lactating dairy cows to maintain their feed efficiency while changing the energy density of the diet by changing its concentration in starch and fibre. A total of 60 lactating Holstein cows, including 33 primiparous cows, were first fed a high starch diet-low fibre (diet S+F-), then switched over to a low starch diet-high fibre (diet S-F+). To know if diet affect feed efficiency, we compared the ability of feed efficiency to be maintained within a diet over subsequent lactation stages, known as repeatability of feed efficiency, with its ability to be maintained across diets, known as reproducibility of feed efficiency. To do so we used two indicators: the estimation of the error of repeatability/reproducibility, which is commonly used in metrology, and the coefficient of correlation of concordance (CCC), which is used in biology. The effect of diet change could also lead to a change in cows sorting behaviour which could potentially affect feed efficiency if for example the most efficient cows select more concentrate than the least efficient. We therefore analysed the relationship between the differences in individual feed refusals composition and the differences in feed efficiency. To do so, the composition of each feed refusal was described with its near infra-red (NIR) spectroscopy and was performed on each individual feed ingredient, diet and refusals and used as composition variable. The variability of the NIR spectra of the refusals was described with its principal components thanks to a principal component analysis (PCA). The Pearson correlation was estimated to check the relationship between feed efficiency and refusals composition, i.e. sorting behaviour.

ResultsThe error of reproducibility of feed efficiency across diets was 2.95 MJ/d. This error was significantly larger than the errors of repeatability estimated within diet, which were 2.01 MJ/d within diet S-F+ and 2.40 MJ/d within diet S+F-. The CCC was 0.64 between feed efficiency estimated within diet S+F- and feed efficiency estimated within diet S-F+. This CCC was smaller than the one observed for feed efficiency estimated within diet between two subsequent lactation stages (CCC = 0.72 within diet S+F- and 0.85 within diet S-F+). Feed efficiency was poorly correlated to the first two principal components, which explained 90% of the total variability of the NIR spectra of the individual refusals. This suggests that feed sorting behaviour did not explain differences in feed efficiency.

ConclusionsFeed efficiency was significantly less reproducible across diets than repeatable within the same diet over subsequent lactation stages, but cows ranking for feed efficiency was not significantly affected by diet change. This loss in repeatability across diets could be due to a more pronounced feed sorting subsequent to the change in diet composition. However, the differences in sorting behaviour between cows were not associated to feed efficiency differences in this trial neither with the S+F- diet nor with the S-F+ diet. Those results have to be confirmed on diets having different forage to concentrate ratios to ensure that the least and most efficient cows will not change. The collapse of genetic incompatibilities in hybridizing populations Diverging species are often genetically incompatible upon hybridization. Such incompatibilities are considered important in keeping the integrity of species from the disruption of hybrids. However, recent empirical work has shown that not all incompatibilities are gene-flow-proof, and they can collapse due to continuing hybridization. Counterintuitively, many studies found that incompatible alleles are already segregating within species, whereas they should go extinct quickly in a randomly mating population. Due to the complexity of multilocus epistasis, few general principles explain behaviors of incompatibilities under gene flow both within and between species. In the current work, we argue that the redundancy of genetic mechanisms can robustly determine the dynamics of intrinsic incompatibilities under gene flow. While higher genetic redundancy decreases the stability of incompatibilities during hybridization, it also increases the tolerance of incompatibility polymorphism within each species. We treat two general classes of incompatibilities. In the redundant class, similar to the classical Dobzhansky-Muller system, the collapse is continuous and eventually approaches quasi-neutral polymorphism between broadly-sympatric species, often as a result of isolation-by-distance. In the non-redundant class, analogous to the shifting-balance process, incompatibilities collapse abruptly with spatial traveling waves. We obtained simulated and analytical results for several incompatibility models to demonstrate the differences between the two classes. As both redundant and non-redundant genetic mechanisms of incompatibilities are common, the proposed conceptual framework may help understand the abundance of incompatibilities in natural populations. Citation needed?Wikipedia bibliometrics during the first waveof the COVID-19 pandemic. BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. The ribose methylation enzyme FTSJ1 has a conserved role in neuron morphology and learning performance FTSJ1 is a conserved human 2-O-methyltransferase (Nm-MTase) that modifies several transfer RNAs (tRNAs) at position 32 and the wobble position 34 in the AntiCodon Loop (ACL). Its loss of function has been linked to Non-Syndromic X-Linked Intellectual Disability (NSXLID), and more recently to cancers. However, the molecular mechanisms underlying these pathologies are currently unclear. Here we report a novel FTSJ1 pathogenic variant from a NSXLID patient. Using blood cells derived from this patient and other affected individuals carrying FTSJ1 mutations, we performed an unbiased and comprehensive RiboMethSeq analysis to map the ribose methylation (Nm) on all tRNAs and identify novel targets. In addition, we performed a transcriptome analysis in these cells and found that several genes previously associated with intellectual disability and cancers were deregulated. We also found changes in the miRNA population that suggest potential cross-regulation of some miRNAs with these key mRNA targets. Finally, we show that differentiation of FTSJ1-depleted human neuronal progenitor cells (NPC) into neurons displays long and thin spine neurites compared to control cells. These defects are also observed in Drosophila and are associated with long term memory deficit in this organism. Altogether, our study adds insight into FTSJ1 pathologies in human by the identification of novel FTSJ1 targets and the defect in neuron morphology. Simulation of foraging behavior using a decision-making agent with Bayesian and inverse Bayesian inference: Temporal correlations and power laws in displacement patterns It has been stated that in human migratory behavior, the step length series may have temporal correlation and that there is some relationship between this time dependency and the fact that the frequency distribution of step length follows a power-law distribution. Furthermore, the frequency of occurrence of the step length in some large marine organisms has been found to switch between power-law and exponential distributions, depending on the difficulty of prey acquisition. However, to date it has not been clarified how the aforementioned three phenomena arise: the positive correlation created in the step length series, the relation between the positive correlation of the step length series and the form of an individuals step length distribution, and the switching between power-law and exponential distributions depending on the abundance of prey. This study simulated foraging behavior using the Bayesian decision-making agent simultaneously performing both knowledge learning and knowledge-based inference to analyze how the aforementioned three phenomena arise. In the agent with learning and inference, past experiences were stored as hypotheses (knowledge) and they were used in current foraging behavior; at the same time, the hypothesis continued to be updated based on new experiences. The simulation results show that the agent with both learning and inference has a mechanism that simultaneously causes all the phenomena. TRACT revisited: an algebraic solution for determining overall rotational correlation times from cross-correlated relaxation rates Accurate rotational correlation times ({tau}c) are critical for quantitative analysis of fast timescale NMR dynamics. As molecular weights increase, the classic derivation of{tau} c using transverse and longitudinal relaxation rates becomes increasingly unsuitable due to the non-trivial contribution of remote dipole-dipole interactions to longitudinal relaxation. Derivations using cross-correlated relaxation experiments, such as TRACT, overcome these limitations but are erroneously calculated in 65% of the citing literature. Herein, we developed an algebraic solutions to the Goldman relationship that facilitate rapid, point-by-point calculations for straightforward identification of appropriate spectral regions where global tumbling is likely to be dominant. The rigid-body approximation of the Goldman relationship has been previously shown to underestimate TRACT-based rotational correlation time estimates. This motivated us to develop a second algebraic solution that employs a simplified model-free spectral density function including an order parameter term that could, in principle, be set to an average backbone S2 {approx} 0.9 to further improve the accuracy of{tau} c estimation. These solutions enabled us to explore the boundaries of the Goldman relationship as a function of the H-N internuclear distance (r), difference of the two principal components of the axially-symmetric 15N CSA tensor ({Delta}{delta}N), and angle of the CSA tensor relative to the N-H bond vector ({theta}). We hope our algebraic solutions and analytical strategies will increase the accuracy and application of the TRACT experiment. TFAP2 paralogs facilitate chromatin access for MITF at pigmentation genes but inhibit expression of cell-cell adhesion genes independently of MITF In developing melanocytes and in melanoma cells, multiple paralogs of the Activating-enhancer-binding Protein 2 family of transcription factors (TFAP2) contribute to expression of genes encoding pigmentation regulators, but their interaction with Microphthalmia transcription factor (MITF), a master regulator of these cells, is unclear. Supporting the model that Tfap2 facilitates MITFs ability to activate expression of pigmentation genes, single-cell seq analysis of zebrafish embryos revealed that pigmentation genes are only expressed in the subset of mitfa- expressing cells that also express Tfap2 paralogs. To test this model in SK-MEL-28 melanoma cells we deleted the two TFAP2 paralogs with highest expression, TFAP2A and TFAP2C, creating TFAP2 knockout (TFAP2-KO) cells. We then assessed gene expression, chromatin accessibility, binding of TFAP2A and of MITF, and the chromatin marks H3K27Ac and H3K27Me3 which are characteristic of active enhancers and silenced chromatin, respectively. Integrated analyses of these datasets indicate TFAP2 paralogs directly activate enhancers near genes enriched for roles in pigmentation and proliferation, and directly repress enhancers near genes enriched for roles in cell adhesion. Consistently, compared to WT cells, TFAP2-KO cells proliferate less and adhere to one another more. TFAP2 paralogs and MITF co-operatively activate a subset of enhancers, with the latter necessary for MITF binding and chromatin accessibility. By contrast, TFAP2 paralogs and MITF do not appear to co-operatively inhibit enhancers. These studies reveal a mechanism by which TFAP2 profoundly influences the set of genes activated by MITF, and thereby the phenotype of pigment cells and melanoma cells. Computational and experimental studies of the breathing motion of a protein loop: implications in PfAMA1-PfRON2 late-stage binding event Protein-protein interactions are important targets for various drug discovery campaigns. One such promising and therapeutically pertinent protein-protein complex is PfAMA1-PfRON2 involved in malarial parasite invasion into human red blood cells. A thorough understanding of the interactions between these macromolecular binding partners is crucial for designing better therapeutics against this age-old disease. Although crystal structures of several PfAMA1-PfRON2 complexes are available, the mechanism of how domain II loop associates with PfRON2 is not clear. The current work investigates how the domain II loop of PfAMA1 exerts its effect on the alpha helix of the PfRON2, thus influencing the overall kinetics of this intricate recognition phenomenon. To this end, we have computationally simulated the dynamics and free energetics of domain II loop closing processes and identified a set of key amino acid residues of PfRON2 helix which are essential for binding. The subsequent evaluation of the binding affinity of Ala-substituted PfRON2 peptide ligands by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) validates the relative importance of the residues in context. Together, the combination of computational and experimental investigation reveals that the domain II loop of PfAMA1 is in fact responsible for arresting the PfRON2 molecule from egress, K2027 and D2028 of PfRON2 being the determinant residues for the capturing event. Our study provides a comprehensive understanding of the molecular recognition event between PfAMA1 and PfRON2, specifically in the post binding stage, which could potentially open up new avenues to drug discovery against malaria.

TOC GRAPHIC

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/463826v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1c26022org.highwire.dtl.DTLVardef@356357org.highwire.dtl.DTLVardef@10ceb04org.highwire.dtl.DTLVardef@1195633_HPS_FORMAT_FIGEXP  M_FIG C_FIG Platelet transcriptome yields progressive markers in chronic myeloproliferative neoplasms and identifies putative targets of therapy Predicting disease progression remains a particularly challenging endeavor in chronic degenerative disorders and cancer, thus limiting early detection, risk stratification, and preventive interventions. Here, profiling the spectrum of chronic myeloproliferative neoplasms (MPNs) as a model, we identify the blood platelet transcriptome as a proxy for highly sensitive progression biomarkers that also enables prediction of advanced disease via machine learning algorithms. Using RNA sequencing (RNA-seq), we derive disease-relevant gene expression in purified platelets from 120 peripheral blood samples constituting two time-separated cohorts of patients diagnosed with one of three MPN subtypes at sample acquisition - essential thrombocythemia, ET (n=24), polycythemia vera, PV (n=33), and primary or post ET/PV secondary myelofibrosis, MF (n=42), and healthy donors (n=21). The MPN platelet transcriptome reveals an incremental molecular reprogramming that is independent of patient driver mutation status or therapy and discriminates each clinical phenotype. Leveraging this dataset that shows a characteristic progressive expression gradient across MPN, we develop a machine learning model (Lasso-penalized regression) and predict advanced subtype MF at high accuracy and under two conditions of external validation: i) temporal: our two Stanford cohorts, AUC-ROC of 0.96; and ii) geographical: independently published data of an additional n=25 MF and n=46 healthy donors, AUC-ROC of 0.97). Lasso-derived signatures offer a robust core set of < 5 MPN transcriptome markers that are progressive in expression. Mechanistic insights from our data highlight impaired protein homeostasis as a prominent driver of MPN evolution, with persistent integrated stress response. We also identify JAK inhibitor-specific signatures and other interferon, proliferation, and proteostasis-associated markers as putative targets for MPN-directed therapy. Our platelet transcriptome snapshot of chronic MPNs demonstrates a proof of principle for disease risk stratification and progression beyond genetic data alone, with potential utility in other progressive disorders.

HighlightsLeveraging two independent and mutually validating MPN patient cohorts, we identify progressive transcriptomic markers that also enable externally validated prediction in MPNs.

Our platelet RNA-Seq data identifies impaired protein homeostasis as prominent in MPN progression and offers putative targets of therapy.

VISUAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=135 HEIGHT=200 SRC="FIGDIR/small/435190v3_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@72dc16org.highwire.dtl.DTLVardef@cf5096org.highwire.dtl.DTLVardef@b3d460org.highwire.dtl.DTLVardef@3c0ddc_HPS_FORMAT_FIGEXP  M_FIG C_FIG PIN-FORMED1 polarity in the shoot is insensitive to the polarity of neighbouring cells In plants, epidermal cells are planar-polarized along an axis marked by the asymmetric localization patterns of several proteins including PIN-FORMED1 (PIN1), which facilitates the directional efflux of the plant hormone auxin to pattern phyllotaxis (Heisler et al., 2010; Mansfield et al., 2018; Reinhardt et al., 2003). While PIN1 polarity is known to be regulated non-cell autonomously via the MONOPTEROS (MP) transcription factor, how this occurs has not been determined (Bhatia et al., 2016). Here we use mosaic expression of the serine threonine kinase PINOID (PID) to test whether PIN1 polarizes according to the polarity of neighbouring cells. Our findings reveal that PIN1 is insensitive to the polarity of PIN1 in neighbouring cells arguing against auxin flux or extracellular auxin concentrations acting as a polarity cue, in contrast to previous model proposals (Abley et al., 2016; Stoma et al., 2008). Use of lignocellulosic residue from second-generation ethanol production to enhance methane production through co-digestion This is a pioneer study evaluating the methane (CH4) production potential from residues of integrated 1st (vinasse and filter cake) and 2nd (deacetylation pretreatment liquor from straw) generation (1G2G) sugarcane biorefinery, providing a fully chemical characterization of them and their relation with the anaerobic digestion (AD) process. Small-scale assays provided fundamentals for basing the co-digestion optimization by assessing the optimal co-substrates synergistic conditions. Biochemical Methane Potential (BMP) tests showed co-digestion enhanced CH4 yield of isolated substrates, reaching up to 605 NmLCH4 gVS-1. The association of vinasse and deacetylation liquor as co-substrates increased the BMP by ~38% mostly by nutritionally benefiting the methanogenic activity. The kinetic analysis confirmed that the deacetylation liquor was the co-substrate responsible for improving the CH4 production in the co-digestion systems due to the highest CH4 conversion rate. The alkaline characteristic of the liquor (pH~12) also prevented alkalizing from being added to the co-digestion, an input that normally makes the process economically unfeasible to implement on an industrial scale due to the large quantities required for buffering the reactor. The filter cake had the lowest BMP (262 NmLCH4 gVS-1) and digestibility ([&le;] 40%), further limited by the required stirring to improve the mass transfer of biochemical reactions. The present study drives towards more sustainable use of vinasse, the most voluminous waste from the sugarcane industry, and lignin-rich residues derived from pre-treatment alkaline methods, aiming at an energy-efficient utilization, by at least 16% when compared to the traditional vinasse AD. The experimental and modeling elements from this work indicated the lignin-rich liquor is the main responsible for putting the co-digestion as a disruptive technological arrangement within the 1G2G sugarcane biorefineries, reinforcing the biogas production as the hub of the bioeconomy in the agroindustrial sector. Common coupling map advances GPCR-G protein selectivity Two-thirds of human hormones and one-third of clinical drugs act on membrane receptors that couple to G proteins to achieve appropriate functional responses. While G protein transducers from literature are annotated in the Guide to Pharmacology database, two recent large-scale datasets now expand the receptor-G protein  couplome. However, these three datasets differ in scope and reported G protein couplings giving different coverage and conclusions on GPCR-G protein signaling. Here, we report a common coupling map uncovering novel couplings supported by both large-scale studies, the selectivity/promiscuity of GPCRs and G proteins, and how the co-coupling and co-expression of G proteins compare to the families from phylogenetic relationships. The coupling map and insights on GPCR-G protein selectivity will catalyze advances in receptor research and cellular signaling towards the exploitation of G protein signaling bias in design of safer drugs. 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. A general role for MIA3/TANGO1 in secretory pathway organization and function. Complex machinery is required to drive secretory cargo export from the endoplasmic reticulum, an essential process in eukaryotic cells. In vertebrates, the Mia3 gene encodes two major forms of Transport ANd Golgi Organization Protein 1 (TANGO1S and TANGO1L). Here, using genome engineering of human cells, light microscopy, secretion assays, genomics, and proteomics we show that disruption of the longer form, TANGO1L, results in relatively minor defects in secretory pathway organization and function including limited impacts on procollagen secretion. In contrast, loss of both long and short forms results in major defects in cell organization and secretion. These include a failure to maintain the localization of ERGIC53 and SURF4 to the ER-Golgi Intermediate Compartment and dramatic changes to the ultrastructure of the ER-Golgi interface. Disruption of TANGO1 expression also causes significant changes in early secretory pathway gene and protein expression. Disruption of both TANGO1L and TANGO1S expression impairs secretion not only of large proteins, including procollagens, but of all types of secretory cargo including small soluble proteins. Our data support a general role for Mia3/TANGO1 in maintaining both secretory pathway structure and function in vertebrate cells. Dramatic consequences of reducing erythrocyte membrane cholesterol on Plasmodium falciparum Cholesterol is the most abundant lipid in the erythrocyte. During its blood stage development, the malaria parasite establishes an active cholesterol gradient across the various membrane systems within the infected erythrocyte. Interestingly, some antimalarial compounds have recently been shown to disrupt cholesterol homeostasis in intraerythrocytic stages of Plasmodium falciparum. These studies point to the importance of cholesterol for parasite growth. Previously, reduction of cholesterol from the erythrocyte membrane by treatment with methyl-{beta}-cyclodextrin (M{beta}CD) was shown to inhibit parasite invasion and growth. In addition, M{beta}CD treatment of trophozoite stage P. falciparum was shown to result in parasite expulsion from the host cell. We have revisited these phenomena by using live video microscopy, ultrastructural analysis, and response to antimalarial compounds. By using time-lapse video microscopy of fluorescently tagged parasites, we show that M{beta}CD treatment for just 30 min causes dramatic expulsion of the trophozoite stage parasites. This forceful expulsion occurs within 10 sec. Remarkably, the plasma membrane of the host cell from which the parasite has been expelled does not appear to be compromised. The parasitophorous vacuolar membrane (PVM) continued to surround the extruded parasite, but the PVM appeared damaged. Treatment with antimalarial compounds targeting PfATP4 or PfNCR1 prevented M{beta}CD-mediated extrusion of the parasites, pointing to a potential role of cholesterol dynamics underlying the expulsion phenomena. We also confirmed the essential role of erythrocyte plasma membrane cholesterol for invasion and growth of P. falciparum. This defect can be partially complemented by cholesterol and desmosterol but not with epicholesterol, revealing stereospecificity underlying cholesterol function. Overall, our studies advance previous observations and reveal unusual cell biological features underlying cholesterol depletion of infected erythrocyte plasma membrane.

ImportanceMalaria remains a major challenge in much of the world. Symptoms of malaria are caused by the growth of parasites belonging to Plasmodium spp. inside the red blood cells (RBC), leading to their destruction. The parasite depends upon its host for much of its nutritional need. Cholesterol is a major lipid in the RBC plasma membrane, which is the only source of this lipid for malarial parasites. We have previously shown that certain new antimalarial compounds disrupt cholesterol homeostasis in P. falciparum. Here we use live time-lapse video microscopy to show dramatic expulsion of the parasite from the host RBC when the cholesterol content of the RBC is reduced. Remarkably, this expulsion is inhibited by the antimalarials that disrupt lipid homeostasis. We also show stereospecificity of cholesterol in supporting parasite growth inside RBC. Overall, these results point to a critical role of cholesterol in physiology of malaria parasites. Genetic targeting of Card19 is linked to disrupted Ninj1 expression, impaired cell lysis, and increased susceptibility to Yersinia infection Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1{beta} release following caspase activation can be uncoupled in certain cell types or in response to particular stimuli, a state termed hyperactivation. However, the factors and mechanisms that regulate terminal cell lysis downstream of GSDMD cleavage remain poorly understood. In the course of studies to define regulation of pyroptosis during Yersinia infection, we identified a line of Card19-deficient mice (Card19lxcn) whose macrophages were protected from cell lysis and showed reduced apoptosis and pyroptosis, yet had wild-type levels of caspase activation, IL-1 secretion, and GSDMD cleavage. Unexpectedly, CARD19, a mitochondrial CARD-containing protein, was not directly responsible for this, as two independently-generated CRISPR/Cas9 Card19 knockout mice showed no defect in macrophage cell lysis, and expression of CARD19 in Card19lxcn macrophages did not restore cell lysis. Card19 is located on chromosome 13, adjacent to Ninj1, which was recently reported to regulate cell lysis downstream of GSDMD activation. Intriguingly, RNA-seq and western blotting revealed that Card19lxcn BMDMs are hypomorphic for NINJ1 expression, and reconstitution of Ninj1 in Card19lxcn immortalized BMDMs restored cell lysis. Card19lxcn mice exhibited significantly increased susceptibility to Yersinia infection, demonstrating that cell lysis itself plays a key role in protection against bacterial infection. Our findings identify genetic targeting of Card19 being responsible for off-target effects on the adjacent Ninj1 gene, thereby disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control during Yersinia infection.

Author SummaryProgrammed cell death is critical for regulating tissue homeostasis and host defense against infection. Pyroptosis is an inflammatory form of programmed cell death that couples cell lysis with release of inflammatory cytokines. Cell lysis is triggered by activation of particular intracellular pore forming proteins, but how regulation of cell lysis occurs is not well understood. Genetic targeting of Card19 on chromosome 13 resulted in decreased expression of the adjacent gene, Ninj1 which was recently found to regulate terminal lysis events in response to cell death-inducing stimuli. We found that macrophages from Card19-deficient mice were resistant to multiple forms of cell death in response to a variety of inflammatory stimuli, including canonical and non-canonical inflammasome activation, as well as triggers of cell-extrinsic apoptosis. Notably, Card19-deficient mice were more susceptible to Yersinia infection, indicating that cell lysis contributes to control of bacterial infections. Our data provide new insight into the impact of terminal cell lysis on control of bacterial infection and highlight the role of additional factors that regulate lytic cell death downstream of gasdermin cleavage. Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in Betacoronavirus replication As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their mRNAs, protect them from degradation by cellular 5 exoribonucleases, and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bi-functional replicase subunit harboring an N-terminal 3'-to-5' exoribonuclease (ExoN) domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14s enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome-CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.

Significance StatementThe ongoing SARS-CoV-2 pandemic emphasizes the urgent need to develop efficient broad-spectrum anti-CoV drugs. The structure-function characterization of conserved CoV replicative enzymes is key to identifying the most suitable drug targets. Using a multidisciplinary comparative approach and different betacoronaviruses, we characterized the key conserved residues of the nsp14 (N7-guanine)-methyltransferase, a poorly defined subunit of the CoV mRNA-synthesizing machinery. Our study highlights the unique structural features of this enzyme and establishes its essential role in betacoronavirus replication, while identifying two residues that are critical for the replication of the four betacoronaviruses tested, including SARS-CoV-2. Localization of signaling receptors maximizes cellular informationacquisition in spatially-structured natural environments Cells in natural environments like tissue or soil sense and respond to extracellular ligands with intricately structured and non-monotonic spatial distributions, sculpted by processes such as fluid flow and substrate adhesion. In this work, we show that spatial sensing and navigation can be optimized by adapting the spatial organization of signaling pathways to the spatial structure of the environment. We develop an information-theoretic framework for computing the optimal spatial organization of a sensing system for a given signaling environment. We find that receptor localization maximizes information acquisition in simulated natural contexts, including tissue and soil. Receptor localization extends naturally to produce a dynamic protocol for continuously redistributing signaling receptors, which when implemented using simple feedback, boosts cell navigation efficiency by 30-fold. Broadly, our work shows how cells can maximize the fidelity of information transfer by adapting the spatial organization of signaling molecules to the spatial structure of the environment. Long-range cortical synchronization supports abrupt visual learning Visual plasticity declines sharply after the critical period, yet we easily learn to recognize new faces and places even as adults. Such learning is often characterized by a "moment of insight", an abrupt and dramatic improvement in recognition. The mechanisms that support abrupt learning are unknown, but one hypothesis is that they involve changes in synchronization between brain regions. To test this hypothesis, we used a behavioral task in which non-human primates rapidly learned to recognize novel images and to associate them with specific responses. Simultaneous recordings from inferotemporal and prefrontal cortices revealed a transient synchronization of neural activity between these areas that peaked around the moment of insight. Synchronization was strongest between inferotemporal sites that encoded images and reward-sensitive prefrontal sites. Moreover, its magnitude intensified gradually over image exposures, suggesting that abrupt learning culminates from the search for informative signals within a circuit linking sensory information to task demands. The obstacles and potential clues of prime editing applications in tomato, a dicot plant Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a "priming" extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. Modifying the pegRNA sequences by shortening or introducing mismatches to the primer binding sequences (PBS) in order to reduce their melting temperatures (Tm) did not enhance the PE efficiency at the SlBMP21, SlALC and SlALS1 loci. Our data show challenges of PE approach in tomato, indicating a further improvement of the PE system for the successful applications such as use of improved expression systems. Our work provides an important clue for the successful application of the PE approach in crop improvement. Fatal COVID-19 and non-COVID-19 Acute Respiratory Distress Syndrome is Associated with Incomplete Alveolar Type 1 Epithelial Cell Differentiation from the Transitional State Without Fibrosis 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. Communities of tropical soil fungi differ between burned and unburned forest, with corresponding changes in plant community composition, litter and soil chemistry Fire is predicted to be more severe and frequent in forests of the Australian Monsoon Tropics over the coming decades. The way in which groups of ecologically important soil fungi respond to disturbance caused by fire has not been studied in Australian tropical forest ecosystems. Ectomycorrhizal (EM) fungi are important tree symbionts and saprotrophic fungi drive soil nutrient cycles. We analysed both publicly-available environmental DNA sequence data as well as soil chemistry data to test a hypothesis that burned areas in a contiguous tropical forest have different community composition and diversity of EM and saprotrophic soil fungi relative to nearby unburned sites. We tested this hypothesis by measuring community-level taxonomic composition, fungal diversity, species richness and evenness. We determined whether changes in fungal communities were associated with fire-altered soil chemical/physical properties, vegetation types, or the direct effect of fire. Soil fungi differed in abundance and community phylogenetic structure between forest sites that had experienced fire, and sites dominated by unburned forest. EM community composition differed between unburned and burned sites, which had more mycorrhizal hosts including Corymbia intermedia, Acacia flavescens and Acacia midgleyi. Lower diversity of saprotrophic fungi was correlated with lower soil nutrient levels and different litter composition at burned sites. Pyrophilic, truffle-like EM fungi that rely on mycophagous mammals for dispersal were abundant at recently burned sites. We conclude that EM fungi show different patterns of diversity in burned tropical forest, likely driven by changing plant communities, whereas differences in saprotrophic fungal communities of burned sites may be driven by by reduced soil nutrient levels and altered litter composition.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY Protein contact map prediction using multiple sequence alignment dropout and consistency learning for sequences with fewer homologs The prediction of protein contact map needs enough normalized number of effective sequence (Nf) in multiple sequence alignment (MSA). When Nf is small, the predicted contact maps are often not satisfactory. To solve this problem, we randomly selected a small part of sequence homologs for proteins with large Nf to generate MSAs with small Nf. From these MSAs, input features were generated and were passed through a consistency learning network, aiming to get the same results when using the features generated from the MSA with large Nf. The results showed that this method effectively improves the prediction accuracy of protein contact maps with small Nf. ZIKV disrupts placental ultrastructure and drug transporter expression in mice Congenital Zika virus (ZIKV) infection can induce fetal brain abnormalities. Here, we investigated whether maternal ZIKV infection affects placental physiology and metabolic transport potential and impacts the fetal outcome, regardless of viral presence in the fetus at term. Low (103 PFU-ZIKVPE243; low ZIKV) and high (5x107 PFU-ZIKVPE243; high ZIKV) virus titers were injected into immunocompetent (ICompetent C57BL/6) and immunocompromised (ICompromised A129) mice at gestational day (GD) 12.5 for tissue collection at GD18.5 (term). High ZIKV elicited fetal death rates of 66% and 100%, whereas low ZIKV induced fetal death rates of 0% and 60% in C57BL/6 and A129 dams, respectively. All surviving fetuses exhibited intrauterine growth restriction (IUGR) and decreased placental efficiency. High-ZIKV infection in C57BL/6 and A129 mice resulted in virus detection in maternal spleens and placenta, but only A129 fetuses presented virus RNA in the brain. Nevertheless, pregnancies in both strains produced fetuses with decreased head sizes (p<0.05). Low-ZIKV-A129 dams had higher IL-6 and CXCL1 levels (p<0.05), and their placentas showed increased CCL-2 and CXCL-1 contents (p<0.05). In contrast, low-ZIKV-C57BL/6 dams had an elevated CCL2 serum level and increased type I and II IFN expression in the placenta. Notably, less abundant microvilli and mitochondrial degeneration were evidenced in the placental labyrinth zone (Lz) of ICompromised and high-ZIKV-ICompetent mice but not in low-ZIKV-C57BL/6 mice. In addition, decreased placental expression of the drug transporters P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and the lipid transporter Abca1 was detected in all ZIKV-infected groups, but Bcrp and Abca1 were only reduced in ICompromised and high-ZIKV ICompetent mice. Our data indicate that gestational ZIKV infection triggers specific proinflammatory responses and affects placental turnover and transporter expression in a manner dependent on virus concentration and maternal immune status. Placental damage may impair proper fetal-maternal exchange function and fetal growth/survival, likely contributing to congenital Zika syndrome.