Cell Intrinsic Control of Death and Proliferation of Lymphocytes in Immune Response and Tumor Cells by pH Many biological processes are controlled by cell death and proliferation. The present study found that intracellular pH positively correlated with proliferation but negatively with apoptosis. Acid treatments lowered intracellular pH and preferentially depleted highly proliferating lymphocytes, whereas alkaline treatments increased intracellular pH and enhanced lymphocyte proliferation. Likewise, acid treatments induced apoptosis and alkaline treatments maintained proliferation of tumor cells. These findings support a unified theory of regulation of cell death and proliferation where an intracellular pH balance controls both events, and the mitochondria act as the pH-stats of the cell. Accordingly, the Warburg effect is explained as necessary for proliferating cells to have a high intracellular pH environment to accelerate proliferation and avoid low pH-induced death.
Measuring mitochondrial electron transfer complexes in previously frozen cardiac tissue from the offspring of sow: A model to assess exercise-induced mitochondrial bioenergetics changes Measuring the mitochondrial electron transfer complex (ETC) profile from previously frozen heart tissue samples from offspring born to an exercised sow provided descriptive data about exercise-induced mitochondrial biochemical changes in heart tissue from the offspring born to the exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

SUMMARYPreparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.
Determining significant correlation between pairs of extant characters in a small parsimony framework When studying the evolutionary relationships among a set of species, the principle of parsimony states that a relationship involving the fewest number of evolutionary events is likely the correct one. Due to its simplicity, this principle was formalized in the context of computational evolutionary biology decades ago by, e.g., Fitch and Sankoff. Because the parsimony framework does not require a model of evolution, unlike maximum likelihood or Bayesian approaches, it is often a good starting point when no reasonable estimate of such a model is available.

In this work, we devise a method for determining if pairs of discrete characters are significantly correlated across all most parsimonious reconstructions, given a set of species on these characters, and an evolutionary tree. The first step of this method is to use Sankoffs algorithm to compute all most parsimonious assignments of ancestral states (of each character) to the internal nodes of the phylogeny. Correlation between a pair of evolutionary events (e.g., absent to present) for a pair of characters is then determined by the (co-) occurrence patterns between the sets of their respective ancestral assignments. The probability of obtaining a correlation this extreme (or more) under a null hypothesis where the events happen randomly on the evolutionary tree is then used to assess the significance of this correlation. We implement this method: parcours (PARsimonious CO-occURrenceS) and use it to identify significantly correlated evolution among vocalizations and morphological characters in the Felidae family.

The parcours tool is freely available at https://github.com/murraypatterson/parcours Deficits in seizure threshold and other behaviors in adult mice without gross neuroanatomic injury after late gestation transient prenatal hypoxia Intrauterine hypoxia is a common cause of brain injury in children resulting in a broad spectrum of long-term neurodevelopmental sequela, including life-long disabilities that can occur even in the absence of severe neuroanatomic damage. Postnatal hypoxia-ischemia rodent models are commonly used to understand the effects of ischemia and transient hypoxia on the developing brain. Postnatal models, however, have some limitations. First, they do not test the impact of placental pathologies on outcomes from hypoxia. Second, they primarily recapitulate severe injury because they provoke substantial cell death, which is not seen in children with mild hypoxic injury. Lastly, they do not model preterm hypoxic injury. Prenatal models of hypoxia in mice may allow us to address some of these limitations to expand our understanding of developmental brain injury. The published rodent models of prenatal hypoxia employ multiple days of hypoxic exposure or complicated surgical procedures, making these models challenging to perform consistently in mice. Furthermore, large animal models suggest that transient prenatal hypoxia without ischemia is sufficient to lead to significant functional impairment to the developing brain. However, these large animal studies are resource-intensive and not readily amenable to mechanistic molecular studies. Therefore, here we characterized the effect of late gestation (embryonic day 17.5) transient prenatal hypoxia (5% inspired oxygen) on long-term anatomical and neurodevelopmental outcomes in mice. Late gestation transient prenatal hypoxia increased hypoxia-inducible factor 1 alpha protein levels (a marker of hypoxic exposure) in the fetal brain. Hypoxia exposure predisposed animals to decreased weight at postnatal day 2, which normalized by day 8. However, hypoxia did not affect gestational age at birth, litter size at birth, or pup survival. No differences in fetal brain cell death or long-term gray or white matter changes resulted from hypoxia. Animals exposed to prenatal hypoxia did have several long-term functional consequences, including sex-dichotomous changes. Hypoxia exposure was associated with a decreased seizure threshold and abnormalities in hindlimb strength and repetitive behaviors in males and females. Males exposed to hypoxia had increased anxiety-related deficits, whereas females had deficits in social interaction. Neither sex developed any motor or visual learning deficits. This study demonstrates that late gestation transient prenatal hypoxia in mice is a simple, clinically relevant paradigm for studying putative environmental and genetic modulators of the long-term effects of hypoxia on the developing brain. Exploitation of the genetic variability in the genotype of Egyptian Wheat by studding Molecular Markers, Molecular Docking and Nano Composites. Regulation of leaf and stem rust This manuscript has been withdrawn by the authors as it was submitted and made public without the full consent of all the authors. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. Cellulomonas zeae sp. nov., Lelliottia zeae sp. nov., Paraburkholderia zeae sp. nov. and Sphingomonas zeigerminis sp. nov., four new bacterial species isolated from Zea mays and other hosts Four novel bacterial species collected from healthy tissues of corn, rice and soybean plants in the United States are described. These include Cellulomonas zeae sp. nov. isolated from corn in Indiana; Lelliottia zeae sp. nov. from corn in Indiana and rice in Arkansas; Paraburkholderia zeae sp. nov. isolated from corn in Iowa; and Sphingomonas zeigerminis sp. nov. from corn in Mississippi and soy in Arkansas. No pathogenic strains are known for any of the novel species based on genome comparisons to assemblies in GenBank. High performance single-cell gene regulatory network inference atscale: The Inferelator 3.0 MotivationGene regulatory networks define regulatory relationships between transcription factors and target genes within a biological system, and reconstructing them is essential for understanding cellular growth and function. Methods for inferring and reconstructing networks from genomics data have evolved rapidly over the last decade in response to advances in sequencing technology and machine learning. The scale of data collection has increased dramatically; the largest genome-wide gene expression datasets have grown from thousands of measurements to millions of single cells, and new technologies are on the horizon to increase to tens of millions of cells and above.

ResultsIn this work, we present the Inferelator 3.0, which has been significantly updated to integrate data from distinct cell types to learn context-specific regulatory networks and aggregate them into a shared regulatory network, while retaining the functionality of the previous versions. The Inferelator is able to integrate the largest single-cell datasets and learn cell-type specific gene regulatory networks. Compared to other network inference methods, the Inferelator learns new and informative Saccharomyces cerevisiae networks from single-cell gene expression data, measured by recovery of a known gold standard. We demonstrate its scaling capabilities by learning networks for multiple distinct neuronal and glial cell types in the developing Mus musculus brain at E18 from a large (1.3 million) single-cell gene expression dataset with paired single-cell chromatin accessibility data.

AvailabilityThe inferelator software is available on GitHub (https://github.com/flatironinstitute/inferelator) under the MIT license and has been released as python packages with associated documentation (https://inferelator.readthedocs.io/). Nutrient metabolism regulates insulin granule formation in the pancreatic islet β-cell via ER redox homeostasis Defects in the pancreatic {beta}-cells secretion system are well-described in Type 2 diabetes (T2D) and include impaired proinsulin processing and a deficit in mature insulin-containing secretory granules; however, the cellular mechanisms underlying these defects and the contribution of hyperglycemia to this process remain poorly understood. Here, we used an in situ fluorescent pulse-chase strategy and proximity labeling-based quantitative proteomics analysis to study proinsulin trafficking and demonstrate a direct link to glucose metabolism via the production of redox intermediates that facilitate proinsulin export from the ER. We show that ER export of proinsulin is delayed in T2D models resulting in decreased insulin granule formation and further demonstrate this process can be regulated by NADPH and reducing equivalent availability. Together, these data highlight a critical role for nutrient metabolism and mitochondrial dysfunction in the maladaptive remodeling of the {beta}-cells secretory pathway in the decline of {beta}-cell function in T2D. Second Heart Field-derived Cells Contribute to Angiotensin II-mediated Ascending Aortopathies BackgroundThe ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies.

MethodsAscending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry assisted proteomics, and molecular features of SHF-derived cells were determined by single cell transcriptomic analyses. Genetic deletion of either low-density lipoprotein receptor-related protein 1 (Lrp1) or transforming growth factor-{beta} receptor 2 (Tgfbr2) in SHF- derived cells was conducted to examine the impact of SHF-derived cells on vascular integrity.

ResultsPathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas following AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion, prior to overt pathology, evoked downregulation of SMC proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single cell transcriptomics results identified plasminogen activator inhibitor 1 (PAI1) as the most increased protein in SHF-derived SMCs and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies.

ConclusionSHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and TGF-{beta} signaling associated with increases of aortic PAI1.



O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/930917v6_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@185e3d9org.highwire.dtl.DTLVardef@131d702org.highwire.dtl.DTLVardef@11afa42org.highwire.dtl.DTLVardef@242786_HPS_FORMAT_FIGEXP  M_FIG C_FIG Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABS- SHF-derived SMCs and fibroblasts associate with AngII-induced aortic pathologies.
- AngII induces a distinct fibroblast sub-cluster that is less abundant for mRNAs related to major extracellular components and TGF{beta} ligands and receptors, but more abundant for proliferative genes.
- TGFBR2 deletion in SHF-derived cells are embryonic lethal with significant dilatation of the outflow tract in mice.
- SHF-specific deletion of LRP1 leads to aortic pathologies in mice, supporting the importance of SHF-derived cells in maintaining ascending aortic wall integrity.


What are the clinical implications?- Heterogeneity of the embryonic origins of SMCs and fibroblasts contributes to complex mechanisms of vasculopathy formation, which should be considered when investigating the pathogenesis of thoracic aortopathies. Comparative Analysis of non-coding small-RNAs in Pseudomonas aeruginosa Keratitis Strains with Different Antibiotic Susceptibility Pseudomonas aeruginosa, is a gram-negative bacterium causes opportunistic or nosocomial infections in immunocompromised individuals. In recent years, a steady increase in human corneal infections of P. aeruginosa has been reported with increased multi-drug resistance (MDR) or extensively drug resistance (XDR). Several non-coding sRNAs, has been identified to regulate various physiological processes in P. aeruginosa, including biofilm formation, quorum sensing. However, the regulatory mechanism of sRNAs in MDR/XDR pathways of P. aeruginosa keratitis strains is not yet studied. In this study, we identified bacterial sRNAs in publicly available P. aeruginosa keratitis genomes and investigated their regulatory role in MDR/XDR pathways using bioinformatic analysis. Totally, 46 P. aeruginosa keratitis strains from different geographical regions were included. Of 46, Eight (30%) out of Twenty-seven and Nine (52) out of Nineteen P. aeruginosa strains from India and Australia were identified as not-MDR. Whereas, 10 (38%) Indian and 9 (47%) Australian strains were identified as MDR. Eight Indian strains were identified as XDR. Out of 46 strains, 23 (50%) carried ExoU, 21(45%) carried ExoS and two (5%) strains carried both ExoU and ExoS, exotoxins for their virulence. The sRNA, SPA0021 was identified in 18 MDR/XDR and 6 not-MDR strains along with UCBPP-PA14. Interestingly, majority of the imipenem resistant P. aeruginosa keratitis strains from the present study was found to be carried SPA0023 sRNA (18 out of 30 strains). The outer membrane porin protein OprD, identified as binding target of SPA0023. Negative regulation or inactivation of OprD, reported in increased imipenem resistance in P. aeruginosa. Mutation analysis revealed that SPA0023 carrying P. aeruginosa keratitis strains contains a lesser number of amino acid changes in OprD protein than other strains. These findings indicate, imipenem resistance in SPA0023 carried strains might arose from the negative regulation or inhibition of OprD by SPA0023. However, functional studies are warranted with large number of P. aeruginosa keratitis strains to confirm the negative regulation of OprD by SPA0023 and imipenem resistance. Feedbacks between size and density determine rapid eco-phenotypic dynamics O_LIBody size is a fundamental trait linked to many ecological processes--from individuals to ecosystems. Although the effects of body size on metabolism are well-known, the potential reciprocal effects of body size and density are less clear. Specifically, 1) whether changes in body size or density more strongly influence the other and 2) whether coupled rapid changes in body size and density are due to plasticity, rapid evolutionary change, or a combination of both.
C_LIO_LIHere, we address these two issues by experimentally tracking population density and mean body size in the protist Tetrahymena pyriformis as it grows from low density to carrying capacity. We then use Convergent Cross Mapping time series analyses to infer the direction, magnitude, and causality of the link between body size and ecological dynamics. We confirm the results of our analysis by experimentally manipulating body size and density while keeping the other constant. Last, we fit mathematical models to our experimental time series that account for purely plastic change in body size, rapid evolution in size, or a combination of both, to gain insight into the processes that most likely explain the observed dynamics.
C_LIO_LIOur results indicate that changes in body size more strongly influence changes in density than the other way around, but also show that there is reciprocity in this effect (i.e., a feedback). We show that a model that only accounts for purely plastic change in size most parsimoniously explains observed, coupled phenotypic and ecological dynamics.
C_LIO_LITogether, these results suggest 1) that body size can shift dramatically through plasticity, well within ecological timescales, 2) that rapid changes in body size may have a larger effect on ecological dynamics than the reverse, but 3) phenotypic and ecological dynamics influence each as populations grow. Overall, we show that rapid plastic changes in functional traits like body size may play a fundamental -but currently unrecognized- role in familiar ecological processes such as logistic population growth.
C_LI The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method The integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR, but is typically assessed under conditions that fail to provide information on the individual mtDNA strands. Using denaturing gel electrophoresis, we show that commonly-used isolation procedures generate mtDNA containing several single-strand breaks per strand. Through systematic comparison of DNA isolation methods, we identify a procedure yielding the highest integrity of mtDNA that we demonstrate displays improved performance in downstream assays. Our results highlight the importance of isolation method choice, and serve as a resource to researchers requiring high-quality mtDNA from solid tissues. The N764K and N856K mutations in SARS-CoV-2 Omicron BA.1 S protein generate potential cleavage sites for SKI-1/S1P protease Spike (S) protein is a key protein in coronaviruses life cycle. SARS-CoV-2 Omicron BA.1 variant of concern (VoC) presents an exceptionally high number of 30 substitutions, 6 deletions and 3 insertions in the S protein. Recent works revealed major changes in the SARS-CoV-2 Omicron biological properties compared to earlier variants of concern (VoCs). Here, these major changes could be explained, at least in part, by the mutations N764K and/or N856K in S2 subunit. These mutations were not previously detected in other VoCs. N764K and N856K generate two potential cleavage sites for SKI-1/S1P serine protease, known to cleave viral envelope glycoproteins. The new sites where SKI-1/S1P could cleave S protein might impede the exposition of the internal fusion peptide for membrane fusion and syncytia formation. Based on the human protein atlas, SKI-1/S1P protease is not found in lung tissues (alveolar cells type I/II and endothelial cells), but present in bronchus and nasopharynx. This may explain why Omicron has change of tissue tropism. Viruses have evolved to use several host proteases for cleavage/activation of envelope glycoproteins. Mutations that allow viruses to change of protease may have a strong impact in host range, cell and tissue tropism, and pathogenesis. Engineered bacteria detect tumor DNA Advances in bacterial engineering have catalysed the development of living cell diagnostics and therapeutics1-3, including microbes that respond to diseases such as gut inflammation4, intestinal bleeding5, pathogens6 and hypoxic tumors7. Bacteria can easily access the entire gastrointestinal tract via oral administration8, and they can produce outputs that can be noninvasively measured in stool4 or urine7. Cellular memory, such as bistable switches4,9,10 or genomic rearrangement11, has been used to allow bacteria to store information over time. However, living biosensors have not yet been engineered to detect specific DNA sequences or mutations from outside the cell. Here, we engineer naturally competent Acinetobacter baylyi to detect donor DNA from the genomes of colorectal cancer (CRC) cells and organoids. We characterize the functionality of the biosensors in vitro with co-culture assays and then validate in vivo with sensor bacteria delivered orally or rectally into mice injected with orthotopic donor CRC organoids. We observe horizontal gene transfer from the tumor to the sensor bacteria in vivo, allowing their detection in stool. The sensor bacteria achieved 100% discrimination between mice with and without CRC using both delivery methods. Our findings establish a framework for biosensing applications that require the detection of mutations or organisms within environments that are difficult to sample. In addition, the platform can be readily expanded to include in situ production and delivery of therapeutic payloads at the detection site. HSP90 modulates T2R bitter taste receptor nitric oxide production and innate immune responses in human airway epithelial cells and macrophages Bitter taste receptors (T2Rs) are G protein-coupled receptors (GPCRs) expressed in various cell types including ciliated airway epithelial cells and macrophages. T2Rs in these two airway innate immune cell types are activated by bitter products, including some secreted by Pseudomonas aeruginosa, leading to Ca2+-dependent activation of endothelial nitric oxide (NO) synthase (eNOS). NO enhances mucociliary clearance and has direct antibacterial effects in ciliated epithelial cells and increases phagocytosis by macrophages. Using biochemistry and live cell imaging, we explored the role of heat shock protein 90 (HSP90) in regulating T2R-dependent NO pathways in primary sinonasal epithelial cells, primary monocyte-derived macrophages, and a human bronchiolar cell line (H441). We used immunofluorescence to show that H441 cells express eNOS and certain T2Rs and that the bitterant denatonium benzoate activates NO production in an HSP90-dependent manner in cells grown either as submerged cultures and at air liquid interface. In primary sinonasal epithelial cells, we determined that HSP-90 inhibition reduces T2R-stimulated NO production and ciliary beating which are crucial for pathogen clearance. In primary monocyte-derived macrophages, we found that HSP-90 is integral to T2R-stimulated NO production and phagocytosis of FITC-labeled Escherichia coli and pHrodo-Staphylococcus aureus. Our study demonstrates that HSP90 serves an innate immune role by regulating NO production downstream of T2R signaling by augmenting eNOS activation without impairing upstream calcium signaling. These findings suggest that HSP90 plays an important role in airway antibacterial innate immunity and may be an important target in airway diseases like chronic rhinosinusitis, asthma, or cystic fibrosis. Analysis of science journalism reveals gender and regional disparities in coverage Science journalism is a critical way in which the public can remain informed and benefit from new scientific findings. Such journalism also shapes the publics view of the current state of scientific findings and legitimizes experts. Those covering science can only cite and quote a limited number of sources. Sources may be identified by the journalists research or by recommendations by other scientists. In both cases, biases may influence who is identified and ultimately included as an expert. To analyze possible biases in science journalism, we analyzed 22,001 non-research articles published by Nature. We chose to analyze Nature non-research articles since its research articles provide a natural comparator. Our analysis considered two possible sources of disparity: gender and name origin. To explore these sources of disparity, we extracted cited authors names as well as extracted names of quoted speakers. While citations and quotations within a piece do not reflect the entire information-gathering process, they can provide insight into the demographics of visible sources. We then used the extracted names to predict gender and name origin of the cited authors and speakers.

In order to appropriately quantify the level of difference, we must identify a suitable reference set for comparison. We chose first and last authors within primary research articles in Nature and a subset of Springer Nature articles in the same time period as our comparator. In our analysis, we found a skew towards male quotation in Nature science journalism-related articles. However, quotation is trending toward equal representation at a faster rate than first and last authorship in academic publishing. Interestingly, we found that the gender disparity in Nature quotes was column-dependent, with the "Career Features" column reaching gender parity. Our name origin analysis found a significant over-representation of names with predicted Celtic/English origin and under-representation of names with a predicted East Asian origin. This finding was observed both in extracted quotes and journal citations, but dampened in citations. Caspase-1-driven neutrophil pyroptosis promotes an incomplete NETosis upon Pseudomonas aeruginosa infection Multiple neutrophil death programs contribute to host defense against infections. Although expressing all necessary components, neutrophils specifically fail to undergo pyroptosis, a lytic form of cell death triggered by the activation of the pro-inflammatory complex inflammasome. In the light of the arm race, we hypothesized that intrinsic neutrophil pyroptosis resistance might be bypassed in response to specific microbial species. We show that Pseudomonas aeruginosa (P. aeruginosa) stimulates Caspase-1-dependent pyroptosis in human and murine neutrophils. Mechanistically, activated NLRC4 inflammasome supports Caspase-1-driven Gasdermin-D (GSDMD) activation, IL-1{beta} cytokine release and neutrophil pyroptosis. Furthermore, GSDMD activates Peptidyl Arginine Deaminase-4 which drives an "incomplete NETosis" where neutrophil DNA fills the cell cytosol but fails crossing plasma membrane. Finally, we show that neutrophil Caspase-1 account for IL-1{beta} production and contributes to various P. aeruginosa strains spread in mice. Overall, we demonstrate that neutrophils are fully competent for Caspase-1-dependent pyroptosis, which drives an unsuspected "incomplete NETosis".

SummaryNeutrophils play an essential roles against infections. Although multiple neutrophil death programs contribute to host defense against infections, they fail to undergo pyroptosis, a pro-inflammatory form of cell death. Upon Infections, pyroptosis can be induced in macrophages or epithelial cells upon activation of pro-inflammatory complexes, inflammasomes that trigger Caspase-1-driven Gasdermin dependent plasma membrane lysis. In the light of host-microbe interactions, we hypothesized that yet to find microbial species might hold the capacity to overcome neutrophil resistance to inflammasome-driven pyroptosis. Among several bacterial species, we describe that the bacterium Pseudomonas aeruginosa specifically engages the NLRC4 inflammasome, which promotes Caspase-1-dependent Gasdermin-D activation and subsequent neutrophil pyroptosis. Furthermore, inflammasome-driven pyroptosis leads to DNA decondensation and expansion into the host cell cytosol but not to the so called Neutrophil Extracellular Trap (NET) release as DNA fails breaching the plasma membrane. Finally, in vivo P. aeruginosa infections highlight that Caspase-1-driven neutrophil pyroptosis is functional and is detrimental upon P. aeruginosa infection. Altogether, our results unexpectedly underline neutrophil competence for Caspase-1-dependent pyroptosis, a process that contributes to host susceptibility to P. aeruginosa infection. A novel two-generation approach to the population dynamics of gregarious parasitoids Parasitoids, as important natural enemies, help maintain balance in natural ecosystems. Their population dynamics is generally predicted from the number of individuals. Here, using gregarious parasitoids as models, we show that this traditional approach omits one important parameter: the mothers manipulation of offspring fertility due to the clutch size-body size- fertility correlation. As a result of this correlation, when females deliberately adjust the number of offspring laid in a host, they determine not only the number but also the body sizes and reproductive potentials of those offspring. For the first time in the model species Anaphes flavipes, we determined the parasitoids offspring fertility from clutch size. Using this, we experimentally clarified the advantage of specific clutch size combinations and we show that identical fertility in the F1 generation can lead to distinctly different fertility values in the F2 generation. Even with the same number of hosts, lower fertility in the F1 generation can cause higher fertility in the F2 generation. Based on these results, we propose a novel two-generation approach which includes the clutch size-body size-fertility correlation. Our novel approach provides a new perspective for determining the individual fitness levels of gregarious parasitoids with new options for the modelling of parasitoid population dynamics. Deep learning can predict multi-omic biomarkers from routine pathology images: A systematic large-scale study We assessed the pan-cancer predictability of multi-omic biomarkers from haematoxylin and eosin (H&E)-stained whole slide images (WSI) using deep learning (DL) throughout a systematic study. A total of 13,443 DL models predicting 4,481 multi-omic biomarkers across 32 cancer types were trained and validated. The investigated biomarkers included a broad range of genetic, transcriptomic, proteomic, and metabolic alterations. Furthermore, established markers relevant for prognosis, molecular subtypes and clinical outcomes were included. Overall, we established the general feasibility of predicting multi-omic markers directly from routine histology images with DL across solid cancer types, where 50% of the models could perform at an area under the curve (AUC) of more than 0.633 (with 25% of the models having an AUC larger than 0.711). A wide range of biomarkers were detectable from routine histology images across all investigated cancer types, with a mean AUC of at least 0.62 in almost all malignancies. Strikingly, we observed that biomarker predictability was mostly consistent and not dependent on sample size and class ratio, suggesting a degree of true predictability inherent in histomorphology. Together, the results of our study show the potential of DL to predict a multitude of biomarkers across the omics spectrum using only routine slides. This paves the way for accelerating diagnosis and developing more precise treatments for cancer patients. Differences in gait stability and acceleration characteristics between healthy young and older females Our aim was to evaluate differences in gait acceleration intensity, variability and stability of feet and trunk between older females and young females using inertial sensors. Twenty older females (OF; mean age 68.4, SD 4.1 years) and eighteen young females (YF; mean age 22.3, SD 1.7 years) were asked to walk straight for 100 meters at their preferred speed, while wearing inertial sensors on heels and lower back. We calculated spatiotemporal measures, foot and trunk acceleration characteristics and their variability, as well as trunk stability using the local divergence exponent (LDE). Two-way analysis of variance (including the factors foot and age), Students t-test, and Mann-Whitney U test were used to compare statistical differences of measures between groups. Cohens d effects were calculated for each variable. Foot maximum vertical acceleration and amplitude, trunk-foot vertical acceleration attenuation, as well as their variability were significantly smaller in OF than in YF. In contrast, trunk mediolateral acceleration amplitude, maximum vertical acceleration, and amplitude, as well as their variability were significantly larger in OF than in YF. Moreover, OF showed lower stability (i.e., higher LDE values) in mediolateral acceleration, mediolateral and vertical angular velocity of the trunk. Even though we measured healthy older females, these participants showed lower vertical foot accelerations with higher vertical trunk acceleration, lower trunk-foot vertical acceleration attenuation, less gait stability, and more variability of the trunk, and hence, were more likely to fall. These findings suggest that instrumented gait measurements may help for early detection of changes or impairments in gait performance, even before this can be observed by clinical eye or gait speed. Atomic Force Microscopy Reveals Distinct Protofilament-scale Structural Dynamics in Depolymerizing Microtubule Arrays The dynamic reorganization of microtubule-based cellular structures, such as the spindle and the axoneme, fundamentally depends on the dynamics of individual polymers within multimicrotubule arrays. A major class of enzymes, implicated in both the complete demolition and fine size control of microtubule-based arrays, are depolymerizing kinesins. How different depolymerases differently remodel microtubule arrays is poorly understood. A major technical challenge in addressing this question is that existing optical or electron-microscopy methods lack the spatial-temporal resolution to observe the dynamics of individual microtubules within larger arrays. Here we use Atomic Force Microscopy (AFM) to image depolymerizing arrays at single microtubule and protofilament resolution. We discover previously unseen modes of microtubule array destabilization by conserved depolymerases. We find that the kinesin-13 MCAK mediates asynchronous protofilament depolymerization and lattice-defect propagation, whereas the kinesin-8 Kip3p promotes synchronous protofilament depolymerization. Unexpectedly, MCAK can depolymerize the highly stable axonemal doublets, but Kip3p cannot. We propose that distinct protofilament-level activities underlie the functional dichotomy of depolymerases, resulting in either large-scale destabilization or length regulation of microtubule arrays. Our work establishes AFM as a powerful strategy to visualize microtubule dynamics within arrays and reveals how nanometer-scale substrate specificity leads to differential remodeling of micron-sized cytoskeletal structures.

SIGNIFICANCE STATEMENTOne cannot help but marvel at the precise organization of microtubule polymers in cellular structures such as the axoneme and the spindle. However, our understanding of the biochemical mechanisms that sculpt these arrays comes largely from in vitro experiments with a small number (one or two) of microtubules. This is somewhat akin to studying the architecture of multilane highways by studying one-lane streets. Here we directly visualize depolymerizing microtubule arrays at individual microtubule and protofilament resolution using Atomic Force Microscopy. Our results reveal differences in microtubule depolymerase activity and provide insights into how these differences in enzymatic activity on the nanometer scale can result in the differential remodeling of multi-microtubule arrays on the micron-length scale. Optogenetic dissection of actomyosin-dependent mechanics underlying tissue fluidity Rapid epithelial tissue flows are essential to building and shaping developing embryos. However, it is not well understood how the mechanical properties of tissues and the forces driving them to flow are jointly regulated to accommodate rapid tissue remodeling. To dissect the roles of actomyosin in the mechanics of epithelial tissue flows, here we use two optogenetic tools, optoGEF and optoGAP, to manipulate Rho/Rho-kinase signaling and actomyosin contractility in the germband epithelium, which flows via convergent extension movements during Drosophila body axis elongation. The ability to perturb actomyosin in the tissue allows us to analyze the effects of actomyosin on cell rearrangements, tissue tensions, and tissue mechanical properties. We find that either optogenetic activation or deactivation of Rho1 signaling and actomyosin contractility at the apical surface of the germband disrupts cell rearrangements and tissue-level flows. Rho1 activation leads to poorly oriented rearrangements that are associated with a redistribution of myosin II from the junctional to the medial-apical domain, whereas Rho1 deactivation leads to fewer, slower cell rearrangements that are associated with decreased junctional and medial myosin. By probing mechanical tensions in the tissue using laser ablation and inferring tissue mechanical properties from cell packings, we find that actomyosin influences both the anisotropic forces that drive tissue flow and the mechanical properties of the tissue that resist flow, leading to complex relationships between actomyosin activity and tissue fluidity. Moreover, our results link the subcellular distribution of myosin II to tissue tension and cell packings, revealing how junctional and medial myosin have differential roles in promoting and orienting cell rearrangements during tissue flow. Live Analysis of Position Effect Variegation (PEV) in Drosophila Reveals Different Modes of Action for HP1a and Su(var)3-9 AO_SCPLOWBSTRACTC_SCPLOWPosition Effect Variegation (PEV) results from the juxtaposition of euchromatic and heterochromatic components of eukaryotic genomes, silencing genes near the new euchromatin/heterochromatin junctions. Silencing is itself heritable through S phase, giving rise to distinctive random patterns of cell clones expressing the genes intermixed with clones in which the genes are silenced. Much of what we know about epigenetic inheritance in the soma stems from work on PEV aimed at identifying the components of the silencing machinery and its mechanism of inheritance. The roles of two central gene activities - the Su(var)3-9-encoded histone H3-Lysine-9 methyltransferase and the Su(var)205-encoded methyl-H3-Lysine-9 binding protein HP1a - have been inferred from terminal phenotypes, leaving considerable gaps in understanding how PEV behaves through development. Here, we investigate the PEV phenotypes of Su(var)3-9 and Su(var)205 mutations in live developing tissues. We discovered that mutation in Su(var)205 compromises the initial establishment of PEV in early embryogenesis. Later gains of heterochromatin-induced gene silencing are possible, but are unstable and lost rapidly. In contrast, a strain with mutation in Su(var)3-9 exhibits robust silencing early in development, but fails to maintain it through subsequent cell divisions. Our analyses show that while the terminal phenotypes of these mutations may appear identical, they have arrived at them through different developmental trajectories. We discuss how our findings expand and clarify existing models for epigenetic inheritance of heterochromatin-induced gene silencing.

SO_SCPLOWIGNIFICANCEC_SCPLOWCurrent concepts of epigenetic inheritance are exemplified by Position Effect Variegation, a phenomenon whereby heterochromatin can repress genes in clonal cell lineages. Heterochromatin is required for genome protection as it silences toxic transposable elements and prevents instability of repeat sequences. Histone H3 modified by methylation of Lysine-9 and HP1a are critical components of heterochromatin. Using live cell analysis of PEV in mutants in strains with mutations in Su(var)3-9, which encodes the histone methyltransferase, and Su(var)205, which encodes HP1a, we describe an unexpected dynamism in PEV, challenging current models of epigenetics, and revealing unexpectedly different modes of action of these two fundamental components of heterochromatin. 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 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. Model sharing in the human medial temporal lobe Effective planning involves knowing where different actions will take us. However natural environments are rich and complex, leading to an exponential increase in memory demand as a plan grows in depth. One potential solution to this problem is to share the neural state transition functions used for planning between similar contexts. Here, we asked human participants to perform a sequential decision making task designed so that knowledge could be shared between some contexts but not others. Computational modelling showed that participants shared a model of state transitions between contexts where appropriate. fMRI data identified the medial temporal lobe as a locus for learning of state transitions, and within the same region, correlated BOLD patterns were observed in contexts where state transition information were shared. Finally, we show that the transition model is updated more strongly following the receipt of positive compared to negative outcomes, a finding that challenges conventional theories of planning which assume knowledge about our environment is updated independently of outcomes received. Together, these findings propose a computational and neural account of how information relevant for planning can be shared between contexts.