Reduced glutathione decreases cell adhesion and increases cell volume. Glutathione is the most abundant thiol in animal cells. Reduced glutathione (GSH) is a major intracellular antioxidant neutralizing free radicals and detoxifying electrophiles. It plays important roles in many cellular processes, including cell differentiation, proliferation, and apoptosis. In the present study we demonstrate that extracellular concentration of reduced glutathione markedly increases cell volume within few hours, in a dose-response manner. Pre-incubation of cells with BSO, the inhibitor of {gamma}-glutamylcysteine synthetase, responsible for the first step in intracellular glutathione synthesis did not change the effect of reduced glutathione on cell volume suggesting a mechanism limited to the interaction of extracellular reduced glutathione on cell membrane. Similarly, inhibition of {gamma}-glutamylcyclotransferase involved in intracellular glutamate production had no effect on the action of reduced glutathione. Oxidized glutathione exerted no effect on cell volume. Results show that reduced GSH decreases cell adhesion resulting in an increased cell volume. Since many cell types are able to export GSH, the present results suggest that this could be a fundamental self-regulation of cell volume, giving the cells a self-control on their adhesion proteins.
Single-Cell Multiomics Reveals Distinct Cell States at the Top of the Human Hematopoietic Hierarchy The advent of single cell (Sc) genomics has challenged the dogma of haematopoiesis as a tree-like structure of stepwise lineage commitment through distinct and increasingly restricted progenitor populations. Instead, analysis of ScRNA-seq has proposed that the earliest events in human hematopoietic stem cell (HSC) differentiation are characterized by only subtle molecular changes, with hematopoietic stem and progenitor cells (HSPCs) existing as a continuum of low-primed cell-states that gradually transition into a specific lineage (CLOUD-HSPCs). Here, we combine ScRNA-seq, ScATAC-seq and cell surface proteomics to dissect the heterogeneity of CLOUD-HSPCs at different stages of human life. Within CLOUD-HSPCs, pseudotime ordering of both mRNA and chromatin data revealed a bifurcation of megakaryocyte/erythroid and lympho/myeloid trajectories immediately downstream a subpopulation with an HSC-specific enhancer signature. Importantly, both HSCs and lineage-restricted progenitor populations could be prospectively isolated based on correlation of their molecular signatures with CD35 and CD11A expression, respectively. Moreover, we describe the changes that occur in this heterogeneity as hematopoiesis develops from neonatal to aged bone marrow, including an increase of HSCs and depletion of lympho-myeloid biased MPPs. Thus, this study dissects the heterogeneity of human CLOUD-HSPCs revealing distinct HSPC-states of relevance in homeostatic settings such as ageing.
A rat model of pregnancy in the male parabiont Male pregnancy is a unique phenomenon in syngnathidae which refers to embryo or fetus incubation by males. However, whether male mammalian animals have the potential to conceive and maintain pregnancy remains unclear. Here, we constructed a rat model of pregnancy in the male parabiont using a four-step strategy. First, a heterosexual parabiotic pair was produced by surgically joining a castrated male rat and a female rat. After 8 weeks, uterus transplantation was performed on the male parabiont. After recovery, blastocyst-stage embryos were transplanted to the grafted uterus of the male parabiont and the native uterus of the female parabiont. Caesarean section was performed at embryonic day 21.5. The success rate of the model was 3.68%, and pups were only delivered from the male parabiont under pregnant blood exposure. Our results reveal the potential for rat embryonic development in male parabionts, which may have a profound impact on reproductive biology.

One-sentence summaryA rat model of pregnancy was constructed in the male parabiont using a four-step strategy.



O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/447686v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1f0694eorg.highwire.dtl.DTLVardef@91997forg.highwire.dtl.DTLVardef@f47086org.highwire.dtl.DTLVardef@7a33f_HPS_FORMAT_FIGEXP  M_FIG C_FIG Variants with the N501Y mutation extend SARS-CoV-2 host range to mice, with contact transmission Receptor recognition is a major determinant of viral host range, infectivity and pathogenesis. Emergences have been associated with serendipitous events of adaptation upon encounters with novel hosts, and the high mutation rate of RNA viruses may explain their frequent host shifts. SARS-CoV-2 extensive circulation in humans results in the emergence of variants, including variants of concern (VOCs) with diverse mutations notably in the spike, and increased transmissibility or immune escape. Here we show that, unlike the initial and Delta variants, the three VOCs bearing the N501Y mutation can infect common laboratory mice. Contact transmission occurred from infected to naive mice through two passages. This host range expansion likely results from an increased binding of the spike to the mouse ACE2. Together with the observed contact transmission, it raises the possibility of wild rodent secondary reservoirs enabling the emergence of new variants. Define protein variant functions with high-complexity mutagenesis libraries and enhanced mutation detection software ASMv1.0 Open reading frame (ORF) variant libraries have advanced our ability to query the functions of a large number of variants of a protein simultaneously in a single experiment. Variant libraries targeting full-length ORFs typically consists of all possible single-amino-acid substitutions and a stop codon at each amino-acid position. Because a variant differs from the template ORF by a single codon variation, variant quantification presents the most profound challenge to this technology. Efforts such as dividing a library into sub-libraries for direct sequencing, or tag-directed subassembly are practical only for small ORFs. Our approach, however, features an enhanced variant-calling tool that processes massive shotgun sequencing data, allowing one to screen a single variant library for ORFs sized up to [~]3600 bases. The variant-calling tool detects variants reliably, and also presents variant annotations in datafiles enabling analyses that have reshaped our strategies governing library design, screen deconvolution, sequencing and sequencing data analysis. A new method for the sampling and preservation of placental specimens in low-resource settings for the identification of P. falciparum and analysis of nucleic acids Collection, preservation, and shipment of histological specimens in low-resource settings is challenging. We present a novel method that achieved excellent preservation of placental specimens from rural Mali by using formalin fixation, ethanol dehydration, and long-term storage in a solar-powered freezer. Sample preservation success was 92%, permitting evaluation of current and past malaria infection, anemia, placental maturity, and inflammation. Using RNAscope(R) hybridization we were able to visualize cell-specific gene expression patterns in the formalin-fixed paraffin-embedded (FFPE) specimens. Additionally, our method entailed mirrored sampling from the two cut faces of a cotyledon, one for the FFPE workflows and the other for storage in RNAlater and RNA-seq. "Adopt a Microorganism" methodology enhances the discourse richness of high school students in both hybrid learning and remote education The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook(R), represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, "Adopt a Microorganism", was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of Sao Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students discourse richness in microbiology even without face-to-face traditional classes. Comparative toxicogenomics of glyphosate and Roundup herbicides by mammalian stem cell-based genotoxicity assays and molecular profiling in Sprague-Dawley rats Whether glyphosate-based herbicides (GBHs) are more potent than glyphosate alone at activating cellular mechanisms, which drive carcinogenesis remains controversial. As GBHs are more cytotoxic that glyphosate, we reasoned they may also be more capable of activating carcinogenic pathways. We tested this hypothesis by comparing the effects of glyphosate with Roundup GBHs both in vitro and in vivo. First, glyphosate was compared with representative GBHs namely MON 52276 (EU), MON 76473 (UK) and MON 76207 (USA) using the mammalian stem cell-based ToxTracker system. Here, MON 52276 and MON 76473, but not glyphosate and MON 76207, activated oxidative stress and unfolded protein responses. Second, molecular profiling of liver was performed in female Sprague-Dawley rats exposed to glyphosate or MON 52276 (both at 0.5, 50, 175 mg/kg bw/day glyphosate) for 90 days. MON 52276 but not glyphosate increased hepatic steatosis and necrosis. MON 52276 and glyphosate altered the expression of genes in liver reflecting TP53 activation by DNA damage and circadian rhythm regulation. Genes most affected in liver were similarly altered in kidneys. Small RNA profiling in liver showed decreased amounts of miR-22 and miR-17 from MON 52276 ingestion. Glyphosate decreased mir-30 while miR-10 levels were increased. DNA methylation profiling of liver revealed 5,727 and 4,496 differentially methylated CpG sites between the control and glyphosate and MON 52276 exposed animals respectively. Apurinic/apyrimidinic DNA damage formation in liver was increased with glyphosate exposure. Altogether, our results show that Roundup formulations cause more biological changes linked with carcinogenesis than glyphosate. Changes to the mtDNA copy number during yeast culture growth We show that the mtDNA copy number in growing cultures of the yeast Saccharomyces cerevisiae increases by a factor of up to 4, being lowest ([~]10 per haploid genome) and stable during rapid fermentative growth, and highest at the end of the respiratory phase. When yeast are grown on glucose, the onset of the mtDNA copy number increase coincides with the early stages of the diauxic shift, and the increase continues through respiration. A lesser yet still substantial copy number increase occurs when yeast are grown on a nonfermentable carbon source, i.e. when there is no diauxic shift. The mtDNA copy number increase during and for some time after the diauxic shift is not driven by an increase in cell size. The copy number increase occurs in both haploid and diploid strains, but is markedly attenuated in a diploid wild isolate that is a ready sporulator. Strain-to-strain differences in mtDNA copy number are least apparent in fermentation and most apparent in late respiration or stationary phase. While changes in mitochondrial morphology and function were previously known to accompany changes in physiological state, it had not been previously shown that the mtDNA copy number changes substantially over time in a clonal growing culture. The mtDNA copy number in yeast is therefore a highly dynamic phenotype. 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. 3D Echo Planar Time-resolved Imaging (3D-EPTI) for ultrafast multi-parametric quantitative MRI Multi-parametric quantitative MRI has shown great potential to improve the sensitivity and specificity of clinical diagnosis and to enhance our understanding of complex brain processes, but suffers from long scan time especially at high spatial resolution. To address this long-standing challenge, we introduce a novel approach, termed 3D Echo Planar Time-resolved Imaging (3D-EPTI), which significantly increases the acceleration capacity of MRI sampling, and provides high acquisition efficiency for multi-parametric MRI. This is achieved by exploiting the spatiotemporal correlation of MRI data at multiple timescales through new encoding strategies within and between efficient continuous readouts. Specifically, an optimized spatiotemporal CAIPI encoding within the readouts combined with a radial-block sampling strategy across the readouts enables an acceleration rate of 800 folds in the k-t space. A subspace reconstruction was employed to resolve thousands of artifact-free high-quality multi-contrast images spaced at a time interval of ~1 ms. We have demonstrated the ability of 3D-EPTI to provide robust and repeatable whole-brain simultaneous T1, T2, T2*, PD and B1+ mapping at high isotropic resolution within minutes (e.g., 1-mm isotropic resolution in 3 minutes), and to enable submillimeter multi-parametric imaging to study detailed brain structures.

HighlightsO_LIUltra-fast acquisition for 3D multi-parametric quantitative MRI.
C_LIO_LISimultaneous T1 T2 T2* PD and B1+ mapping.
C_LIO_LI3-minute scan at 1-mm isotropic resolution with whole-brain coverage.
C_LIO_LIMulti-parametric mapping at 700-m isotropic resolution in 10 minutes.
C_LIO_LIRepeatable quantification and cortical-depth analysis.
C_LI 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. SufB intein splicing in Mycobacterium tuberculosis is influenced by two remote conserved N-extein Histidines Inteins are auto-processing domains that implement a multi-step biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in [Fe-S] cluster assembly protein SufB from Mycobacterium tuberculosis. Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wild-type precursor protein. Structural analysis and molecular dynamics simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N-terminal cleavage reaction. His-38 is stabilized by the solvent-exposed His-5, and can impact N-S acyl shift by direct interaction with the catalytic Cys1. Development of inteins as biotechnological tools or as pathogen specific novel antimicrobial targets requires a more complete understanding of such unexpected roles of conserved extein residues in protein splicing. Design, Mutate, Screen: High-throughput creation of genetic clocks with different period-amplitude characteristics Directed evolution has become an invaluable tool in protein engineering and has also greatly influenced the construction of synthetic gene circuits. The ability to generate diversity at precise targets for directed evolution approaches has improved vastly, allowing researchers to create large, specific mutant libraries with relative ease. Screening approaches for large mutant libraries have similarly come a long way, especially when the desired behavior can easily be tested for with static, single time-point assays. For more complex gene circuits with dynamic phenotypes that change over time, directed evolution approaches to controlling and tuning circuit behavior have been hindered by the lack of sufficiently high-throughput screening methods to isolate variants with desired characteristics. Here we utilize directed mutagenesis and multiplexed microfluidics to develop a workflow for creating, screening and tuning dynamic gene circuits that operate at the population level. Specifically, we create a mutant library of an existing oscillator, the synchronized lysis circuit, and tune its dynamics while uncovering principles regarding its behavior. Lastly, we utilize this directed evolution workflow to construct a new synchronized genetic oscillator that exhibits robust dynamics over long time scales. The joint evolution of animal movement and competition strategies Competition typically takes place in a spatial context, but eco-evolutionary models rarely address the joint evolution of movement and competition strategies. Here we investigate a spatially explicit producer-scrounger model where consumers can either forage on a heterogeneous resource landscape or steal resource items from conspecifics (kleptoparasitism). We consider three scenarios: (1) a population of foragers in the absence of kleptoparasites; (2) a population of consumers that are either specialized on foraging or on kleptoparasitism; and (3) a population of individuals that can fine-tune their behavior by switching between foraging and kleptoparasitism depending on local conditions. By means of individual-based simulations, we study the joint evolution of movement and competition strategies, and we investigate the implications on the resource landscape and the distribution of consumers over this landscape. In all scenarios and for all parameters considered, movement and competition strategies evolved rapidly and consistently across replicate simulations. The evolved movement and resource exploitation patterns differ considerably across the three scenarios. For example, foragers are attracted by conspecifics in scenario (1), while they are repelled by conspecifics in scenario (2). Generally the movement strategies of kleptoparasites differ markedly from those of foragers, but even within each class of consumers polymorphisms emerge, corresponding to pronounced differences in movement patterns. In all scenarios, the distribution of consumers over resources differs substantially from  ideal free predictions. We show that this is related to the intrinsic difficulty of moving effectively on a depleted landscape with few reliable cues for movement. Our study emphasises the advantages of a mechanistic approach when studying competition in a spatial context, and suggests how evolutionary modelling can be integrated with current work in animal movement ecology. Sablefish (Anoplopoma fimbria Pallas, 1814) plasma biochemistry and hematology reference intervals including blood cell morphology Plasma biochemistry and hematology reference intervals are integral health assessment tools in all medical fields, including aquatic animal health. As sablefish (Anoplopoma fimbria) are becoming aquaculturally and economically more important, this manuscript provides essential reference intervals (RI) for their plasma biochemistry and hematology along with reference photomicrographs of blood cells in healthy, fasted sablefish. Blood cell morphology can differ between fish species. In addition, blood cell counts and blood chemistry can vary between fish species, demographics, water conditions, seasons, diets, and culture systems, which precludes the use of RIs from other fish species. For this study, blood was collected for plasma biochemistry and hematology analysis between June 20 and July 18, 2019, from healthy, yearling sablefish, hatched and reared in captivity on a commercial diet. Overnight fast of 16-18 hours did not sufficiently reduce lipids in the blood, which led to visible lipemia and frequent rupture of blood cells during analysis. Therefore, sablefish should be fasted for 24 to 36 hours before blood is collected to reduce hematology artifacts or possible reagent interference in plasma biochemistry analysis. Lymphocytes were the most dominant leukocytes (98%), while eosinophils were rare, and basophils were not detected in sablefish. Neutrophils were very large cells with Dohle bodies. In mammals and avian species, Dohle bodies are usually signs of toxic change from inflammation, but no such association was found in these fish. In conclusion, lipemia can interfere with sablefish blood analysis, and available removal methods should be evaluated as fasting for up to 36 h might not always be feasible. Also, more studies are required to establish RI for different developmental stages and rearing conditions. Somewhere I belong: phylogeny and morphological evolution in a species-rich lineage of ectoparasitic flatworms infecting cichlid fishes AO_SCPLOWBSTRACTC_SCPLOWA substantial portion of biodiversity evolved through adaptive radiation. However, the effects of explosive speciation on species interactions remain poorly understood. Metazoan parasites infecting radiating host lineages could improve our knowledge because of their intimate host relationships. Yet limited molecular, phenotypic, and ecological data discourage multivariate analyses of evolutionary patterns and encourage the use of discrete characters. Here, we assemble new molecular, morphological, and host range data widely inferred from a species-rich lineage of parasites (Cichlidogyrus, Platyhelminthes: Monogenea) infecting cichlid fishes to address data scarcity. We infer a multi-marker (28S/18S rDNA, ITS1, COI mtDNA) phylogeny of 58/137 species and characterise major lineages through synapomorphies inferred from mapping morphological characters. We predict the phylogenetic position of species without DNA data through shared character states, a combined molecular-morphological phylogenetic analysis, and a classification analysis with support vector machines. Based on these predictions and a cluster analysis, we assess the systematic informativeness of continuous characters, search for continuous equivalents for discrete characters, and suggest new characters for morphological traits not analysed to date. We also model the attachment/reproductive organ and host range evolution using the data of 136/137 described species and multivariate phylogenetic comparative methods (PCMs). We show that discrete characters can mask phylogenetic signals but can be key for characterising species groups. Regarding the attachment organ morphology, a divergent evolutionary regime for at least one lineage was detected and a limited morphological variation indicates host and environmental parameters affecting its evolution. However, moderate success in predicting phylogenetic positions, and a low systematic informativeness and high multicollinearity of morphological characters call for a revaluation of characters included in species characterisations. spatialGE: Quantification and visualization of the tumor microenvironment heterogeneity using spatial transcriptomics SummarySpatially-resolved transcriptomics promises to increase our understanding of the tumor microenvironment and improve cancer prognosis and therapies. Nonetheless, analytical methods to explore associations between the spatial heterogeneity of the tumor and clinical data are not available. Hence, we have developed spatialGE, a software that provides visualizations and quantification of the tumor microenvironment heterogeneity through gene expression surfaces, spatial heterogeneity statistics (SThet) that can be compared against clinical information, spot-level cell deconvolution, and spatially-informed clustering (STclust), all using a new data object to store data and resulting analyses simultaneously.

Availability and implementationThe R package and tutorial/vignette are available at https://github.com/FridleyLab/spatialGE. A script to reproduce the analyses in this manuscript is available in Supplementary information.

ContactFridley.Lab@Moffitt.org or Brooke.Fridley@Moffitt.org

Supplementary informationAvailable at Bioinformatics online.



O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/454023v2_figG1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@191cbe7org.highwire.dtl.DTLVardef@e1b6c7org.highwire.dtl.DTLVardef@a6f68forg.highwire.dtl.DTLVardef@1853601_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOGraphical abstractC_FLOATNO Overview of spatialGE features. A. The STList data object from spatialGE can be creared from several sources, including comma- or tab-separated files containing gene counts and spatial coordinates. The object can also be created directly from Visium outputs, Seurat objects, or GeoMx outputs. B. Users can optionally provide a metadata file, containing information associated with each sample (one row per sample, or per ROI if GeoMx data). C. Methods for quality control of data are provided by spatialGE, including visualizations of counts and genes per spot, as well as filtering of spots or genes within user-determined thresholds. D. A novel method (STclust) performs spatially informed clustering of spots and tissue domain identification. E. spatialGE provides different types of data visualization, including gene expression at each spot ("quilt plots"), as well as adaptation of spatial interpolation ("kriging") to spatial transcriptomics data (transcriptomic surface). F. spatialGE also leverages spatial statistics (Morans I, Gearys C, Getis-Ord Gi) to quantitatively describe heterogeneity within the tumor microenvironment and to explore associations between spatial heterogeneity and clinical oucomes. G. Gene expression deconvolution can also be applied to each spot to detect immune cell types (xCell) and classification of spots as tumor or stroma (ESTIMATE).

C_FIG Antibacterial potency of Type VI amidase effector toxins is dependent on substrate topology and cellular context Members of the bacterial T6SS amidase effector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance (NMR) and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions. Repetition effects in action planning reflect effector- but not hemisphere-specific coding Action choices are influenced by future and recent past action states. For example, when performing two actions in succession, response times (RT) to initiate the second action are reduced when the same hand is used. These findings suggest the existence of effector-specific processing for action planning. However, given that each hand is primarily controlled by the contralateral hemisphere, the RT benefit might actually reflect effector-independent, hemisphere-specific rather than effector-specific repetition effects. Here, participants performed two consecutive movements, each with a hand or a foot, in one of two directions. Direction was specified in an egocentric reference frame (inward, outward) or in an allocentric reference frame (left, right). Successive actions were initiated faster when the same limb (e.g., left hand - left hand), but not when the other limb of the same body side (e.g., left foot - left hand) executed the second action. The same-limb advantage was evident even when the two movements involved different directions, whether specified egocentrically or allocentrically. Corroborating evidence from computational modeling lends support to the claim that repetition effects in action planning reflect persistent changes in baseline activity within neural populations that encode effector-specific action plans. Chimeric mRNA based COVID-19 vaccine induces protective immunity against Omicron and Delta The emerging SARS-CoV-2 variants of concern (VOCs) exhibit enhanced transmission and immune escape, reducing the efficacy and effectiveness of the two FDA-approved mRNA vaccines. Here, we explored various strategies to develop novel mRNAs vaccines to achieve safer and wider coverage of VOCs. Firstly, we constructed a cohort of mRNAs that feature a furin cleavage mutation in the spike (S) protein of predominant VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2). Not present in the mRNA vaccines currently in use, the mutation abolished the cleavage between the S1 and S2 subunits, potentially enhancing the safety profile of the immunogen. Secondly, we systematically evaluated the induction of neutralizing antibodies (nAb) in vaccinated mice, and discovered that individual VOC mRNAs elicited strong neutralizing activity in a VOC-specific manner. Thirdly, the IgG produced in mice immunized with Beta-Furin and Washington (WA)-Furin mRNAs showed potent cross-reactivity with other VOCs, which was further corroborated by challenging vaccinated mice with the live virus of VOCs. However, neither WA-Furin nor Beta-Furin mRNA elicited strong neutralizing activity against the Omicron variant. Hence, we further developed an Omicron-specific mRNA vaccine that restored protection against the original and the sublineages of Omicron variant. Finally, to broaden the protection spectrum of the new Omicron mRNA vaccine, we tested the concept of bivalent immunogen. Instead of just fusing two RBDs head-to-tail, we for the first time constructed an mRNA-based chimeric immunogen by introducing the RBD of Delta variant into the entire S antigen of Omicron. The resultant chimeric mRNA was capable of inducing potent and broadly acting nAb against Omicron (both BA.1 and BA.2) and Delta, which paves the way to develop new vaccine candidate to target emerging variants in the future. 3D Reconstructions of Mouse Skeletal Muscle Reveal a Decrease in the MICOS Complex and Altered Mitochondrial Networks Background: Skeletal muscle gradually loses mass, strength, endurance, and oxidative capacity during aging. Studies of bioenergetics and protein turnover show that mitochondria mediate this decline in function. Mitochondria cristae are essential for the production of ATP. While mitochondrial aging is associated with endoplasmic reticulum stress, fragmented mitochondria, and decreased mitochondrial capacity, the genes associated with morphological changes in mitochondria during aging still requires further elucidation Further, it is not completely understood how 3D mitochondrial networks and the specialization of mitochondria are altered during aging. Methods: We measured changes in mitochondrial morphology and mitochondrial connectivity during the aging of the mouse gastrocnemius muscle through serial block facing-scanning electron microscopy and 3D reconstruction. Nanotunnels were also measured through 3D reconstruction. CRISPR/Cas9 KO was performed to examine changes in mitochondria upon loss of the mitochondrial contact site and cristae organizing system (MICOS) complex and optic atrophy 1 (OPA-1). Metabolomics were used to find key metabolite pathways changed upon off of the MICOS complex. Results: We found changes in mitochondrial network configuration, nanotunneling, size, shape, number, contact sites, cristae organizing system (MICOS) dynamics and gene expression in skeletal muscle during aging. We also found an association of OPA-1 and the MICOS complex in the gastrocnemius with mitochondrial aging. Further, the loss of the MICOS complex was linked with decreased oxidative capacity, and altered mitochondrial metabolism. Conclusions: To our knowledge, studies of mitochondrial changes in skeletal muscle throughout aging remains limited. 3D reconstructions of nanotunnels elucidated novel patterns of mitochondrial aging in skeletal muscle. MICOS proteins decreased with age and mitochondrial morphology was similar between aged skeletal muscle and that of young mice with MICOS protein loss. In tandem, our data suggest a relationship between the MICOS complex and aging, which could be potentially linked to disease states with additional 3D reconstruction. Differential impact of mutations in cell cycle checkpoint kinases on breast cancer presentation and prognosis. Cell cycle checkpoint kinases ATM/Chk2 and ATR/Chk1 are important tumor suppressors that are activated by DNA damage and other stressors to arrest replication. Germline variants in ATM and CHEK2 associate with increased incidence of estrogen receptor positive (ER+)/HER2- breast cancer and some evidence suggests that somatic inactivation of ATM/Chk2 induces therapy resistance in ER+/HER2- breast cancer patients. However, the relative contributions of germline and somatic inactivation of each of the four cell cycle checkpoint kinases to ER+/HER2- and ER-/PR-/HER2- (or triple negative breast cancer; TNBC) breast cancer incidence and outcomes has not yet been systematically assessed. Here, we comprehensively analyze associations between mutations in each cell cycle checkpoint kinase gene with age at diagnosis, tumor characteristics, and patient survival outcomes using six breast cancer datasets. Overall, we find that of all the cell cycle checkpoint kinases, CHEK2 (p=0.001) is the only gene that is preferentially mutated in ER+/HER2- breast cancer samples relative to TNBCs. CHEK2 mutations also associate with younger age at diagnosis of metastatic ER+/HER2- breast cancer (median age 41.5 years compared to 50 years for CHEK2 wildtype patients, p=0.03) that is highly PR positive (10% increase in PR positivity in metastatic vs primary CHEK2 mutant tumors, as opposed to a 25% decrease in PR positivity in CHEK2 wildtype tumors, p=0.03). These associations appear to be driven by germline variants, rather than somatic mutations. Whether germline or somatic, deleterious (truncating, frameshift or splice site) mutations in CHEK2 associate with rapid disease progression in response to standard first line endocrine therapy when compared to tumors with wildtype CHEK2 in two independent datasets (HR=6.15, p=0.01). Conversely, neither ATM nor ATR mutations are enriched in ER+/HER2- breast cancers relative to TNBC. However, co-incident mutation of ATR and TP53 is 2-fold enriched in this subset (p=0.002), and further enriched in metastatic (3.3%) vs primary (1%) ER+/HER2- breast cancers (p=0.002). This co-incident mutation of ATR and TP53 associates with decreased PR positivity in primary ER+/HER2- breast cancer diagnoses (p<0.0001) and significantly worse metastasis-free survival than mutations in either ATR or TP53 alone in two independent datasets (HR=2.01, p=0.007). This association is not specific to ER+/HER2- breast cancer but is observable in ER- breast cancer patients as well. These results provide the first systematic analysis of the contribution of germline and somatic mutations in cell cycle checkpoint kinases to tumor characteristics affecting patient prognosis and treatment response. They demonstrate a unique role for CHK2 in ER signaling and response to endocrine therapy, and a more universal role for ATR in combination with TP53 in metastatic progression across breast cancer subtypes. These findings provide rationale for streamlined testing of checkpoint kinase mutations as prognostic markers of metastatic potential and/or predictive markers of responsiveness to endocrine treatment in breast cancer patients. Characterization of methylation profiles in spontaneous preterm birth placental villous tissue Preterm birth is a global public health crisis which results in significant neonatal and maternal mortality. Yet little is known regarding the molecular mechanisms of idiopathic spontaneous preterm birth, and we have few diagnostic markers for adequate assessment of placental development and function. Previous studies of placental pathology and our transcriptomics studies suggest a role for placental maturity in idiopathic spontaneous preterm birth. It is known that placental DNA methylation changes over gestation. We hypothesized that if placental hypermaturity is present in our samples, we would observe a unique idiopathic spontaneous preterm birth DNA methylation profile potentially driving the gene expression differences we previously identified in our placental samples. Our results indicate the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern suggesting hypermaturity. Only seven significant differentially methylated regions fitting the idiopathic spontaneous preterm birth specific (relative to the controls) profile were identified, indicating unusually high similarity in DNA methylation between idiopathic spontaneous preterm birth and term birth samples. We identified an additional 1,718 significantly methylated regions in our gestational age matched controls were the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern, again indicating a striking level of similarity between the idiopathic spontaneous preterm birth and term birth samples. Pathway analysis of these regions revealed differences in genes within the WNT and Cadherin signaling pathways, both of which are essential in placental development and maturation. Taken together, these data demonstrate that the idiopathic spontaneous preterm birth samples are molecularly more mature than expected given their respective gestational age which likely impacts birth timing. Resetting of the 24-nt siRNA landscape in rice zygotes BackgroundThe zygote, a totipotent stem cell, constitutes a critical stage of the life cycle of sexually reproducing organisms. It is produced by the fusion of two differentiated cells -- the egg and sperm, which in plants have radically different siRNA transcriptomes from each other and from multicellular embryos. Due to technical challenges, the epigenetic changes that accompany the zygotic transition are poorly understood.

ResultsHere, we characterized the small RNA transcriptome of rice zygotes. We found widespread redistribution of 24-nt siRNAs relative to gametes, including absence of sperm signature siRNAs, reduction at egg signature siRNA loci, and upregulation at seedling signature siRNA loci. Loci with reduced siRNAs in zygote relative to egg were gene-distal and heterochromatic, while loci with increased siRNAs relative to egg had a similar genomic distribution to canonical siRNA loci. Although both egg and zygote siRNA loci had higher mCHH level in wildtype than in drm2 embryo, zygote but not egg siRNA loci were associated with hypermethylation in mature embryo. A small fraction of siRNA loci (~1%) called siren loci accounted for 60% of all siRNAs within zygote siRNA loci, that likely arose from maternal carryover as they had similarly abundant siRNAs in egg; these siren loci were not associated with embryo hypermethylation.

ConclusionsTaken together, our results indicate re-distribution of siRNAs in rice zygotes towards the canonical vegetative profile, that are consistent with the initiation of resetting of the gametic epigenome before the first embryonic division. Cell type-specific inference of differential expression in spatial transcriptomics Spatial transcriptomics enables spatially resolved gene expression measurements at near single-cell resolution. There is a pressing need for computational tools to enable the detection of genes that are differentially expressed (DE) within specific cell types across tissue context. We show that current approaches cannot learn cell type-specific DE due to changes in cell type composition across space and the fact that measurement units often detect transcripts from more than one cell type. Here, we introduce a statistical method, Cell type-Specific Inference of Differential Expression (C-SIDE), that identifies cell type-specific patterns of differential gene expression while accounting for localization of other cell types. We model spatial transcriptomics gene expression as an additive mixture across cell types of general log-linear cell type-specific expression functions. This approach provides a unified framework for defining and identifying gene expression changes in a wide-range of relevant contexts: changes due to pathology, anatomical regions, physical proximity to specific cell types, and cellular microenvironment. Furthermore, our approach enables statistical inference across multiple samples and replicates when such data is available. We demonstrate, through simulations and validation experiments on Slide-seq and MER-FISH datasets, that our approach accurately identifies cell type-specific differential gene expression and provides valid uncertainty quantification. Lastly, we apply our method to characterize spatially-localized tissue changes in the context of disease. In an Alzheimers mouse model Slide-seq dataset, we identify plaque-dependent patterns of cellular immune activity. We also find a putative interaction between tumor cells and myeloid immune cells in a Slide-seq tumor dataset. We make our C-SIDE method publicly available as part of the open source R package https://github.com/dmcable/spacexr.