title: An environmentally-responsive transcriptional state modulates cell identities during root development

abstract: Roots are fundamental organs for plant development and response to their environment: they anchor the plant to its growth substrate, uptake nutrients and water vital to plant growth, and can sense and respond to a variety of biotic and abiotic stresses. The architecture of root systems and their growth are known to be strongly affected by the environmental conditions found in the soil. However, the acquisition of cell identities at the root meristem is still mainly viewed as ontogenetically driven, where a small number of stem cells generate all the cell types through stereotyped divisions followed by differentiation, along a simple developmental trajectory. The extent to which environmental cues precisely shape and affect these developmental trajectories remains an open question. We used single-cell RNA-seq, combined with spatial mapping, to deeply explore the trajectories of cell states at the tip of Arabidopsis roots, known to contain multiple developing lineages. Surprisingly, we found that most lineage trajectories exhibit a stereotyped bifid topology with two developmental trajectories rather than one. The formation of one of the trajectories is driven by a strong and specific activation of genes involved in the responses to various environmental stimuli, that affects only of a subset of the cells in multiple cell types simultaneously, revealing another layer of patterning of cell identities in the root that is independent of cell ontogeny. We demonstrate the robustness of this environmentally responsive transcriptional state by showing that it is present in a mutant where cell type identities are greatly perturbed, as well as in different Arabidopsis ecotypes. We also show that the root can adapt the proportion of cells that acquire this particular state in response to environmental signals such as nutrient availability. The discovery of this cell state reveals new layers of cell identity that may underpin the adaptive potential of plant development.


title: An environmentally responsive transcriptional state modulates cell identities during root development

category: plant biology


title: Modernizing the NEURON Simulator for Sustainability, Portability, and Performance

abstract: The need for reproducible, credible, multiscale biological modeling has led to the development of standardized simulation platforms, such as the widely-used NEURON environment for computational neuroscience. Developing and maintaining NEURON over several decades has required attention to the competing needs of backwards compatibility, evolving computer architectures, the addition of new scales and physical processes, accessibility to new users, and efficiency and flexibility for specialists. In order to meet these challenges, we have now substantially modernized NEURON, providing continuous integration, an improved build system and release workflow, and better documentation. With the help of a new source-to-source compiler of the NMODL domain-specific language we have enhanced NEURONs ability to run efficiently, via the CoreNEURON simulation engine, on a variety of hardware platforms, including GPUs. Through the implementation of an optimized in-memory transfer mechanism this performance optimized backend is made easily accessible to users, providing training and model-development paths from laptop to workstation to supercomputer and cloud platform. Similarly, we have been able to accelerate NEURONs reaction-diffusion simulation performance through the use of just-in-time compilation. We show that these efforts have led to a growing developer base, a simpler and more robust software distribution, a wider range of supported computer architectures, a better integration of NEURON with other scientific workflows, and substantially improved performance for the simulation of biophysical and biochemical models.

category: neuroscience


title: Control of contextual memory through interneuronal α5-GABAA receptors

abstract: {gamma}-aminobutyric acid type A receptors that incorporate 5 subunits (5-GABAARs) are highly enriched in the hippocampus and they are strongly implicated in the control of learning and memory. Receptors located on pyramidal neuron dendrites have long been considered responsible, but here we report that selective elimination of 5-GABAARs from either interneurons or pyramidal cells prevented the general anesthetic etomidate from suppressing contextual learning. Using Ca2+ imaging of hippocampal neuronal activity in freely exploring mice, we also found that etomidate interfered with the development of place cells and spatial engrams in wild type mice but not in interneuron-specific 5-GABAAR knockout mice. These findings show that 5-GABAARs on interneurons as well as pyramidal neurons mediate etomidate-induced amnesia.


title: Highly Connected and Highly Variable: A Core Brain Network during Resting State Supports Propofol-induced Unconsciousness

abstract:
Leading theories of consciousness make diverging predictions for where and how neural activity gives rise to subjective experience. The Global Neuronal Workspace theory (GNW) states that consciousness is instantiated through global broadcasting of information across the prefrontal-parietal regions, whereas the integrated information theory (IIT) postulates that consciousness requires the posterior cortex to produce maximally irreducible integrated information. As both theories seem to partially agree on that the neural correlates of consciousness (NCC) require globally integrated brain activity across a network of functionally specialized modules, it is not known yet whether brain regions with such functional configurations would align with the NCC distribution predicted by the GNW or the IIT. We scanned resting-state fMRI data from 21 subjects during wakefulness, propofol-induced sedation and anesthesia. Graph-theoretical analysis were conducted on awake fMRI data to search for the NCC candidates as brain regions that exhibit both high rich-clubness and high modular variability. Another independent dataset of 10 highly-sampled subjects were used to validate the NCC distribution at individual-level. Brain module-based dynamic analysis was conducted to estimate temporal stability of the NCC candidates. Alterations in functional connectivity and modular variability from awake to propofol-induced anesthesia were assessed to test the involvement of the NCC candidates in conscious processing. NCC candidates that are characterized by both high functional interconnectivity and high modular variability were identified to locate in prefrontal and temporoparietal cortices, which covered brain structures predicted by the GNW as well as the IIT. The identified NCC was found to mainly attributed to higher-order cognitive functions, and associated with genes enriched in synaptic transmission. Dynamic analysis revealed two discrete reoccurring brain states, which were characterized by their difference in temporal stability -- the state dominated by the NCC candidates appearred to be temporally more stable than the other state predominately composed of primary sensory/motor regions, suggesting that the identified NCC members could sustain conscious contents as metastable network representations. Finally, we showed that the prefrontal GNW regions and posterior IIT regions within the identified NCC was differentially modulated in terms of functional connectedness and modular variability in response to loss of consciousness induced by propofol anesthesia. This work offers a framework to search for neural correlates of consciousness by charting the brain network topology, and provides new insights in understanding the distinct roles of the frontoparietal and posterior network in underpinning human consciousness.

HighlightsStudies suggest that there are neural correlates of consciousness (NCC) we experience subjectively everyday. By overlapping regions with both high functional interconnectivity (rich-clubness) and high modular variability, we identified the putative NCC distributed in prefrontal and temporoparietal cortices, attributed to higher-order cognitive functions, and associated with genes enriched in synaptic transmission. We further revealed that the NCC members appeared to sustain conscious contents as metastable network representations in a reoccurring NCC dominant state. The identified NCC architecture was significantly modulated in terms of functional connectedness and modular varibility during propofol anesthesia, demonstrating its critical role in supporting consciousness. These findings testify to the NCCs abilities in information integration and differentiation, and provide novel insights in reconciling the ongoing discussion of the contribution of anterior versus posterior regions in supporting human consciousness.


title: Reduced childhood social attention in an autism model marmoset predicts impaired social skills and inflexible behavior in adulthood

abstract: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social and communication impairments and restricted and repetitive behavior. Although currently no established cure exists for ASD, early intervention to the deficits of attention to other individuals is expected to reduce the progression of ASD symptoms in later life. In order to confirm this hypothesis and improve early therapeutic interventions, it is desirable to develop an animal model of ASD in which social attention is impaired in childhood and ASD-like social behavior is observed in adulthood. However, rodent models of ASD have difficulty in recapitulating the deficit of gaze-based social attention. In this study, we examined the direction of gaze towards other conspecifics during childhood and puberty in a three-chamber test setting using an ASD model of marmoset produced by maternal exposure to valproic acid (VPA). We also conducted a reversal learning test in an adult VPA-exposed marmoset as an indicator of perseveration, a core symptom of ASD that has not previously been investigated in this model. The results showed that time spent gazing at other conspecifics was reduced in VPA-exposed marmosets in childhood, and adults displayed rigidity of response. In a longitudinal study using the same animals, deficits in social attention in childhood correlated well with ASD-like social disturbance (inequity aversion and third-party reciprocity) and inflexible behavior in adulthood. Since VPA-exposed marmosets exhibit these diverse ASD-like behaviors that are coherent from childhood to adulthood, VPA-exposed marmosets will provide a valuable opportunity to elucidate mechanisms for early intervention and contribute to the development of early therapies.


title: Ultrastructural analysis of wildtype and RIM1α knock-out active zones in a large cortical synapse

abstract: Rab3A-interacting molecule (RIM) is crucial for fast Ca2+-triggered synaptic vesicle (SV) release in presynaptic active zones (AZ). While loss of RIM1 impairs long-term plasticity at hippocampal giant mossy fiber boutons (MFB), it remains unclear how AZ ultrastructure is altered. We investigated MFB AZ architecture in 3D using electron tomography of rapid cryo-immobilized acute brain slices in RIM1-/- and wild-type mice. In RIM1-/-, AZs are larger with increased synaptic cleft heights and with a three-fold reduced number of tightly docked SVs (0-2nm). The distance of tightly docked SVs to the AZ center is increased from 110 to 195 nm, and the width of their electron dense material between outer SV membrane and AZ membrane is reduced. Furthermore, the SV pool in RIM1-/- is more heterogeneous. Thus, RIM1, beside its role in tight SV docking, is crucial for synaptic architecture and vesicle pool organization in MFBs.


title: Effects of dopamine D2 and opioid receptor antagonism on the trade-off between model-based and model-free behavior in healthy volunteers

abstract: Our daily behaviour requires a flexible arbitration between actions we do out of habit and actions that are directed towards a specific goal. Drugs that target opioid and dopamine receptors are notorious for inducing maladaptive habitual drug consumption, yet how the opioidergic and dopaminergic neurotransmitter systems contribute to the arbitration between habitual and goal-directed behaviour is poorly understood. By combining pharmacological challenges with a well-established decision-making task and a novel computational model, we show that the administration of the D2 dopamine receptor antagonist amisulpride led to an increase in goal-directed or  model-based relative to habitual or  model-free behaviour, whereas the non-selective opioid receptor antagonist naltrexone had no appreciable effect. These findings highlight the distinct functional contributions of dopamine and opioid receptors to goal-directed and habitual behaviour and support the notion that D2 receptor antagonists promote stabilisation of goal-relevant information.


title: Polypyrimidine Tract Binding Protein 1 regulates the activation of mouse CD8 T cells

abstract: We show that the RNA-binding protein Polypyrimidine Tract Binding Protein 1 (PTBP1) is dispensable for the development of naive mouse CD8 T cells, but is necessary for the optimal expansion and production of effector molecules by antigen-specific CD8 T cells in vivo. PTBP1 has an essential role in regulating the early events following activation of the naive CD8 T cell leading to IL-2 and TNF production. It is also required to protect activated CD8 T cells from apoptosis. PTBP1 controls alternative splicing of over 400 genes in naive CD8 T cells in addition to regulating the abundance of [~]200 mRNAs. PTBP1 is required for the nuclear accumulation of c-Fos, NFATc2 and NFATc3, but not NFATc1. This selective effect on NFAT proteins correlates with PTBP1-promoted expression of the shorter A{beta}1 isoform and exon 13 skipped A{beta}2 isoform of the catalytic A-subunit of calcineurin phosphatase. These findings reveal a crucial role for PTBP1 in regulating CD8 T cell activation.

category: immunology


title: Neonatal Peyer's patch cDC activation as a pacemaker of postnatal immune maturation

abstract: Marked differences exist between the mucosal immune system of the neonate and adult host. The pronounced influence of the enteric microbiota in adults suggests a causal relationship between postnatal colonization and immune maturation. However, using metagenomic, metaproteomic, and functional immunological analyses we demonstrate an early presence of bacteria and immunogenic microbial antigens preceding immune maturation in the small intestine, the primary inductive site of intestinal immunity. Instead, transcriptomic, flow cytometric and histological analysis indicated neonatal Peyers patch (PP) mononuclear phagocytes (MNP) as rate limiting factor of postnatal immune maturation. Despite the early presence of MNPs, conventional dendritic cells (cDC) of type 1, 2a and 2b exhibited significant age-dependent differences in tissue distribution and cellular composition. Single cell transcriptional profiling and functional assays revealed decreased antimicrobial and antigen processing/presentation capacity, an overall retarded cell maturation and reduced antigen uptake. In cDC2a this resulted in a reduced proportion of CCR7+ migratory cells and a consequent defect in CD4 T cell priming. Interestingly, transcriptional profiling of neonatal DC subsets identified reduced expression of type I interferon (IFN)-stimulated genes (ISG). Type I IFN induction by oral administration of the TLR7 agonist R848 accelerated MNP maturation and enhanced cognate antigen CD4 T cell priming. However, humoral responses to oral vaccination in the presence of R848 were significantly reduced. Together, our results identify PP MNP maturation as pacemaker of postnatal mucosal immune priming, indicate the biological role of delayed maturation and demonstrate that targeted interventional strategies allow manipulation of mucosal responses in early life.


title: Development of a dual fluorescent reporter system in Clostridioides difficile reveals a division of labor between virulence and transmission gene expression

abstract:
The bacterial pathogen Clostridioides difficile causes gastroenteritis through its production of toxins and transmits disease through its production of resistant spores. Toxin and spore production are energy-expensive processes that are regulated by multiple transcription factors in response to many nutritional inputs. While toxin and sporulation genes are both heterogeneously expressed in only a subset of C. difficile cells, the relationship between these two sub-populations remains unclear. To address whether C. difficile coordinates the generation of these sub-populations, we developed a dual transcriptional reporter system that allows toxin and sporulation gene expression to be simultaneously visualized at the single-cell level using chromosomally-encoded mScarlet and mNeonGreen fluorescent transcriptional reporters. We then adapted an automated image analysis pipeline to quantify toxin and sporulation gene expression in thousands of individual cells in different media conditions and genetic backgrounds. These analyses revealed that toxin and sporulation gene expression rarely overlap during growth on agar plates, but broth culture increases this overlap in a manner dependent on the multifunctional RstA transcriptional regulator. Our results suggest that certain growth conditions promote a "division of labor" between transmission and virulence gene expression, highlighting how these subpopulations are influenced by environmental inputs. Given that recent work has revealed population-wide heterogeneity for numerous cellular processes in C. difficile, we anticipate that our dual reporter system will be broadly useful for determining the overlap in these subpopulations.

IMPORTANCEClostridioides difficile is an important nosocomial pathogen that causes severe diarrhea by producing toxins and is transmitted by producing spores. While both processes are crucial for C. difficile disease, only a subset of cells express toxins and/or undergo sporulation. Whether C. difficile coordinates the relationship between these energy-expensive processes remains unknown. We developed a dual fluorescent reporter system coupled with an automated image analysis pipeline to rapidly characterize expression two genes of interest across thousands of bacterial cells. Using this reporter system, we discovered that toxin and sporulation gene expression appear to undergo a "division of labor" in certain growth conditions, particularly during growth on agar plates. Since C. difficile specializes into subpopulations for numerous vital cellular processes, this novel dual reporter system will enable future studies aimed at understanding how C. difficile coordinates various subpopulations throughout its infectious disease cycle.

category: microbiology


title: The Ferrous Iron Transporter FeoB1 is Essential for Clostridioides difficile Toxin Production and Pathogenesis in Mice

abstract:
Ferrous iron is the dominant form of iron in the oxygen-limited large intestine, the site for Clostridioides difficile infection (CDI). We investigated the extent to which C. difficile requires the ferrous iron transporter 1 (FeoB1), a main permease for iron acquisition, using in vitro and in vivo approaches. Construction of feoB1 deletion mutant in C. difficile R20291 (i.e., R20291{Delta}feoB1) decreased intracellular iron content by ~25%. This was accompanied by reduced synthesis of TcdA and TcdB toxins and downregulation of tcdA and tcdB genes, which was reflected by a ~1000-fold reduction in cytopathy against Vero cells. Complementation with WT feoB1 restored toxin production. R20291{Delta}feoB1 was avirulent in mice with underlying colitis. In this model, mice are prone to develop severe CDI, but mice infected with the mutant lacked diarrheal symptoms, had lesser inflammatory responses, bacterial bioburdens and cecal toxin titers. Transcriptional and biochemical analyses revealed {Delta}feoB1 altered metabolic pathways that negatively impact toxin production, including downregulation of the oxidative branch of the Kreb cycle and an associated cellular accumulation of pyruvate. Hence FeoB1 influences multiple bioenergetic and redox pathways, which in turn inhibits toxin biosynthesis. Targeting FeoB1 could be a potential therapeutic strategy to impede the colonization and pathogenesis of C. difficile, even in the setting of colitis where CDI is more severe and intestinal bleeding may occur.

AUTHOR SUMMARYClostridioides difficile is a major cause of antibiotic associated diarrhea that result in significant morbidity and mortality in hospitals. The ability of C. difficile to cause disease and damage the gut is directly related to the production of toxin A and toxin B. Without these toxins, C. difficile is not virulent. There is need to identify drug targets and therapeutic strategies that prevent C. difficile from producing toxins A and B. For C. difficile to colonize the large bowel, it must acquire ferrous iron, which is thought to be the main form of iron within the oxygen-limited large bowel environment. We show that genetic deletion of the ferrous iron transporter FeoB1 stops C. difficile from producing significant amounts of toxins under in vitro conditions. Importantly, the mutant R20291{Delta}feoB1 was avirulent when tested in a mouse model that favors the development of severe infections. Avirulence of R20291{Delta}feoB1 was related to the strain having significant metabolic changes that negatively impact toxin production. For example, cells could not properly metabolize pyruvate, which became accumulated in cells; pyruvate is known to reduce the production of toxins A and B. Our study provides new insights into how FeoB1 affects C. difficile colonization and disease development, indicating FeoB1 could be a drug target to stop toxin production in this bacterium.


title: The importance of m6A topology in chicken embryo mRNA; a precise mapping of m6A at the conserved chicken β-actin zipcode

abstract: N6-Methyladenosine (m6A) in mRNA regulates almost every stage in the mRNA life cycle, and the development of methodologies for the high throughput detection of methylated sites in mRNA using m6A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIPSeq) or m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) have revolutionized the m6A research field. Both of these methods are based on immunoprecipitation of fragmented mRNA. However, it is well documented that antibodies often have nonspecific activities, thus verification of identified m6A sites using an antibody-independent method would be highly desirable. We mapped and quantified the m6A site in the chicken {beta}-actin zipcode based on the data from chicken embryo MeRIPSeq results and our RNA-Epimodification Detection and Base-Recognition (RedBaron) antibody independent assay. We also demonstrated that methylation of this site in the {beta}-actin zipcode enhances ZBP1 binding in vitro, whilst methylation of a nearby adenosine abolishes binding. This suggests that m6A may play a role in regulating localised translation of {beta}-actin mRNA, and the ability of m6A to enhance or inhibit a reader proteins RNA binding highlights the importance of m6A detection at nucleotide resolution.

category: molecular biology


title: Elevated p21 (CDKN1a) mediates β-thalassemia erythroid apoptosis but its loss does not improve β-thalassemic erythropoiesis

abstract:
{beta}-thalassemias are common hemoglobinopathies due to mutations in the {beta}-globin gene that lead to hemolytic anemias. Premature death of {beta}-thalassemic erythroid precursors results in ineffective erythroid maturation, increased production of erythropoietin (Epo), expansion of erythroid progenitor compartment, extramedullary erythropoiesis and splenomegaly. However, the molecular mechanism of erythroid apoptosis in {beta}-thalassemia is not well understood. Using a mouse model of {beta}-thalassemia (Hbbth3/+), we show that dysregulated expression of Foxo3 transcription factor and its upstream pro-apoptotic regulator TP53 is implicated in {beta}-thalassemia erythroid apoptosis. In Foxo3-/- /Hbbth3/+ mice, erythroid apoptosis is significantly reduced while erythroid cell maturation, red blood cell and hemoglobin production are substantially improved. However, persistence of elevated reticulocytes and splenomegaly suggests that ineffective erythropoiesis is not resolved in Foxo3-/-/Hbbth3/+. We next focused on cell cycle inhibitor Cdkn1a (p21) and show that p21 that is a target of both Foxo3 and TP53 is markedly upregulated in both mouse and patients-derived {beta}-thalassemic erythroid precursors. To address the contribution of p21 to {beta}-thalassemia pathophysiology, we generated p21-/- /Hbbth3/+ mice. Double mutant p21/Hbbth3/+ mice exhibited embryonic lethality with only a fraction of mice surviving to weaning. Notably, studies in adult mice showed apoptosis and circulating Epo were greatly reduced in erythroid compartments of surviving p21-/- /Hbbth3/+ relative to Hbbth3/+ mice, while ineffective erythroid cell maturation, extramedullary erythropoiesis and splenomegaly were not modified. These combined results indicate that while lack of Foxo3 reduces apoptosis and improves anemia, diminished p21-mediated apoptosis is insufficient to improve red blood cell production in Hbbth3/+ mice. They also suggest that a molecular network constituted by p21, FOXO3 and TP53, control erythroid cell survival and differentiation in {beta}-thalassemia. Overall, these studies provide a new framework for investigating ineffective erythropoiesis in {beta}-thalassemia.

Key PointsO_LIElevated p21 mediates {beta}-thalassemia erythroid cell apoptosis
C_LIO_LILoss of Foxo3 or p21 reduces {beta}-thalassemia erythroid cell apoptosis but does not improve {beta}-thalassemia ineffective erythropoiesis
C_LIO_LIA network of Foxo3, p21 and TP53 controls {beta}-thalassemia erythroid apoptosis
C_LIO_LIApoptosis may be uncoupled from ineffective erythropoiesis in {beta}-thalassemia
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category: developmental biology


title: The role of TGFβ signaling in Gli1+ tendon and enthesis cells

abstract: The development of musculoskeletal tissues such as tendon, enthesis, and bone relies on proliferation and differentiation of mesenchymal progenitor cells. Gli1+ cells have been described as putative stem cells in several tissues and are presumed to play critical roles in tissue formation and maintenance. For example, the enthesis, a fibrocartilage tissue that connects tendon to bone, is mineralized postnatally by a pool of Gli1+ progenitor cells. These cells are regulated by hedgehog signaling, but it is unclear if TGF{beta} signaling, necessary for tenogenesis, also plays a role in their behavior. To examine the role of TGF{beta} signaling in Gli1+ cell function, the receptor for TGF{beta}, TbR2, was deleted in Gli1-lineage cells in mice at P5. Decreased TGF{beta} signaling in these cells led to defects in enthesis formation by P56, including deficient bone morphometry underlying the enthesis and decreased mechanical properties. In vitro experiments using Gli1+ cells isolated from mouse tail tendons demonstrated that TGF{beta} controls cell proliferation and differentiation through canonical and non-canonical pathways and that TGF{beta} directly controls the tendon transcription factor scleraxis by binding to its distant enhancer. These results have implications in the development of treatments for tendon and enthesis pathologies.


title: Dynamic nucleosome organization after fertilization reveals regulatory factors for mouse zygotic genome activation

abstract: Chromatin remodeling is essential for epigenome reprogramming after fertilization. However, the underlying mechanisms of chromatin remodeling remain to be explored. Here, we investigated the dynamic changes in nucleosome occupancy and positioning in pronucleus-stage zygotes using ultra low-input MNase-seq. We observed distinct features of inheritance and reconstruction of nucleosome position in both paternal and maternal genomes. Genome-wide de novo nucleosome occupancy in the paternal genome was observed as early as 1 hour after the injection of sperm into ooplasm. The nucleosome positioning pattern was continually rebuilt to form nucleosome depletion regions (NDRs) at promoters and transcription factor (TF) binding sites with differential dynamics in paternal and maternal genomes. NDRs formed more quickly on the promoters of genes involved in zygotic genome activation (ZGA), and this formation is closely connected with histone acetylation, but not transcription elongation or DNA replication. Importantly, we found that NDR establishment on the binding motifs of specific TFs might be associated with their potential pioneer functions in ZGA. Further investigations suggested that the predicted factors MLX and RFX1 played important roles in regulating minor and major ZGA, respectively. Our data not only elucidate the nucleosome positioning dynamics in both male and female pronuclei following fertilization, but also provide an efficient method for identifying key transcription regulators during development.


title: Oncomodulin (OCM) uniquely regulates calcium signaling in neonatal cochlear outer hair cells.

abstract: In cochlear outer hair cells (OHCs), a network of Ca2+ channels, pumps and Ca2+-binding proteins (CaBPs) regulates the localization, spread, and magnitude of free Ca2+ ions. During early postnatal development, OHCs express three prominent mobile EF-hand CaBPs: oncomodulin (OCM), -parvalbumin (APV) and sorcin. We have previously shown that deletion of Ocm (Ocm-/-) gives rise to progressive cochlear dysfunction in young adult mice. Here, we show that changes in Ca2+ signaling begin early in postnatal development of Ocm-/- mice. While mutant OHCs exhibit normal electrophysiological profiles compared to controls, their intracellular Ca2+ signaling is altered. The onset of OCM expression at postnatal day 3 (P3) causes a developmental change in KCl-induced Ca2+ transients in OHCs and leads to slower KCl-induced Ca2+ transients than those elicited in cells from Ocm-/- littermates. We compared OCM buffering kinetics with other CaBPs in animal models and cultured cells. In a double knockout of Ocm and Apv (Ocm-/-;Apv-/-), mutant OHCs show even faster Ca2+ kinetics, suggesting that APV may also contribute to early postnatal Ca2+ signaling. In transfected HEK293T cells, OCM slows Ca2+ kinetics more so than either APV or sorcin. We conclude that OCM controls the intracellular Ca2+ environment by lowering the amount of freely available [Ca2+]i in OHCs and in transfected HEK293T cells. We propose that OCM plays an important role in shaping the development of early OHC Ca2+ signals through its inimitable Ca2+ buffering capacity.


title: Pervasive Under-Dominance in Gene Expression as Unifying Principle of Biomass Heterosis in Arabidopsis

abstract: Heterosis, the generally superior performance in hybrids compared to their inbred parents, is one of the most enigmatic biological phenomena. Many different explanations have been put forward for heterosis, which begs the question whether common principles underpinning it do exist at all. We performed a systematic transcriptomic study in Arabidopsis thaliana involving 141 random crosses, to search for the general principles, if any, that heterotic hybrids share. Consistent additive expression in F1 hybrids was observed for only about 300 genes enriched for roles in stress response and cell death. Regulatory rare-allele burden affects the expression level of these genes but does not correlate with heterosis. Non-additive gene expression in F1 hybrids is much more common, with the vast majority of genes (over 90%) being expressed below parental average. These include genes that are quantitatively correlated with biomass accumulation in both parents and F1 hybrids, as well as genes strongly associated with heterosis. Unlike in the additive genes, regulatory rare allele burden in this non-additive gene set is strongly correlated with growth heterosis, even though it does not covary with the expression level of these genes. Together, our study suggests that while additive complementation is an intrinsic property of F1 hybrids, the major driver of growth in hybrids derives from the quantitative nature of non-additive gene expression, especially under-dominance and thus lower expression in hybrids than predicted from the parents.

category: genomics


title: Characterizing the interplay of rubisco and nitrogenase enzymes in anaerobic-photoheterotrophically grown Rhodopseudomonas palustris CGA009 through a genome-scale metabolic and expression model

abstract:
Rhodopseudomonas palustris CGA009 (R. palustris) is a gram negative purple non-sulfur bacteria that grows phototrophically or chemotrophically by fixing or catabolizing a wide array of substrates including lignin breakdown products (e.g., p-coumarate) for its carbon and nitrogen requirements. It can grow aerobically or anaerobically and can use light, inorganic, and organic compounds for energy production. Due to its ability to convert different carbon sources into useful products in anaerobic mode, this study, for the first time, reconstructed a metabolic and expression (ME-) model of R. palustris to investigate its anaerobic-photoheterotrophic growth. Unlike metabolic (M-) models, ME-models include transcription and translation reactions along with macromolecules synthesis and couple these reactions with growth rate. This unique feature of the ME-model led to nonlinear growth curve predictions which matched closely with experimental growth rate data. At the theoretical maximum growth rate, the ME-model suggested a diminishing rate of carbon fixation and predicted malate dehydrogenase and glycerol-3 phosphate dehydrogenase as alternate electron sinks. Moreover, the ME-model also identified ferredoxin as a key regulator in distributing electrons between major redox balancing pathways. Since ME-models include turnover rate for each metabolic reaction, it was used to successfully capture experimentally observed temperature regulation of different nitrogenases. Overall, these unique features of the ME-model demonstrated the influence of nitrogenases and rubiscos on R. palustris growth and predicted a key regulator in distributing electrons between major redox balancing pathways, thus establishing a platform for in silico investigation of R. palustris metabolism from a multi-omics perspective.

IMPORTANCEIn this work, we reconstructed the first ME-model for a purple non-sulfur bacterium (PNSB). Using the ME-model, different aspects of R. palustris metabolism were examined. First, the ME-model was used to analyze how reducing power entering the R. palustris cell through organic carbon sources gets partitioned into biomass, carbon dioxide fixation, and nitrogen fixation. Furthermore, the ME-model predicted electron flux through ferredoxin as a major bottleneck in distributing electrons to nitrogenase enzymes. Next, the ME-model characterized different nitrogenase enzymes and successfully recapitulated experimentally observed temperature regulations of those enzymes. Identifying the bottleneck responsible for transferring electron to nitrogenase enzymes and recapitulating the temperature regulation of different nitrogenase enzymes can have profound implications in metabolic engineering, such as hydrogen production from R. palustris. Another interesting application of this ME-model can be to take advantage of its redox balancing strategy to gain understanding on regulatory mechanism of biodegradable plastic production precursors, such as polyhydroxybutyrate (PHB).

category: systems biology


title: Dorsolateral Striatal Selection and Frontal Cortex Inhibition for a Selective Detection Task in Mice

abstract: A learned sensory-motor behavior engages multiple brain regions, including the neocortex and the basal ganglia. How these brain regions coordinate to affect sensory selection (for target stimuli) and inhibition (for distractor stimuli) remains unknown. Here, we performed laminar electrophysiological recordings and muscimol inactivation in frontal cortex and dorsolateral striatum to determine the representations within and functions of each region during selective detection performance. From the recording experiments, in deep layers of frontal cortex and dorsolateral striatum we observed similar encoding of sensory and motor features. However, muscimol inactivation of each region resulted in drastically different outcomes. Inactivation of target-aligned striatum substantially reduced behavioral responses to all task stimuli without disrupting the ability to respond, suggesting essential roles in sensory selection. In contrast, inactivation of distractor-aligned frontal cortex increased responses to distractor stimuli, indicating essential roles in distractor inhibition. Overall, these data suggest distinct functions of frontal cortex and dorsolateral striatum in this task, despite having similar neuronal representations.


title: Transcytosis-mediated anterograde transport of TrkA receptors is necessary for sympathetic neuron development and function

abstract: In neurons, many membrane proteins, synthesized in cell bodies, must be efficiently delivered to axons to influence neuronal connectivity, synaptic communication, and repair. Previously, we found that axonal targeting of TrkA neurotrophin receptors in sympathetic neurons occurs via an atypical transport mechanism called transcytosis, which relies on TrkA interactions with PTP1B, a protein tyrosine phosphatase. Here, we generated TrkAR685A mice, where TrkA receptor signaling is preserved, but its PTP1B-dependent transcytosis is disrupted, to show that this mode of axonal transport is essential for sympathetic neuron development and autonomic function. TrkAR685A mice have decreased axonal TrkA levels in vivo, developmental loss of sympathetic neurons, and reduced innervation of targets. Postnatal TrkAR685A mice exhibit reduced pupil size and eyelid ptosis, indicative of sympathetic dysfunction. These findings establish the necessity of transcytosis in supplying TrkA receptors to sympathetic axons and highlight the physiological relevance of this axon targeting mechanism in the nervous system.


title: Mesoscale corticocortical connectivity corresponds with intracortical microstimulation-evoked neural activity

abstract:
BackgroundCortical stimulation has been a versatile technique for examining the structure and function of cortical regions as well as for implementing novel therapies. While stimulation has been used to examine the local spread of neural activity, it may also enable longitudinal examination of mesoscale interregional connectivity. Recent studies have used focal intracortical microstimulation with optical imaging to show cross-region spread of neural activity, but the exact neural mechanisms elucidated by these modalities is uncertain.

ObjectiveHere, we sought to use intracortical microstimulation (ICMS) in conjunction with recordings of multi-unit action potentials to assess the mesoscale effective connectivity within sensorimotor cortex.

MethodsNeural recordings were made from multielectrode arrays placed into sensory, motor, and premotor regions during surgical experiments in three squirrel monkeys. During each recording, single-pulse ICMS was repeatably delivered to a single region. Mesoscale effective connectivity was calculated from ICMS-evoked changes in multi-unit firing.

ResultsMulti-unit action potentials were able to be detected on the order of 1 ms after each ICMS pulse. Across sensorimotor regions, short-latency (<2.5 ms) ICMS-evoked neural activity strongly correlated with known anatomic connections. Additionally, ICMS-evoked responses remained stable across the experimental period, in spite of small changes in electrode locations and anesthetic state.

ConclusionsThese results show that monitoring ICMS-evoked neural activity is a viable way to longitudinally assess effective connectivity, enabling studies comparing the time course of connectivity changes with the time course of changes in behavioral function.

HighlightsO_LIShort-latency neural responses to single-pulse intra-cortical microstimulation (ICMS) was evaluated as a surrogate for anatomical connectivity.
C_LIO_LIAcross three new-world monkeys, the strength of neural responses strongly correlated with known anatomical connections.
C_LIO_LINeural responses to stimulation were repeatable across the duration of the experiments and were invariant to minor deviations in electrode positions and anesthetic state.
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title: Cardio-audio synchronization elicits prediction in auditory sequences during human wakefulness and sleep

abstract: The human brain can infer temporal regularities across cardiac and auditory inputs. Here, we investigated whether cardio-audio regularity processing occurs during sleep, when the balance between bodily and environmental stimulus processing may be altered. Using electroencephalography and electrocardiography in healthy volunteers (N=26) during wakefulness and sleep, we measured the response to unexpected sound omissions within three regularity conditions: synchronous, where sound and heartbeat are temporally coupled, isochronous, with fixed sound-to-sound intervals, and a third control condition without specific regularity. The synchronous and isochronous sequences induced a modulation of the omission-evoked neural response in wakefulness and N2 sleep, the latter accompanied by a reorganization of the background oscillatory activity. Cardio-audio regularity encoding was further demonstrated by a heartbeat deceleration upon omissions in all vigilance states, higher in the synchronous compared to the other regularity types. The human brain processes cardio-audio temporal relationships to predict future auditory events across distinct vigilance stages.


title: Dynamic causal modelling shows a prominent role of local inhibition in alpha power modulation in higher visual cortex

abstract:
During resting-state EEG recordings, alpha activity is more prominent over the posterior cortex in eyes-closed (EC) conditions compared to eyes-open (EO). In this study, we characterized the difference in spectra between EO and EC conditions using dynamic causal modelling. Specifically, we investigated the role of intrinsic and extrinsic connectivity--within the visual cortex--in generating EC-EO alpha power differences over posterior electrodes. The primary visual cortex (V1) and the bilateral middle temporal visual areas (V5) were equipped with bidirectional extrinsic connections using a canonical microcircuit. The states of four intrinsically coupled subpopulations--within each occipital source--were also modelled. Using Bayesian model selection, we tested whether modulations of the intrinsic connections in V1, V5 or extrinsic connections (or a combination thereof) provided the best evidence for the data. In addition, using parametric empirical Bayes (PEB), we estimated group averages under the winning model. Bayesian model selection showed that the winning model contained both extrinsic connectivity modulations, as well as intrinsic connectivity modulations in all sources. The PEB analysis revealed increased extrinsic connectivity during EC. Overall, we found a reduction in the inhibitory intrinsic connections during EC. The results suggest that the intrinsic modulations in V5 played the most important role in producing EC-EO alpha differences, suggesting an intrinsic disinhibition in higher order visual cortex, during EC resting state.

Author summaryOne of the strongest signals that can be measured using EEG are so called alpha rhythms. These are neural oscillations that fall within the 8-12Hz frequency range. Alpha rhythms are most prominent when the eyes are closed and are seen at the electrodes placed at the back of the head. In this study, we studied the mechanism of alpha rhythms changes when going from eyes-open to an eyes-closed state. We used a biologically plausible model including different neural populations. We focused on modelling connections within and between different neural sources of the visual cortex and how they are modulated when going from eyes-open to an eyes closed state. We found evidence that inhibitory neurons play an important role in alpha rhythms.


title: Periventricular nodular heterotopia is functionally coupled with the neocortex during resting and task states

abstract: Periventricular nodular heterotopia (PVNH) is a well-defined developmental disorder characterized by failed neuronal migration, which forms ectopic neuronal nodules along the ventricular walls. Previous studies mainly focus on clinical symptoms caused by the PVNH tissue, such as seizures. However, little is known about whether and how neurons in the PVNH tissue functionally communicate with neurons in the neocortex. To probe this, we applied magnetoencephalography (MEG) and stereo-electroencephalography (sEEG) recordings to patients with PVNH during resting and task states. By estimating frequency-resolved phase coupling strength of the source-reconstructed neural activities, we found that the PVNH tissue was spontaneously coupled with the neocortex in the  to {beta} frequency range, which was consistent with the synchronization pattern within the neocortical network. Furthermore, the coupling strength between PVNH and sensory areas effectively modulated the local neural activity in sensory areas. In both MEG and sEEG visual experiments, the PVNH tissue exhibited visual evoked responses, with a similar pattern and latency as the ipsilateral visual cortex. These findings demonstrate that PVNH is functionally integrated into cognition-related cortical circuits, suggesting a co-development perspective of ectopic neurons after their migration failure.


title: The Flexible Linker of Decorin Binding Protein A from Borrelia burgdorferi is a Target of Antibodies in Lyme disease

abstract:
Decorin binding protein A (DbpA) is a surface adhesin expressed by Borrelia burgdorferi, the causative agent of Lyme disease. While DbpA is one of the most immunogenic B. burgdorferi antigens in Lyme disease patients, the B cell epitopes recognized over the course of infection have not been defined. In this report we profiled [~]300 human serum samples from early, mid, and late-stage Lyme disease for IgM and IgG reactivity with DbpA and a tiled DbpA 18-mer peptide array derived from B.b. strains B31 and 297. Using ELISA and multiplex immunoassays (MIA), we identified 12 DbpA-derived peptides whose reactivity was elevated in Lyme disease patients as compared to healthy controls. The most reactive peptide ("A7") corresponds to the flexible loop between DbpAB31 -helix 1 and -helix 2, implicated in influencing DbpA binding to heparin and dermatan sulfate. An A7-like peptide is also reportedly a target of antibodies in Lyme neuroborreliosis patients. The remaining peptides, while highly reactive in serum samples across disease stages, likely represent non-native epitopes, as antibody reactivity to a subset of peptides in competition assays was unaffected by the addition of soluble DbpA. Moreover, peptide reactivity of any given patient sample rarely correlated with overall DbpA antibody levels, suggesting the antibodies were raised against DbpA degradation products. Insights into B cell epitopes on DbpA elicited during Lyme disease have important implications for understanding how B.burgdorferi persists in the face of an overwhelming antibody response.

IMPORTANCEThe bacterium, Borrelia burgdorferi, is the causative agent of Lyme disease, the most reported tick-borne illness in the United States. In humans, clinical manifestations of Lyme disease are complex and can persist for months, even in the face of a robust antibody response directed against numerous B. burgdorferi surface proteins, including decorin binding protein A (DbpA), which is involved in early stages of infection. In this study we employed [~]300 serum samples from Lyme disease patients to better understand antibody reactivity with specific regions ("epitopes") of DbpA. We identified one epitope on DbpA recognized by antibodies from almost half of the serum samples tested and that, theoretically, should block DbpAs ability to function in promoting B. burgdorferi colonization of human hosts.


title: Intercellular CRISPR Screens Enhance the Discovery of Cancer Immunotherapy Targets

abstract:
Cancer immunotherapy works through the interplay between immune and cancer cells. Particularly, interactions between cytotoxic T lymphocytes (CTLs) and cancer cells, such as PDCD1 (PD-1) and CD274 (PD-L1), are crucial for removing cancer cells. However, immune checkpoint inhibitors targeting these interactions are effective only to a subset of patients, requiring the development of novel immunotherapy drugs with novel targets.

Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening in either cancer or immune cells has been used to discover regulators of immune cell function as immunotherapeutic targets. However, the method has two main limitations. First, performing CRISPR screens in one cell type alone makes it difficult to identify essential intercellular interactions due to the focus on single genes instead of interactions. Second, pooled screening is associated with high noise levels. Therefore, we propose intercellular CRISPR screens, which perform genome-wide CRISPR screening in every interacting cell type to discover intercellular interactions as immunotherapeutic targets.

Intercellular CRISPR screens use two individual genome-wide CRISPR screens one each in immune and cancer cells to evaluate intercellular interactions that are crucial for killing cancer cells. We used two publicly available genome-wide CRISPR screening datasets obtained while triple-negative breast cancer (TNBC) cells and CTLs were interacting. We analyzed 4825 interactions between 1391 ligands and receptors on TNBC cells and CTLs to assess the effects of intercellular interactions on CTL function by incorporating both CRISPR datasets and the expression levels of ligands and receptors.

Our results showed that intercellular CRISPR screens discovered targets of approved drugs, a few of which were not identifiable using single datasets. To quantitatively evaluate the methods performance, we used data for cytokines and costimulatory molecules because they constitute the majority of immunotherapeutic targets. Combining both CRISPR datasets improved the F1 score of discovering these genes relative to using single CRISPR datasets by more than twice.

Our results indicate that intercellular CRISPR screens can identify novel immune-oncology targets that were not obtained using individual CRISPR screens. The pipeline can be extended to other cancer and immune cell types, such as natural killer cells, to identify important intercellular interactions as potential immunotherapeutic targets.


title: Eukaryotic initiation factor 2α kinases regulate virulence functions, stage conversion, and the stress response in Entamoeba invadens

abstract:
Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. This pathogen possesses a two-stage life cycle consisting of an environmentally stable cyst and a pathogenic amoeboid trophozoite. Since infection is acquired by ingestion of cysts from contaminated food and water, this parasite is prevalent in underdeveloped countries. A reptilian pathogen, Entamoeba invadens, which can encyst in culture, has long-served as a surrogate to study stage conversion. In the host, the Entamoebae must manage stress including nutrient deprivation and host immune pressure. In many systems, the stress response is characterized by down-regulation of translation, which is initiated by the phosphorylation of eukaryotic initiation factor-2 alpha (eIF2). In mammalian cells, this phosphorylation is carried out by a family of eIF2 kinases. A canonical eIF2 translational control system exists in the Entamoebae; however, no eIF2 kinases have been characterized. In this study, we identified two eIF2 kinases in E. invadens, EiIF2K-A and EiIF2K-B. Their identity as eIF2 kinases was validated using a heterologous yeast system. We used an RNAi Trigger-mediated silencing system to reduce expression of EiIF2K-A, which also reduced expression of EiIF2K-B. Parasites with decreased kinase expression exhibited decreased phosphorylation of eIF2 and increased sensitivity to oxidative stress. Diminished kinase expression also correlates with an increased rate of encystation, a decreased the rate of excystation, and an increase in several virulence functions, erythrophagocytosis and adhesion to host cells. Taken together, these data suggest that EiIF2K-A and EiIF2K-B are authentic eIF2 kinases that may regulate the Entamoeba stress response.

ImportanceEntamoeba histolytica is a human pathogen that causes dysentery and affects millions of people worldwide. This parasite possesses a two-stage life cycle: an environmentally stable cyst and the pathogenic trophozoite. Cysts are ingested from contaminated food and water; thus, this parasite in prevalent in underdeveloped countries. Current therapies commonly cause adverse side effects; therefore, new treatments are needed. In the host, Entamoeba experiences stress brought on, in part, by the host immune system. Understanding stage conversion and the stress response of this pathogen may lead to new drug therapies. Using the model organism, E. invadens, we identified two kinases, similar to those involved in stress and stage conversion in other systems. We determined that these kinases may regulate the oxidative stress response, stage conversion, and virulence. This work is significant as it will inform future studies on the life cycle and pathogenicity of the Entamoeba species.


title: Two new Rhizobiales species isolated from root nodules of common sainfoin (Onobrychis viciifolia) show different plant colonization strategies

abstract:
Root nodules of legume plants are primarily inhabited by rhizobial nitrogen-fixing bacteria. Here we propose two new Rhizobiales species isolated from root nodules of common sainfoin (Onobrychis viciifolia), as shown by core-gene phylogeny, overall genome relatedness indices and pan-genome analysis.

Mesorhizobium onobrychidis sp. nov., actively induces nodules, and achieves atmospheric nitrogen and carbon dioxide fixation. This species appears to be depleted in motility genes, and is enriched in genes for direct effects on plant growth performance. Its genome reveals functional and plant growth-promoting signatures like a large unique chromosomal genomic island with high density of symbiotic genetic traits. Onobrychidicola muellerharveyae gen. nov. sp. nov., is described as type species of the new genus Onobrychidicola in Rhizobiaceae. This species comprises unique genetic features and plant growth-promoting traits (PGPTs), which strongly indicate its function in biotic stress reduction and motility. We applied a newly developed bioinformatics approach for in silico prediction of PGPTs (PGPT-Pred), which supports the different lifestyles of the two new species and the plant growth-promoting performance of M. onobrychidis in the greenhouse trial.


title: The Endoplasmic Reticulum Membrane Complex Promotes Proteostasis of GABAA Receptors

abstract:
The endoplasmic reticulum membrane complex (EMC) plays a critical role in the biogenesis of tail-anchored and a subset of multi-pass membrane proteins in the endoplasmic reticulum. However, due to the nearly exclusive expression of neurotransmitter-gated ion channels in the central nervous system, the role of the EMC in their biogenesis is not well understood. In this study, we demonstrated that the EMC positively regulates the surface trafficking and thus function of endogenous {gamma}-aminobutyric acid (GABAA) receptors, the primary inhibitory ion channels in the mammalian brain. Further, among ten EMC subunits, EMC3 and EMC6 have the most prominent effects, indicating a subunit-specific contribution. EMC3 and EMC6 show endogenous interactions with major neuroreceptors, which depends on their transmembrane domains. Overexpression of EMC3 and EMC6 is sufficient to restore the function of epilepsy-associated GABAA receptor variants, suggesting that operating EMC has the potential to ameliorate neurological diseases associated with protein conformational defects.

In briefThe multi-subunit EMC serves as an insertase for a subset of membrane proteins and enables their biogenesis in the endoplasmic reticulum. However, the subunit-specific effect of the EMC on multi-pass neuroreceptors is not well understood. Whittsette et al. demonstrate that EMC3 and EMC6 interact with GABAA receptors and positively regulate their trafficking and function.

HighlightsO_LIEMC3 and EMC6 positively regulate the function of endogenous GABAA receptors.
C_LIO_LIThe EMC interacts with major endogenous neuroreceptors.
C_LIO_LIThe interaction between EMC and GABAA receptors depends on the EMC transmembrane domains.
C_LIO_LIOverexpressing the EMC is sufficient to restore the function of pathogenic GABAA receptor variants.
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category: cell biology


title: Follicle cell contact maintains main body axis polarity in the Drosophila melanogaster oocyte

abstract: In Drosophila melanogaster the anterior-posterior body axis is maternally established and governed by differential localisation of partitioning defective (Par) proteins within the oocyte. At mid-oogenesis, Par-1 accumulates at the posterior end of the oocyte while Par-3/Bazooka is excluded there but maintains its localisation along the remaining oocyte cortex. This mutual exclusion leads to a polarised microtubule network and accumulation of posterior determinant oskar later in oogenesis. Reciprocal biochemical interactions between Par proteins can explain their cortical exclusion and domain formation - for example, Par-1 excludes Par-3 by phosphorylation. However, past studies have proposed the need for somatic cells at the posterior end to initiate oocyte polarization by providing a trigger signal. To date, despite modern screening approaches and genetic manipulation, neither the molecular identity nor the nature of the signal is known. Here, we provide the first evidence that mechanical contact of posterior follicle cells with the oocyte cortex causes the posterior exclusion of Bazooka and maintains oocyte polarity. We show that Bazooka prematurely accumulates exclusively where posterior follicle cells have been mechanically detached or ablated. This occurs before Par-1 is removed suggesting that phosphorylation of Bazooka by Par-1 is not sufficient to maintain Bazooka exclusion. Furthermore, we provide evidence that posterior follicle cell contact maintains Par-1 localisation and microtubule cytoskeleton polarity in the oocyte. Our observations suggest that cell-cell contact mechanics modulates Par protein binding sites at the oocyte cortex.


title: The demand for wildlife not protected by the CITES multilateral treaty

abstract: The international wildlife trade presents severe conservation and environmental security risks. However, no international regulatory framework exists to monitor the trade of species not listed in the appendices of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). We explored the composition and dynamics of internationally regulated versus non-regulated trade, focussing on importations of wild-caught terrestrial vertebrates entering the United States of America (US) from 2009-2018. The prominence of the US in global wildlife imports and its detailed data collection conventions allows a unique opportunity to formally assess this substantial but often overlooked and understudied component of the legal wildlife trade. We found 3.6 times the number of unlisted species in US imports compared with CITES-listed species (1,366 versus 378). CITES-listed species were more likely to face reported conservation threats relative to the unlisted species (71.7% vs 27.5%). Yet, we found 376 unlisted species facing conversation threats, 297 species with unknown population trends and 139 species without an evaluation by the IUCN Red List of Threatened Species. Unlisted species appeared novelly in imports at 5.5 times higher rates relative to CITES-listed species, where unlisted reptiles saw the largest rate of entry, averaging 53 unique species appearing in imports for the first time per year. Overall trade volumes were substantially larger for unlisted imports with approximately 11 times the number of animals relative to CITES-listed imports, however, import volumes were similar when compared at a species-by-species level. We found that the countries that were top exporters for CITES-listed shipments were mostly different from exporters of unlisted species. In highlighting the vulnerabilities of the wild-caught unlisted vertebrate trade entering the US and in the face of increasing global demand, we recommend governments adapt policies to monitor the trade of all wildlife.

category: ecology


title: A new method to estimate the ecological niche through n-dimensional hypervolumes that combines convex hulls and elliptical envelopes

abstract:
O_LIMethods that estimate the niche of a species by calculating a convex hull or an elliptical envelope have become popular due to their simplicity and interpretation, given Hutchinsons conception of the niche as an n-dimensional hypervolume.
C_LIO_LIIt is well known that convex hulls are sensitive to outliers and do not have the ability to differentiate between regions of low and high concentration of presences, while the elliptical envelopes may contain large regions of niche space that are not relevant for the species. Thus, when the goal is to estimate the realized niche of the species, both methods may overestimate the niche.
C_LIO_LIWe present a methodology that combines both the convex hull and the elliptical envelope methods producing an n-dimensional hypervolume that better fits the observed density of species presences, making it a better candidate to model the realized niche. Our method, called the CHE approach, allows defining regions of iso-suitability as a function of the significance levels inherited from the method (Mahalanobis distance model, minimum covariance determinant, or minimum volume ellipsoid) used to fit an initial elliptical envelope from which we then discard regions not relevant for the species by calculating a convex hull.
C_LIO_LIWe applied the CHE approach to a case study of twenty-five species of bats present in the Iberian Peninsula, fitting a hypervolume for each species and comparing them to both the convex hulls and elliptical envelopes obtained with the same data and different values of n. We show that as the number of variables used to define the niche space increases, both the convex hull and elliptical envelope models produce overly large hypervolumes, while the size of the hypervolume fitted with the CHE approach remains stable. As a consequence, similarity measures that account for the niche overlap among different species may be inflated when using convex hulls or elliptical envelopes to model the niche; something that does not occur under the CHE approach.
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title: Coexistence in diverse communities with higher-order interactions

abstract: A central assumption in most ecological models is that the interactions in a community operate only between pairs of species. However, the interaction between two species may be fundamentally changed by the presence of others. Although interactions among three or more species, called higher-order interactions, have the potential to modify our theoretical understanding of coexistence, ecologists lack clear expectations for how these interactions shape community structure. Here, we analytically predict and numerically confirm how the variability and strength of higher-order interactions affect species coexistence. We found that, as higher-order interaction strengths become more variable across species, fewer species coexist, echoing the behavior of pairwise models. If inter-specific higher-order interactions become too harmful relative to self-regulation, coexistence was destabilized, but coexistence was also lost when these interactions were too weak and mutualistic effects became prevalent. Last, we showed that more species rich communities structured by higher-order interactions lose species more readily than their species poor counterparts, generalizing classic results for community stability. Our work provides needed theoretical expectation for how higher-order interactions impact species coexistence in diverse communities.


title: Stabilization of recombinant proteins by fusion with heat-resistant obscure (Hero) proteins

abstract: Although recombinant proteins are widely used in biotechnology and pharmaceutical industries, improving their solubility and stability is often a challenging issue. We recently discovered a class of highly unstructured heat-resistant obscure (Hero) proteins, which function to protect other "client" proteins in trans from various stresses in vitro and in vivo. Here, we show that Hero proteins can enhance the stability of recombinant proteins when fused in cis. Fusion with Hero proteins improved the otherwise challenging production of TAR DNA-binding protein of 43 kDa (TDP-43) in Escherichia coli. Moreover, fusing with Hero proteins strongly protected the activity of firefly luciferase bearing destabilizing mutations under various stress conditions. These data suggest that Hero proteins can be used as versatile stabilization tags for recombinant protein production.

category: biochemistry


title: Highly efficient protein expression of Plasmodium vivax surface antigen, Pvs25 by silkworm, Bombyx mori, and its biochemical analysis.

abstract: Plasmodium vivax ookinete surface protein, Pvs25 is a transmission-blocking vaccine (TBV) candidate for malaria. Pvs25 has four EGF-like domains containing 22 cysteine residues forming 11 intramolecular disulfide bonds and this structural feature makes recombinant expression of Pvs25 difficult. In this study, we report the high expression of recombinant Pvs25 as a soluble form in silkworm, Bombyx mori. The Pvs25 protein was purified from hemolymphs of larvae and pupae by affinity chromatography. In the Pvs25 expressed by silkworm, no isoform with inappropriate disulfide bonds was found, requiring no further purification step which is necessary in case of Pichia pastoris based expressions systems. The Pvs25 from silkworm were confirmed to be the molecularly uniform by sodium dodecyl sulfate gel electrophoresis analysis and size exclusion chromatography analysis. To examine the immunogenicity, the Pvs25 from B. mori, was administered to BALB/c mice by the subcutaneous (s.c.) route with the oil adjuvant. The Pvs25 produced by silkworm induced potent and robust immune response, and the induced antisera correctly recognized P. vivax ookinetes in vitro, demonstrating the potency of Pvs25 from silkworm as a TBV candidate for malaria. This is the first study that to construct a mass production system for malaria TBV antigens by the silkworm to the best of our knowledge.


title: Deep learning on reflectance confocal microscopy improves Raman spectral diagnosis of basal cell carcinoma

abstract:
SignificanceRaman spectroscopy may be useful to assist Mohs micrographic surgery for skin cancer diagnosis; however, the specificity of Raman spectroscopy is limited by the high spectral similarity between tumors and normal tissues structures such as epidermis and hair follicles. Reflectance confocal microscopy (RCM) can provide imaging guidance with morphological and cytological details similar to histology. Combining Raman spectroscopy with deep-learning-aided RCM has the potential to improve the diagnostic accuracy of Raman without requiring additional input from the clinician.

AimWe seek to improve the specificity of Raman for basal cell carcinoma (BCC) by integrating information from RCM images using an Artificial Neural Network.

ApproachA Raman biophysical model was used in prior work to classify BCC tumors from surrounding normal tissue structures. 191 RCM images were collected from the same site as the Raman data and served as inputs to train two ResNet50 networks. The networks selected the hair structure images and epidermis images respectively within all the images corresponding to the positive predictions of the Raman Biophysical Model.

ResultsDeep learning on RCM images removes 54% of false positive predictions from the Raman Biophysical Model result and keeps the sensitivity as 100%. The specificity was improved from 84.8% by using Raman spectra alone to 93.0% by integrating Raman spectra with RCM images

ConclusionsCombining Raman spectroscopy with deep-learning-aided RCM imaging is a promising tool to guide tumor resection surgery.

category: bioengineering


title: Impact of Inert Crowders on Host-Guest Recognition Process

abstract:
Biological environments typically contain high concentrations (300-400 mg/mL) of different macromolecules at volume fractions as large as 30%-40%. Biomolecular processes, especially ubiquitous recognition phenomena, occurring in such crowded heterogeneous media would differ significantly compared to the dilute buffer solutions. Here we quantify the impact of inert crowders on prototypical host-guest recognition process by explicit-solvent molecular dynamics (MD) simulations in atomic resolution. We demonstrate that the crowders, especially when smaller in size, would facilitate the binding process of guest molecule by decreasing the free energy barrier for binding via excluded volume effect and desolvation of the host receptor. However, the extent of crowder-induced stabilization of host-guest complex is found to be significantly higher when the guest molecule is sterically constricted to approach the host along a centrosymmetric direction, contrary to its unrestricted, freely diffusive movement. A kinetic analysis of the recognition process reveals that the origin of relatively stronger crowder impact, during constricted movement of guest molecule, lies in significantly enhanced residence time of the guest inside the host by crowders, compared to freely diffusive ligand movement. Together our results suggest that the extent of im-pact of crowding on recognition processes would be contingent upon presence or absence of constriction on ligand movement.

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category: biophysics


title: Cellular and clinical impact of Protein Phosphatase Enzyme epigenetic silencing in multiple cancer tissues.

abstract: Protein Phosphatase Enzymes (PPE) and protein kinases simultaneously control phosphorylation mechanisms that tightly regulate intracellular signaling pathways and stimulate cellular responses. In human malignancies, PPE and protein kinases are frequently mutated resulting in uncontrolled kinase activity and PPE suppression, leading to cell proliferation, migration and resistance to anti-cancer therapies. Cancer associated DNA hypermethylation at PPE promoters gives rise to transcriptional silencing (epimutations) and is a hallmark of cancer. Despite recent advances in sequencing technologies, data availability and computational capabilities, only a fraction of PPE have been reported as transcriptionally inactive as a consequence of epimutations. Using the Infinium HumanMethylation450 BeadChip, we compared DNA methylation profiles from 705 cancer patients across 5 major tissues and 3 cancer cell models against a cohort of healthy controls. Here, we report epimutations in PPE (and their interacting proteins or PPEIP) are a frequent occurrence in the cancer genome and manifest independent of transcriptional activity. We observed that different tumors have varying susceptibility to epimutations and identify specific cellular signalling networks that are primarily affected by epimutations. Additionally, RNA-seq analysis showed the negative impact of epimutations on most (not all) Protein Tyrosine Phosphatase transcription. Finally, we detected novel clinical biomarkers that inform on patient mortality and anti-cancer treatment sensitivity. We propose that DNA hypermethylation marks at PPEIP frequently contribute to the pathogenesis of malignancies and within the precision medicine space, hold promise as biomarkers to inform on clinical features such as patient survival and therapeutic response.

category: cancer biology


title: Comments on snails of the genus Zoniferella from Ecuador (Mollusca: Achatinidae), with restriction of the type locality "Los Puentes" for several species of Gastropoda and Arachnida

abstract: The genus Zoniferella includes six taxa of land snails from Colombia and Ecuador for which little has been published beyond their original descriptions. In this paper, we present new records of Zoniferella vespera, a species previously known only from its type locality, expanding its range across northwestern Ecuador. We provide the first description of the colouration in life for Zoniferella snails. We comment on the similarities among some species of Zoniferella, suggesting the possibility that Z. vespera and Z. riveti are junior synonyms of Z. albobalteata, and Z. bicingulata is a synonym of Z. riveti var. bizonalis (as Z. bizonalis). Finally, we offer evidence to allow the restriction of the locality of "Los Puentes", type locality of Mesembrinis vesperus Jousseaume, 1887 = Zoniferella vespera (Gastropoda: Achatinidae); Isomeria bourcieri lutea Cousin, 1887 (Gastropoda: Labyrinthidae); Guestieria locardi Jousseaume, 1887 (Gastropoda: Scolodontidae); Proserpinella cousini Jousseaume, 1887 (Gastropoda: Proserpinellidae); Idiophthalma robusta Simon, 1889 (Arachnida: Barychelidae); Eurypelma (Lasiodora) augusti Simon, 1889 = Pamphobeteus augusti (Arachnida: Theraphosidae), Eurypelma (Lasiodora) vespertinus Simon, 1889 = Pamphobeteus vespertinus (Arachnida: Theraphosidae).

category: zoology


title: Thoracic crop formation is spatiotemporally coordinated with flight muscle histolysis during claustral colony foundation of a Lasius japonicus queen

abstract: In a majority of ants, a newly mated queen independently founds a colony and claustrally raises her first brood without foraging outside the nest. During claustral independent colony foundation (ICF) in several ants, the esophagus of the founding queen expands and develops into a thoracic crop, which is then filled with a liquid substrate for larval feeding. It has been suggested that these substrates are converted from her body reserves (e.g., histolyzed flight muscles) or redistributed from a gastral crop. Here, we examined thoracic crop development in Lasius japonicus during claustral ICF. The foundresses claustrally fed their larvae from week 2 to 5 after ICF onset, and the first worker emerged at week 6. The development proceeded as follows: in week 0, foundress dorsal esophagus wall was pleated and thickened. Then, from week 2 to 5, the esophagus expanded dorsally toward where flight muscles had been present, following flight muscle histolysis. Gastral crop expansion followed esophagus expansion. Thus, thoracic crop formation may be spatiotemporally coordinated with flight muscle histolysis in Lasius japonicus queens, and similar developmental regulations might be common in other claustral ICF ants.


title: Parallel Mechanosensory Systems are Required for Negative Gravitaxis in C. elegans

abstract: The ability to sense Earths gravitational pull is essential for orientation, navigation, and proprioception in most organisms. Here, we demonstrate that C. elegans adults and dauers exhibit negative gravitaxis and that this behavior is modulated by other environmental inputs such as light and electromagnetic fields. We found that components of the DEG/ENaC pathway involved in gentle touch sensation - specifically the neuronally-enriched microtubule subunits MEC-7 and MEC-12, but not the related MEC-4 and MEC-10 mechanosensory subunits - are required for negative gravitaxis. In addition, we found that TRPA-1, a receptor protein associated with cold and mechanosensation in both mammals and invertebrates, is critical for this behavior. Finally, we show that PVD neurons, which utilize TRPA-1 to detect harsh cold, are also essential for gravity sensation. Conversely, TRNs are not required for this behavior. These findings implicate a complex, interconnected mechanism for gravity sensation involving an ion channel that is also present in the mammalian vestibular system.


title: Not digested: algal glycans move carbon dioxide into the deep-sea

abstract:
Marine algae annually synthesize gigatons of glycans from carbon dioxide, exporting it within sinking particles into the deep-sea and underlying sea floor, unless those glycans are digested before by bacteria. Identifying algal glycans in the ocean remains challenging with the molecular resolution of conventional analytic techniques. Whether algal glycans are digested by heterotrophic bacteria during downward transport, before they can transfer carbon dioxide from the ocean surface into the deep-sea or the sea floor, remains unknown. In the Red Sea Shaban Deep, where at 1500 m water depth a brine basin acts as a natural sediment trap, we found its high salt and low oxygen concentration accumulated and preserved exported algal glycans for the past 2500 years. By using monoclonal antibodies specific for glycan structures, we detected fucose-containing sulfated polysaccharide, {beta}-glucan, {beta}-mannan and arabinogalactan glycans, synthesized by diatoms, coccolithophores, dinoflagellates and other algae living in the sunlit ocean. Their presence in deep-sea sediment demonstrates these algal glycans were not digested by bacteria. Instead they moved carbon dioxide from the surface ocean into the deep-sea, where it will be locked away from the atmosphere at least for the next 1000 years. Considering their global synthesis, quantity and stability against degradation during transport through the water column, algal glycans are agents for carbon sequestration.

Significance statementAlgae and plants use the greenhouse gas carbon dioxide to synthesize polymeric carbohydrates, or glycans, for energy storage, structural support and as protection against invasion by microbes. Glycans provide protection, are carbon sinks and enable carbon sequestration for as long as they are not digested by bacteria or other organisms, which releases the carbon dioxide back in to the atmosphere. In this study, we show that non-digested algal glycans sink into the deep ocean and into marine sediment. Thus, glycans are more than food for animals and prebiotics for bacteria, they are also molecules that remove carbon dioxide from the atmosphere and transfer it to the deep-sea, where it can be stored for 1000 years and longer.


title: Immunogenicity of an Ad26-based SARS-CoV-2 Omicron Vaccine in Naïve Mice and SARS-CoV-2 Spike Pre-immune Hamsters

abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant sparked concern due to its fast spread and the unprecedented number of mutations in the spike protein that enables it to partially evade spike-based COVID-19 vaccine-induced humoral immunity. In anticipation of a potential need for an Omicron spike-based vaccine, we generated an Ad26 vector encoding an Omicron (BA.1) spike protein (Ad26.COV2.S.529). Ad26.COV2.S.529 encodes for a prefusion stabilized spike protein, similar to the current COVID-19 vaccine Ad26.COV2.S encoding the Wuhan-Hu-1 spike protein. We verified that spike expression by Ad26.COV2.S.529 was comparable to Ad26.COV2.S. Immunogenicity of Ad26.COV2.S.529 was then evaluated in naive mice and SARS-CoV-2 Wuhan-Hu-1 spike pre-immunized hamsters. In naive mice, Ad26.COV2.S.529 elicited robust neutralizing antibodies against SARS-CoV-2 Omicron (BA.1) but not to SARS-CoV-2 Delta (B.1.617.2), while the opposite was observed for Ad26.COV2.S. In pre-immune hamsters, Ad26.COV2.S.529 vaccination resulted in robust increases in neutralizing antibody titers against both SARS-CoV-2 Omicron (BA.1) and Delta (B.1.617.2), while Ad26.COV2.S vaccination only increased neutralizing antibody titers against the Delta variant. Our data imply that Ad26.COV2.S.529 can both expand and boost a Wuhan-Hu-1 spike-primed humoral immune response to protect against distant SARS-CoV-2 variants.


title: Highly Thermotolerant SARS-CoV-2 Vaccine Elicits Neutralising Antibodies Against Delta and Omicron in Mice

abstract: As existing vaccines fail to completely prevent COVID-19 infections or community transmission, there is an unmet need for vaccines that can better combat SARS-CoV-2 variants of concern (VOC). We have previously developed highly thermo-tolerant monomeric and trimeric receptor binding domain derivatives that can withstand 100{degrees}C for 90 minutes and 37{degrees}C for four weeks, and help eliminate cold chain requirements. We show that mice immunised with these vaccine formulations elicit high titres of antibodies that neutralise SARS-CoV-2 variants VIC31 (with Spike: D614G mutation), Delta and Omicron (BA.1.1) VOC. Compared to VIC31, there was an average 14.4-fold reduction in neutralisation against BA.1.1 for the three monomeric antigen-adjuvant combinations, and 16.5-fold reduction for the three trimeric antigen-adjuvant combinations; the corresponding values against Delta were 2.5 and 3.0. Our findings suggest that monomeric formulations are suitable for the upcoming Phase I human clinical trials, and that there is potential for increasing efficacy with vaccine matching to improve responses against emerging variants. These findings are consistent with in silico modelling and AlphaFold predictions which show that while oligomeric presentation can be generally beneficial, it can make important epitopes inaccessible, and also carries the risk of eliciting unwanted antibodies against the oligomerisation domain.


title: Campylobacter jejuni modulates reactive oxygen species production and NADPH oxidase 1 expression in human intestinal epithelial cells

abstract: Campylobacter jejuni is the major bacterial cause of foodborne gastroenteritis worldwide. Mechanistically, how this pathogen interacts with intrinsic defence machinery of human intestinal epithelial cells (IECs) remains elusive. To address this, we investigated how C. jejuni counteracts the intracellular and extracellular reactive oxygen species (ROS) in IECs. Our work shows that C. jejuni differentially regulates intracellular and extracellular ROS production in human T84 and Caco-2 cells. C. jejuni downregulates the transcription and translation of Nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase (Nox1), a key ROS-generating enzyme in IECs and antioxidant defence genes cat and sod1. Furthermore, inhibition of Nox1 by diphenylene iodonium (DPI) and siRNA reduced C. jejuni ability to interact, invade and intracellularly survive within T84 and Caco-2 cells. Collectively, these findings provide mechanistic insight into how C. jejuni modulates the IEC defence machinery.


title: Gut microbe Lactiplantiballus plantarum undergoes different evolutionary trajectories between insects and mammals

abstract: Animals form complex symbiotic associations with their gut microbes, whose evolution is determined by an intricate network of host and environmental factors. In many insects, such as Drosophila melanogaster, the microbiome is flexible, environmentally determined and less diverse than in mammals. In contrast, mammals maintain complex multispecies consortia that are able to colonize and persist in the gastrointestinal tract. Understanding the evolutionary and ecological dynamics of gut microbes in different hosts is challenging. This requires disentangling the ecological factors of selection, determining the timescales over which evolution occurs, and elucidating the architecture of such evolutionary patterns. Here, we employ experimental evolution to track the pace of the evolution of a common gut commensal, Lactiplantibacillus plantarum, within invertebrate (Drosophila melanogaster) and vertebrate (Mus musculus) hosts and their respective diets. While the symbiosis between Drosophila and L. plantarum is mainly determined by host diet, with the host influencing the fitness of its gut microbes only over short timescales, bacterial evolution within the mammalian host follows a divergent evolutionary path. Here, the host and its intrinsic factors play a critical role in selection and influence both the phenotypic and genomic evolution of its gut microbes, as well as the outcome of their symbiosis.


title: The interaction of calcium ions with specific residues in the SARS-CoV fusion peptide and the regulation of viral infectivity

abstract:
Viral envelope fusion with the host cell membrane is dependent on a specific viral fusion peptide (FP) or loop, which becomes exposed during virus entry to drive the process of membrane fusion. In coronaviruses, the FP is a highly conserved domain that sits in the center of spike protein and in SARS-CoV, is adjacent to the S2 proteolytic cleavage site. This peptide contains a hydrophobic LLF motif, as well as several conserved negatively charged amino acids that interact with Ca2+ ions to promote membrane fusion. In this work we perform a systematic mutagenesis study of the negatively charged amino acids within the SARS-CoV fusion peptide (FP1/FP2) and combine this with molecular dynamics simulations to define the membrane interactions that regulate virus infectivity. We show that the E801/D802 amino acid pair in the SARS-CoV FP is predicted to bind to one Ca2+ ion to promote FP-membrane interaction, with a second Ca2+ ion likely pairing residue D812 with either E821 or D825. The D812/D821 residue pair promotes membrane interaction, whereas the D821/D825 is inhibitory to membrane insertion. Taken together, our results demonstrate the dynamic nature of the coronavirus FP region that likely facilitates its interactions with and insertion into the host cell membrane.

Author SummaryCoronaviruses have reemerged as a highly pathogenic virus family through the rise of SARS-CoV, MERS-CoV, and more recently, SARS-CoV-2. As more transmissible variants of SARS-CoV-2 arise, it is imperative that we understand the mechanisms of CoV viral entry to enable the development of effective therapeutics. Recent reviews have suggested the repurposing of FDA-approved calcium channel blockers to treat infection by coronaviruses; however, calciums method of action on viral-host cell fusion events is unknown. We have found that increased calcium availability leads to increased viral infection across the CoV family, suggesting that calcium is involved in mediating the interaction between the viral fusion peptide and the host cell membrane. As such, we hypothesize that the highly conserved fusion peptide interacts directly with calcium and this interaction is required for viral entry and infection. Through mutagenesis studies of specific negatively charged residues in the fusion peptide, we have identified residues that impact viral infectivity. We have also compared the infectivity of wild-type and mutant CoV pseudoparticles in calcium-rich or -depleted environments using chelating drugs. Our data mirrors the residue coordination observed SARS-CoV-2, as both between SARS-CoV and SARS-CoV-2 FPs bind to two calcium ions. These results demonstrate the importance of Ca2+ for CoV FP function during viral entry and opens the possibility of utilizing FDA-approved calcium-blocking drugs as a treatment for COVID-19.


title: A mass spectrometry-based atlas of extracellular matrix proteins across 25 mouse organs

abstract: The extracellular matrix is a critical non-cellular component of multi-cellular organisms containing a variety of proteins, glycoproteins, and proteoglycans which has been implicated in a wide variety of essential biological processes, including development, wound healing, and aging. Due to low solubility, many ECM proteins have been underrepresented in previous proteomics datasets. Using an optimized 3-step decellularization and ECM extraction method involving chaotrope extraction and digestion via hydroxylamine hydrochloride, we have generated coverage of the matrisome across 25 organs. We observe that the top 100 most abundant proteins from the ECM fractions of all tissues are generally present in all tissues, indicating that tissue matrices are principally composed of a shared set of ECM proteins. However, these proteins vary up to 4,000-fold between tissues, resulting in highly unique matrix profiles even with the same primary set of proteins. A data reduction approach was used to reveal related networks of expressed ECM proteins across varying tissues, including basement membrane and collagen subtypes.


title: DNA supercoiling restricts the transcriptional bursting of neighboring eukaryotic genes

abstract:
DNA supercoiling has emerged as a major contributor to gene regulation in bacteria. The impact of DNA supercoiling on transcription dynamics in eukaryotes is less clear. Here, using single-molecule dual-color RNA imaging in budding yeast, we show that transcriptional bursting of the divergent and tandem GAL genes is coupled. Upon topoisomerase degradation, supercoils that buildup from transcription inhibit subsequent transcription at neighboring genes, thereby reducing their simultaneous bursting. GAL gene transcription is inhibited more by negative than by positive supercoiling accumulation. Unlike bacteria, wildtype yeast has sufficient topoisomerase levels to minimize inhibition from supercoils at adjacent genes. Overall, we discover fundamental differences in supercoiling-mediated gene regulation between bacteria and yeast and show that rapid supercoiling release in eukaryotes ensures proper gene expression of neighboring genes.

One sentence summaryTranscription causes twisting of the DNA double helix, which can inhibit transcription of adjacent genes.


title: Notch signaling in germ line stem cells controls reproductive aging in C. elegans

abstract: Reproductive aging in females often occurs early in life, resulting in a substantial post-reproductive lifespan. Despite the medical importance of age-related infertility, relatively little is known about mechanisms that control this age-related decline. C. elegans is a leading system for aging biology due to its short lifespan and powerful experimental tools, and detailed descriptions of molecular and cellular changes in the gonad during reproductive aging were recently reported. Here we show that reproductive aging occurs early in life in multiple species in the genus Caenorhabditis, indicating this is a feature of both female/male and hermaphrodite/male species. In mutants previously established to display delayed reproductive aging (daf-2, eat-2, phm-2), we observed correlations between changes in the distal germline and changes in egg-laying, consistent with the model that distal germline changes are a cause of reproductive aging. By screening for additional mutants that delay reproductive aging, we identified an allele of che-3 with impaired sensory perception that displayed increased progeny production in mid-life, a pattern of reproductive aging distinct from previous mutants. To directly test the role of Notch signaling in the distal germline, we analyzed the effect of ectopic expression of the Notch effector gene SYGL-1. Ectopic expression of SYGL-1 was sufficient to delay reproductive aging, suggesting that an age-related decline in Notch signaling in the distal germline is a root cause of reproductive aging.

category: genetics


title: The Hydractinia Allorecognition Complex encodes a novel family of immunoglobulin superfamily proteins

abstract:
Most colonial marine invertebrates are capable of allorecognition, the ability to distinguish between themselves and conspecifics. One long-standing question is whether invertebrate allorecognition genes are homologous to vertebrate histocompatibility genes. In the cnidarian Hydractinia symbiolongicarpus, allorecognition is controlled by at least two genes, Allorecognition 1 (Alr1) and Allorecognition 2 (Alr2), which encode highly polymorphic cell surface proteins that serve as markers of self. Here, we show that Alr1 and Alr2 are part of a family of 41 Alr genes, all of which reside a single genomic interval called the Allorecognition Complex (ARC). Using sensitive homology searches and highly accurate structural predictions, we demonstrate that the Alr proteins are members of the immunoglobulin superfamily (IgSF) with V-set and I-set Ig domains unlike any previously identified in animals. Specifically, their primary amino acid sequences lack many of the motifs considered diagnostic for V-set and I-set domains, yet they adopt secondary and tertiary structures nearly identical to canonical Ig domains. Thus, the V-set domain, which played a central role in the evolution of vertebrate adaptive immunity, was present in the last common ancestor of cnidarians and bilaterians. Unexpectedly, several Alr proteins also have immunoreceptor tyrosine-based activation motifs (ITAMs) and immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic tails, suggesting they could participate in pathways homologous to those that regulate immunity in humans and flies. This work expands our definition of the IgSF with the addition of a family of unusual members, several of which play a role in invertebrate histocompatibility.

Significance StatementThe immunoglobulin superfamily (IgSF) is one of the largest and most functionally versatile domain families in animal genomes. Although their amino acid sequences can vary considerably, IgSF domains have been traditionally defined by conserved residues at several key positions in their fold. Here, we sequenced an invertebrate histocompatibility complex and discovered a family of IgSF genes with amino acid sequences that lack most of these residues yet are predicted to adopt folds virtually identical to canonical V-set and I-set IgSF domains. This work broadens the definition of the IgSF and shows that the V-set domain was present earlier in animal evolution than previously appreciated.


title: A family of unusual immunoglobulin superfamily genes in an invertebrate histocompatibility complex


title: Zinc pyrithione is a potent inhibitor of PLPro and cathepsin L enzymes with ex vivo inhibition of SARS-CoV-2 entry and replication

abstract:
As SARS-CoV-2 triggered a global health crisis, there is an urgent need to provide patients with safe, effective, accessible, and preferably oral therapeutics for COVID-19 that complement mRNA vaccines. Zinc compounds are widely known for their antiviral properties. Therefore, we have prepared a library of zinc complexes with pyrithione (1-hydroxy-2(1H)-pyridinethione) and its analogues, all of which showed promising in vitro inhibition of cathepsin L, an enzyme involved in SARS-CoV-2 entry, and PLPro, an enzyme involved in SARS-CoV-2 replication both in (sub)micromolar range. Zinc pyrithione 1a is a well-established, commercially available antimicrobial agent and was therefore selected for further evaluation of its SARS-CoV-2 entry and replication inhibition in an ex vivo system derived from primary human lung tissue. Our results suggest that zinc pyrithione complex 1a provides a multitarget approach to combat SARS-CoV-2 and should be considered for repurposing as a potential therapeutic against the insidious COVID-19 disease.

Featured imageIn our study, we show that zinc pyrithione holds immense potential for the development of a possible out-patient treatment for SARS-CoV-2 due to its inhibition of viral entry and replication.

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title: Elaboration of a MALDI-TOF Mass Spectrometry-based Assay of Parkin Activity and High-Throughput screening platform for Parkin Activators

abstract: Parkinsons disease (PD) is a progressive neurological disorder that manifests clinically as alterations in movement (bradykinesia, postural instability, loss of balance, and resting tremors) as well as multiple non-motor symptoms including but not limited to cognitive and autonomic abnormalities. Mitochondrial dysfunction has been linked to sporadic PD and loss-of-function mutations in genes encoding the ubiquitin E3 ligase Parkin and protein kinase, PTEN-induced kinase 1 (PINK1), that regulate mitophagy, are causal for familial and juvenile PD1-3. Among several therapeutic approaches being explored to treat or improve PD patients prognosis, the use of small molecules able to reinstate or boost Parkin activity represents a potential pharmacological treatment strategy4. A major barrier is the lack of high throughput platforms based on robust and accurate quantification of Parkin activity in vitro. Here we present two different and complementary Matrix Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (MALDI-TOF MS) based approaches for the quantification of Parkin E3 ligase activity in vitro. These methods recapitulate distinct aspects of ubiquitin conjugation: Parkin auto-ubiquitylation and Parkin-catalysed discharge on lysine residues. Both approaches are scalable for high-throughput primary screening to facilitate the identification of Parkin modulators.