title: The E3 Ubiquitin Ligase Mindbomb1 controls zebrafish Planar Cell Polarity

abstract:
Vertebrate Delta/Notch signaling involves multiple ligands, receptors and transcription factors. Delta endocytosis - a critical event for Notch activation - is however essentially controlled by the E3 Ubiquitin ligase Mindbomb1 (Mib1). Due to its position at a molecular bottleneck of the pathway, Mib1 inactivation is often used to inhibit Notch signaling. However, recent findings indicate that the importance of Mib1 extends beyond the Notch pathway. We report an essential role of Mib1 in Planar Cell Polarity (PCP).

mib1 null mutants or morphants display impaired gastrulation stage Convergence Extension (CE) movements. Comparison of different mib1 mutants and functional rescue experiments indicate that Mib1 controls CE independently of Notch. In contrast, Mib1-dependent CE defects can be rescued using the PCP downstream mediator RhoA. Mib1 regulates CE through the RING Finger domains that have been implicated in substrate ubiquitination, suggesting that Mib1 may control PCP protein trafficking. Accordingly, we show that Mib1 controls the endocytosis of the PCP component Ryk and that Ryk internalization is required for CE.

Numerous morphogenetic processes involve both Notch and PCP signaling. We show that Mib1, a known Notch signaling regulator, is also an essential PCP pathway component. Care should therefore be taken when interpreting Mib1 loss of function phenotypes.

category: developmental biology


title: Embigin deficiency leads to delayed embryonic lung development and high neonatal mortality

abstract:
Embigin (gp70), a transmembrane glycoprotein, has been shown to regulate hematopoietic stem cell and progenitor cell niche. Still, little is known about its expression and function in other organ systems during development or adulthood. By combining immunofluorescence, RNA sequencing, and in vivo mouse models, we show that embigin is highly expressed during development and in adult lung, kidney, epididymis, skin, and testis. Adult Emb-/- mice have a normal lifespan and fertility without apparent pathologies. In contrast, the Emb-/- embryos are significantly smaller than their WT littermates. Markedly increased mortality of the Emb-/- embryos is seen especially during the neonatal period. Embigin is present in the placenta, but placental morphology and gene expression patterns stay unaltered. At E17.5, Emb-/- mice show defective morphogenesis of the lung, low alkaline phosphatase activity in amniotic fluid, and remarkable activation of genes involved in cell proliferation in the lungs. Thus, lung underdevelopment explains the high neonatal mortality. Our work demonstrates the crucial role of embigin during development, and it paves the way to further characterization of embigin in specific organ systems in development and homeostasis.

Summary statementEmbigin is a basigin-group transmembrane glycoprotein. In vivo mouse model shows that embigin is crucial for embryonic lung development and neonatal survival.


title: Periosteum-derived podoplanin-expressing stromal cells regulate nascent vascularization during epiphyseal marrow development

abstract: Bone marrow development and endochondral bone formation occur simultaneously. During endochondral ossification, periosteal vasculatures and stromal progenitors invade the primary avascular cartilaginous anlage; this induces primitive marrow development. We previously determined that bone marrow podoplanin (PDPN)-expressing stromal cells exist in a perivascular microenvironment, and promote megakaryopoiesis and erythropoiesis. In this study, we aimed to examine the involvement of PDPN-expressing stromal cells in the postnatal bone marrow generation. We found that periosteum-derived PDPN-expressing stromal cells regulate vascularization during postnatal epiphyseal marrow development. Our findings suggest that these cells act as pericytes on the primitive vasculature of the nascent marrow. They invade the cartilaginous epiphysis and regulate marrow development and homeostasis by maintaining vascular integrity. To the best of our knowledge, this is the first study to comprehensively examine how PDPN-expressing stromal cells contribute to marrow development and homeostasis.


title: ECM-integrin signalling instructs cellular position-sensing to pattern the early mouse embryo

abstract: Development entails patterned emergence of diverse cell types within the embryo. In mammals, cells positioned inside the embryo gives rise to the inner cell mass (ICM) that eventually forms the embryo proper. Yet the molecular basis of how these cells recognise their  inside position to instruct their fate is unknown. Here we show that cells perceive their position through extracellular matrix (ECM) and integrin-mediated adhesion. Provision of ECM to isolated embryonic cells induces ICM specification and alters subsequent spatial arrangement between epiblast (EPI) and primitive endoderm (PrE) cells that emerge within the ICM. Notably, this effect is dependent on integrin {beta}1 activity and involves apical to basal conversion of cell polarity. We demonstrate that ECM-integrin activity is sufficient for  inside positional signalling and it is required for proper sorting of EPI/PrE cells. Our findings thus highlight the significance of ECM-integrin adhesion in enabling position-sensing by cells to achieve tissue patterning.


title: Sand dropseed (Sporobolus cryptandrus) - A new pest in Eurasian sand areas?

abstract: For the effective control of an invasive species, gathering as much information as possible on its ecology, establishment and persistence in the subjected communities is of utmost importance. We aimed to review the current distribution and characteristics of Sporobolus cryptandrus (sand dropseed), an invasive C4 grass species of North American origin recently discovered in Hungary. We aimed to provide information on (i) its current distribution paying special attention to its invasion in Eurasia; (ii) the characteristics of the invaded habitats in Central Europe; (iii) seed bank formation and germination characteristics, crucial factors in early establishment; and (iv) the effects of its increasing cover on vegetation composition. Finally, we aimed to (v) point out further research directions that could enable us to understand the invasion success of this potential invasive species. Field surveys uncovered large stands of the species in Central and Eastern Hungary with most of the locations in the former, especially the Kiskunsag region. The species invaded disturbed stands of dry and open sand grasslands, closed dune slack grasslands and it also penetrates into natural open sand grasslands from neighbouring disturbed habitats. Increasing cover of Sporobolus cryptandrus caused a decline in species richness and abundance of subordinate species both in the vegetation and seed banks, but a low density of Sporobolus cryptandrus can even have a weak positive effect on these characteristics. Viable seeds of Sporobolus were detected from all soil layers (2.5 cm layers measured from the surface to 10 cm in depth), which indicates that the species is able to form a persistent seed bank (1,114 to 3,077 seeds/m2 with increasing scores towards higher abundance of the species in vegetation). Germination of Sporobolus cryptandrus was negatively affected by both litter cover and 1 cm deep soil burial. To sum up, Sporobolus cryptandrus can be considered as a transformer invasive species, whose spread forms a high risk for dry sand and steppe grasslands in Eurasia. We can conclude that for the effective suppression of the species it is necessary: (i) to clarify the origin of the detected populations; (ii) to assess its competitive ability including its potential allelopathic effects; (iii) to assess its seed bank formation potential in habitats with different abiotic conditions; and (iv) to assess the possibility of its suppression by natural enemies and management techniques such as mowing or livestock grazing.

category: ecology


title: Meta-analysis of tree diversity effects on the abundance, diversity and activity of herbivores' enemies

abstract:
O_LIThe natural enemies hypothesis predicts that the abundance and diversity of antagonists such as predators and parasitoids of herbivores increases with the diversity of plants, which can lead to more effective top-down control of insect herbivores. However, although the hypothesis has received large support in agricultural systems, fewer studies have been conducted in forest ecosystems and a comprehensive synthesis of previous research is still lacking.
C_LIO_LIWe conducted a meta-analysis of 65 publications comparing the diversity, abundance or activity of various groups of natural enemies (including birds, bats, spiders and insect parasitoids) in pure vs. mixed forest stands. We tested the effects of forest biome, natural enemy taxon and type of study (managed vs experimental forest).
C_LIO_LIWe found a significant positive effect of forest tree diversity on natural enemy abundance and diversity but not on their activity. The effect of tree diversity on natural enemies was stronger towards lower latitudes but was not contingent on the natural enemy taxon level.
C_LIO_LIOverall, our study contributes toward a better understanding of the "natural enemies hypothesis" in forest systems and provides new insights about the mechanisms involved. Furthermore, we outline potential avenues for strengthening forest resistance to the growing threat of herbivorous insects.
C_LI


title: Stable Isotope Tracing In Vivo Reveals A Metabolic Bridge Linking The Microbiota To Host Histone Acetylation

abstract: The gut microbiota influences host epigenetics by fermenting dietary fiber into butyrate. Although butyrate could promote histone acetylation by inhibiting histone deacetylases, it may also undergo oxidation to acetyl-CoA, a necessary cofactor for histone acetyltransferases. Here, we find that epithelial cells from germ-free mice harbor a loss of histone H4 acetylation across the genome except at promoter regions. Using stable isotope tracing in vivo with 13C-labeled fiber, we demonstrate that the microbiota supplies carbon for histone acetylation. Subsequent metabolomic profiling revealed hundreds of labeled molecules and supported a microbial contribution to host fatty acid metabolism, which declined in response to colitis and correlated with reduced expression of genes involved in fatty acid oxidation. These results illuminate the flow of carbon from the diet to the host via the microbiota, disruptions to which may affect energy homeostasis in the distal gut and contribute to the development of colitis.

category: biochemistry


title: Steps to (s)-stereoisomers: Rationalization of a standard Short-chain dehydrogenase for the reduction of multiple pharmaceutical ketone intermediates

abstract: Short-chain dehydrogenases/reductases (SDRs) are a convenient class of enzymes used to synthesize enantiopure alcohols. Several studies describe native or engineered SDRs for converting substrates of interest using cost and time-intensive high-throughput approaches. The classification of SDRs is based on chain length and cofactor binding site. Of these, the shorter  Classical and the longer  Extended enzymes participate in ketoreduction. However, comparative analysis of various modelled SDRs reveals a length independent conserved N-terminal Rossmann fold and a variable C-terminus region for both types. The general hypothesis is that the latter domain influences the enzymes flexibility that may affect the observed promiscuity of the enzyme. We have used a machine learning algorithm on this flexible domain to build a rationale to screen promiscuous candidates. We have built a data set consisting of physicochemical properties derived from the amino-acid composition of enzymes to select closely associated promiscuous mesophilic enzymes. The resulting in vitro studies on pro-pharmaceutical substrates illustrate a direct correlation between the C-terminal lid-loop structure, enzyme melting temperature and the turnover number. We present a walkthrough for exploring promiscuous SDRs for catalyzing enantiopure alcohols of industrial importance.


title: Flexibility of Short-chain dehydrogenase is interconnected to its promiscuity for the reduction of multiple ketone intermediates


title: Comprehensive evaluation of machine learning models and gene expression signatures for prostate cancer prognosis using large population cohorts

abstract: Overtreatment remains the pervasive problem in prostate cancer (PCa) management due to the highly variable and often indolent course. Molecular signatures derived from gene expression profiling have played critical roles in PCa treatment decision-making. Many gene expression signatures have been developed to improve the risk stratification of PCa and some of them have already been translationally applied to clinical practice, however, no comprehensive evaluation was performed to compare the performances of the signatures. In this study, we conducted a systematic and unbiased evaluation of 15 machine learning (ML) algorithms and 30 published PCa gene expression-based prognostic signatures leveraging 10 transcriptomics datasets with 1,558 primary PCa patients from public data repositories. The results revealed that survival analysis models outperformed binary classification models for risk assessment, and the performances of the survival analysis methods - Cox model regularized with ridge penalty (Cox-Ridge) and partial least squares regression for Cox model (Cox-PLS) - were generally more robust than the other methods. Based on the Cox-Ridge algorithm, a few top prognostic signatures that have comparable or even better performances than the commercial panels have been identified. The findings from the study will greatly facilitate the identification of existing prognostic signatures that are promising for further validations in prospective studies and promote the development of robust prognostic models to guide clinical decision-making. Moreover, the study provided a valuable data resource from large primary PCa cohorts, which can be used to develop, validate, and evaluate novel statistical methodologies and molecular signatures to improve PCa management.

category: bioinformatics


title: Meta-analysis of exhausted CD8+ T cells from Homo sapiens and Mus musculus provides robust targets for immunotherapy

abstract: T cell exhaustion is a state of T cell dysfunction during chronic infection and cancer. Antibody-targeting immune checkpoint inhibitors to reverse T cell exhaustion is a promising approach for cancer immunotherapy. However, the therapeutic efficacy of known immune checkpoint inhibitors remains low. To expand the potential effective targets to reverse T cell exhaustion, a meta-analysis was performed to integrate seven exhaustion datasets caused by multiple diseases in both humans and mice. In this study, an overlap of 21 upregulated and 37 downregulated genes was identified in human and mouse exhausted CD8+ T cells. These genes were significantly enriched in exhaustion response-related pathways, such as signal transduction, immune system processes, and regulation of cytokine production. Gene expression network analysis revealed that the well-documented exhaustion genes were defined as hub genes in upregulated genes, such as programmed cell death protein 1 and cytotoxic T-lymphocyte associated protein 4. In addition, a weighted gene co-expression analysis identified 175 overlapping genes that were significantly correlated with the exhaustion trait in both humans and mice. This study found that nine genes, including thymocyte selection associated high mobility group box and CD200 receptor 1, were significantly upregulated and highly related to T cell exhaustion. These genes should be additional robust targets for immunotherapy and T-cell dysfunction studies.


title: Multifaceted roles of SAMHD1 in cancer

abstract: SAMHD1 is discussed as a tumour suppressor protein, but its potential role in cancer has only been investigated in very few cancer types. Here, we performed a systematic analysis of the TCGA (adult cancer) and TARGET (paediatric cancer) databases, the results of which did not suggest that SAMHD1 should be regarded as a bona fide tumour suppressor. SAMHD1 mutations that interfere with SAMHD1 function were not associated with poor outcome, which would be expected for a tumour suppressor. High SAMHD1 tumour levels were associated with increased survival in some cancer entities and reduced survival in others. Moreover, the data suggested differences in the role of SAMHD1 between males and females and between different races. Often, there was no significant relationship between SAMHD1 levels and cancer outcome. Taken together, our results indicate that SAMHD1 may exert pro- or anti-tumourigenic effects and that SAMHD1 is involved in the oncogenic process in a minority of cancer cases. These findings seem to be in disaccord with a perception and narrative forming in the field suggesting that SAMHD1 is a tumour suppressor. A systematic literature review confirmed that most of the available scientific articles focus on a potential role of SAMHD1 as a tumour suppressor. The reasons for this remain unclear but may include confirmation bias and publication bias. Our findings emphasise that hypotheses, perceptions, and assumptions need to be continuously challenged by using all available data and evidence.

category: cancer biology


title: CXCL17 is an endogenous inhibitor of CXCR4 via a novel mechanism of action

abstract: CXCL17 is the most recently described chemokine. It is principally expressed by mucosal tissues, where it facilitates chemotaxis of monocytes, dendritic cells, and macrophages and has antimicrobial properties. CXCL17 is also implicated in the pathology of inflammatory disorders and progression of several cancers, as well as being highly upregulated during viral infections of the lung. However, the exact role of CXCL17 in health and disease is largely unknown, mainly due to a lack of known molecular targets mediating CXCL17 functional responses. Using a range of bioluminescence resonance energy transfer (BRET) based assays, here we demonstrate that CXCL17 inhibits CXCR4-mediated signalling and ligand binding. Moreover, CXCL17 interacts with neuropillin-1, a VEGFR2 co-receptor. Additionally, we find CXCL17 only inhibits CXCR4 ligand binding in intact cells and demonstrate that this effect is mimicked by known glycosaminoglycan binders, surfen and protamine sulfate. This indicates that CXCL17 inhibits CXCR4 by a unique mechanism of action that potentially requires the presence of a glycosaminoglycan containing accessory protein. Altogether, our results reveal that CXCL17 is an endogenous inhibitor of CXCR4 and represents an important discovery in our understanding of the (patho) physiological functions of CXCL17 and regulation of CXCR4 signalling.

category: pharmacology and toxicology


title: Direct neuronal reprogramming by temporal identity factors

abstract: Temporal identity factors are sufficient to reprogram developmental competence of neural progenitors, but whether they could also reprogram the identity of fully differentiated cells is unknown. To address this question, we designed a conditional gene expression system combined with genetic lineage tracing that allows rapid screening of potential reprogramming factors in the mouse retina. Using this assay, we report that co-expression of the early temporal identity transcription factor Ikzf1, together with Ikzf4, another Ikaros family member, is sufficient to directly convert adult Muller glial cells into neuron-like cells in vivo, without inducing a proliferative progenitor state. scRNA-seq analysis shows that the reprogrammed cells share some transcriptional signatures with both cone photoreceptors and bipolar cells. Furthermore, we show that co-expression of Ikzf1 and Ikzf4 can reprogram mouse embryonic fibroblasts to induced neurons by remodeling chromatin and promoting a neuronal gene expression program. This work uncovers general neuronal reprogramming properties for temporal identity factors in differentiated cells, opening new opportunities for cell therapy development.

category: neuroscience


title: Investigating facial information content in the hand area of individuals with a congenital and acquired missing hand

abstract: Cortical remapping after hand loss in the primary somatosensory cortex (S1) is thought to be predominantly dictated by cortical proximity, with adjacent body parts remapping into the deprived area. Traditionally, this remapping has been characterised by changes in the lip representation, which is assumed to be the immediate neighbour of the hand based on electrophysiological research in non-human primates. However, the orientation of facial somatotopy in humans is debated, with contrasting work reporting both an inverted and upright topography. We aimed to fill this gap in the S1 homunculus by investigating the topographic organisation of the face. Using both univariate and multivariate approaches we examined the extent of face-to-hand remapping in individuals with a congenital and acquired missing hand (hereafter one-handers and amputees, respectively), relative to two-handed controls. Participants were asked to move different facial parts (forehead, nose, lips, tongue) during fMRI scanning. We first report evidence for an upright facial organisation in all three groups, with the upper face and not the lips bordering the hand area. We further found little evidence for remapping of all tested facial parts in amputees, with no significant relationship to the chronicity of their PLP. In contrast, we found converging evidence for a complex pattern of face remapping in congenital one-handers across all facial parts, where the location of the cortical neighbour - the forehead - is shown to shift away from the deprived hand area, which is subsequently activated by the lips and the tongue. Together, our findings demonstrate that the face representation in humans is highly plastic, but that this plasticity is restricted by the developmental stage of input deprivation, rather than cortical proximity.


title: Reassessing face topography in primary somatosensory cortex and remapping following hand loss


title: The Dilemma Of Disobedience: An ERP study

abstract: The present experiment aimed at investigating the decision-making and the associated event-related potentials (ERPs) of subordinates under hierarchical pressure. Participants (N = 33) acted as UAV operators and had to decide to crash their defective drone either on a civilian site killing all civilians present on the site or on a military site destroying military material but preventing any human losses. While in the no-command condition, participants decided according to their own preferences, in the command condition they were ordered to protect the military material at the expense of civilians for undisclosed strategic reasons. The results revealed that in the no-command condition participants almost always crashed the drone on the military site (96%), whereas in the command condition they chose to obey orders and sacrifice civilians to protect the military material 33% of the time. In the command condition, participants were longer to make their decisions, mobilizing greater attentional and cognitive resources (i.e., greater P300 responses) to resolve the conflict between their internal moral values and the orders they were given (i.e., greater N200 responses) than in the no-command condition, where they automatically applied the "you shall not kill" rule. Participants also showed a greater negative affective response (i.e., greater P260 amplitudes) after choosing to disobey than to obey orders. This result suggests that disobeying authority could be perceived as a greater moral violation than obeying and sacrificing civilians, suggesting that individuals may sometimes choose to obey malevolent authority to avoid the negative affective reaction triggered by disobedience.


title: Generation, quality control, and analysis of the first genomically humanised knock-in mice for the ALS/FTD genes SOD1, TARDBP (TDP-43), and FUS

abstract:
Amyotrophic lateral sclerosis - frontotemporal dementia spectrum disorder (ALS/FTD) is a complex neurodegenerative disease; up to 10% of cases are familial, usually arising from single dominant mutations in >30 causative genes. Transgenic mouse models that overexpress human ALS/FTD causative genes have been the preferred organism for in vivo modelling. However, while conferring human protein biochemistry, these overexpression models are not ideal for dosage-sensitive proteins such as TDP-43 or FUS.

We have created three next-generation genomically humanised knock-in mouse models for ALS/FTD research, by replacing the entire mouse coding region of Sod1, Tardbp (TDP-43) and Fus, with their human orthologues to preserve human protein biochemistry, with exons and introns intact to enable future modelling of coding or non-coding mutations and variants and to preserve human splice variants. In generating these mice, we have established a new-standard of quality control: we demonstrate the utility of indirect capture for enrichment of a region of interest followed by Oxford Nanopore sequencing for robustly characterising large knock-in alleles. This approach confirmed that targeting occurred at the correct locus and to map homologous recombination events. Furthermore, extensive expression data from the three lines shows that homozygous humanised animals only express human protein, at endogenous levels. Characterisation of humanised FUS animals showed that they are phenotypically normal compared to wildtype littermates throughout their lifespan.

These humanised mouse strains are critically needed for preclinical assessment of interventions, such as antisense oligonucleotides (ASOs), to modulate expression levels in patients, and will serve as templates for the addition of human ALS/FTD mutations to dissect disease pathomechanisms.


title: Atypical development of subcortico-cortical effective connectivity in autism

abstract:
Hypersensitivity, stereotyped behaviors and attentional problems in autism spectrum disorder (ASD) are compatible with inefficient filtering of undesired or irrelevant sensory information at early stages of neural processing. This could stem from delays in the neurotypical development of the functional segregation between cortical and subcortical brain processes, as suggested by previous findings of overconnectivity between primary sensory regions and deep brain nuclei in ASD.

To test this hypothesis, we used dynamic causal modelling to quantify the effect of age on the development of (1) cortical functional segregation from subcortical activity and (2) directional influence of subcortical activity on cortical processing in 166 participants with ASD and 193 typically developing controls (TD) from the Autism Brain Imaging Data Exchange (ABIDE).

We found that in TD participants age was significantly associated with increased functional segregation of cortical sensory processing from subcortical activity, paralleled by a decreased influence of subcortical activity on cortical processing. Instead these effects were highly reduced and mostly absent in ASD participants, suggesting a delayed or arrested development of the segregation between subcortical and cortical sensory processing in ASD.

This atypical configuration of subcortico-cortical connectivity in ASD can result in an excessive amount of unprocessed sensory information relayed to the cortex, which is likely to impact cognitive functioning in everyday situations where it is beneficial to limit the influence of basic sensory information on cognitive processing, such as activities requiring focused attention or social interactions.


title: Disturbance-based management of ecosystem services and disservices in partial nitritation anammox biofilms

abstract: The resistance and resilience provided by functional redundancy, a common feature of microbial communities, is not always advantageous. An example is nitrite oxidation in partial nitritation-anammox (PNA) reactors during wastewater treatment, where suppression of nitrite oxidizers like Nitrospira is sought. In these ecosystems, biofilms provide microhabitats with oxygen gradients, allowing the coexistence aerobic and anaerobic bacteria. We designed a disturbance experiment where PNA biofilms treating water from a high rate activated sludge process removing organic matter (mainstream wastewater), were constantly or intermittently exposed to the effluent of anaerobic sewage sludge digestion dewatering (sidestream wastewater), which has been proposed to inhibit nitrite oxidizers. With increasing sidestream exposure we observed decreased abundance, alpha-diversity, functional versatility, and hence functional redundancy, among Nitrospira in the PNA biofilms, while the opposite patterns were observed for anammox bacteria within Brocadia. At the same time, species turnover was observed for the aerobic ammonia-oxidizing Nitrosomonas populations. The different exposure regimens were associated with metagenomic assembled genomes of Nitrosomonas, Nitrospira, and Brocadia, encoding genes related to N-cycling, substrate usage, and osmotic stress response, possibly explaining the three different patterns by niche differentiation. These findings imply that disturbances can be used to manage the functional redundancy of biofilm microbiomes in a desirable direction, which should be considered when designing operational strategies for wastewater treatment.

category: microbiology


title: Lightsheet Optical Tweezer (LOT) for Optical Manipulation of Microscopic Particles and Live Cells

abstract: A light sheet based optical tweezer (LOT) is developed to trap microscopic dielectric particles and live HeLa cells. The technique requires the generation of a tightly focussed diffraction-limited light sheet which is realized by a combination of cylindrical lens and high NA objective lens. The field pattern generated at the geometrical focus is a tightly focussed line (along x-axis) perpendicular to the beam propagation direction (z-axis). Spherical beads undergoing Brownian motion in the solution are trapped by the gradient potential, and the time (to reach trap-center under the influence of gradient potential) is estimated from the fast CMOS camera (operating at 230frames/sec). High-speed imaging of beads at varying laser power shows a steady increase in the stiffness of LOT with a maximum of 0.00118 pN/nm at 52.5 mW. This is an order less than traditional optical point-traps. The trapped beads displayed free movement along the light-sheet axis (x-axis), exhibiting a single degree of freedom. Subsequently, LOT is used to optically trap and pattern dielectric beads and HeLa cells in a line. We could successfully pattern 8 dielectric beads and 3 HeLa cells in a straight line. We anticipate that LOT can be used to study the 1D-physics of microscopic particles and help understand the patterned growth of live cells.

category: biophysics


title: Transient beta modulates decision thresholds during human action-stopping

abstract: Action-stopping in humans involves bursts of beta oscillations in prefrontal-basal ganglia regions. To determine the functional role of these beta bursts we took advantage of the Race Model framework describing action-stopping. We incorporated beta bursts in three race model variants, each implementing a different functional contribution of beta to action-stopping. In these variants, we hypothesized that a transient increase in beta could 1) modulate decision thresholds, 2) change stop accumulation rates, or 3) promote the interaction between the Stop and the Go process. We then tested the model predictions using EEG recordings in humans performing a Stop-signal task. We found that the model variant in which beta increased decision thresholds for a brief period of time best explained the empirical data. The model parameters fitted to the empirical data indicated that beta bursts involve a stronger decision threshold modulation for the Go process than for the Stop process. This suggests that prefrontal beta influences stopping by temporarily holding the response from execution. Our study further suggests that human action-stopping could be multi-staged with the beta acting as a pause, increasing the response threshold for the Stop process to modulate behavior successfully. Our novel approach of introducing transient oscillations into the race model and testing against human neurophysiological data allowed us to discover potential mechanisms of prefrontal beta, possibly generalizing its role in situations requiring executive control over actions.


title: Aberrant motor contagion of emotions in psychopathy and high-functioning autism

abstract: Psychopathy and autism are both associated with aberrant social interaction and communication, yet only psychopaths are markedly antisocial and violent. Here we compared the functional neural alterations underlying these two different phenotypes with distinct patterns of socioemotional difficulties. We studied 19 incarcerated male offenders with high psychopathic traits, 20 males with high-functioning autism and 19 age-matched healthy controls. All groups underwent functional magnetic resonance imaging while they viewed dynamic happy, angry and disgust facial expressions or listened to laughter and crying sounds. Psychopathy was associated with reduced somatomotor responses to almost all expressions, while subjects with autism demonstrated less marked and emotion-specific alterations in the somatomotor area. These data suggest that psychopathy and autism involve both common and distinct functional alterations in the brain networks involved in socioemotional processing.


title: Microbial diversity and sulfur cycling in an early earth analogue: From ancient novelty to modern commonality

abstract: Life emerged and diversified in the absence of molecular oxygen. The prevailing anoxia and unique sulfur chemistry in the Paleo-, Meso- and Neoarchean, and early Proterozoic eons may have supported microbial communities that are drastically different than those currently thriving on the earths surface. Zodletone spring in southwestern Oklahoma represents a unique habitat where spatial sampling could substitute for geological eons: from the anoxic, surficial light exposed sediments simulating a preoxygenated earth, to overlaid water column where air exposure simulates the relentless oxygen intrusion during the Neo Proterozoic. We document a remarkably diverse microbial community in the anoxic spring sediments, with 340/516 (65.89%) of genomes recovered in a metagenomic survey belonging to 200 bacterial and archaeal families that were either previously undescribed or that exhibit an extremely rare distribution on the current earth. Such diversity is underpinned by the widespread occurrence of sulfite-, thiosulfate, tetrathionate-, and sulfur-reduction, and paucity of sulfate-reduction machineries in these taxa; hence greatly expanding lineages mediating reductive sulfur cycling processes in the tree of life. Analysis of the overlaying water community demonstrated that oxygen intrusion lead to the development of a significantly less diverse community dominated by well-characterized lineages and a prevalence of oxidative sulfur cycling processes. Such transition from ancient novelty to modern commonality underscores the profound impact of the great oxygenation event on the earths surficial anoxic community. It also suggests that novel and rare lineages encountered in current anaerobic habitats could represent taxa once thriving in an anoxic earth, but have failed to adapt to earths progressive oxygenation.


title: Bioactivity, chemical profiling, and 16S rRNA-based phylogeny of haloalkaliphilic Nocardiopsis sp. GhM-HA-6 isolated from the Gulf of Khambhat

abstract:
ObjectivesActinomycetes are well known sources of antibiotics, however; recently the focus of antimicrobial research has been turning towards actinomycetes of extreme environments. Therefore, present work would highlight the isolation, identification and characterization of antimicrobial metabolites produced by marine haloalkaliphilic actinomycetes.

MethodsSaline soil sample was collected from Ghogha coast (Gulf of Khambhat), Bhavnagar, Western India. Isolation was carried out using selective media while identification was done based on morphological, cultural and molecular characterization. The antimicrobial potential was checked by spot inoculation method. Optimization was carried out by the one variable at a time (OVAT) method. The antimicrobial compounds were extracted using ethyl acetate and characterized by GC-MS.

ResultsThe haloalkaliphilic actinomycetes Nocardiopsis sp. GhM-HA-6 was isolated from saline soil of Ghogha coast using starch agar with 10% w/v NaCl and pH 9 and was identified as Nocardiopsis sp. based on morphology, cultural characteristics and 16S rRNA phylogenetic analysis (NCBI Genbank Accession number: KF384492). The organism showed antimicrobial activity against five Gram positive and three Gram negative bacteria while the isolate didnt show any antifungal activity. Results of optimization showed that the highest production of antimicrobial compounds was obtained using starch broth with 0.5% w/v starch, 1% w/v yeast extract, 10% w/v NaCl and pH 9. GC-MS analysis of ethyl acetate extract of the isolate showed presence of total 18 compounds including various antimicrobial compounds like 2, 4-bis (1, 1-dimethylethyl)-Phenol, various types of alkanes and their derivatives.

ConclusionHaloalkaliphilic actinomycete Nocardiopsis sp. GhM-HA-6, from a rarely explored marine habitat, can be a source of antimicrobial compounds with the novel biotechnological applications.


title: Cyp26b1 restrains murine heart valve growth during development

abstract:
Endothelial cells (ECs) are critical to proper heart valve development, directly contributing to the mesenchyme of the cardiac cushions, which progressively transform into mature valves. To date, investigators have lacked useful markers of valve ECs to fully evaluate their contributions during valve morphogenesis. As a result, it has been unclear whether the well-characterized regional differentiation of valves correlates with any endothelial domains in the heart. Furthermore, it has been difficult to ascertain whether endothelial heterogeneity in the heart influences underlying mesenchymal zones in an angiocrine manner. To identify regionally expressed EC genes in the heart valves, we screened publicly available databases and assembled a toolkit of endothelial-enriched genes. We identified Cyp26b1 as one of many endothelial enriched genes found to be expressed in the endocardium of the developing cushions and valves. Here, we show that Cyp26b1 is required for normal heart valve development. Genetic ablation of Cyp26b1 in mouse embryos leads to abnormally thickened aortic valve leaflets, which is due in part to increased endothelial and mesenchymal cell proliferation in the remodeling valves. In addition, Cyp26b1 mutant hearts display ventricular septal defects (VSDs) in a portion of null embryos. We show that loss of Cyp26b1 results in upregulation of retinoic acid (RA) target genes, supporting the observation that Cyp26b1 has RA-dependent roles. Together, this work identifies a novel role for Cyp26b1 in heart valve morphogenesis. Understanding the spatiotemporal expression dynamics of cardiac EC genes will likely prove useful to the investigation of both normal as well as dysfunctional heart valve development.

HIGHLIGHTS{middle dot} A mouse heart valve gene expression atlas can be generated with publicly available online tools, such as Genepaint and other gene expression databases.
{middle dot} Endothelium of developing mouse heart valves is regionally heterogeneous.
{middle dot} Cyp26b1 is expressed in the endocardial/endothelial lining of developing heart valves.
{middle dot} Loss of Cyp26b1 leads to significant enlargement of aortic valves and to ventricular septal defects.
{middle dot} Cyp26b1 represses cell proliferation in valve mesenchyme.
{middle dot} Retinoic acid targets are upregulated in Cyp26b1-/- heart valves, indicating dysregulation of RA metabolism.


title: Long Range PCR-based deep sequencing for haplotype determination in mixed HCMV infections

abstract:
Short read sequencing, which has extensively been used to decipher the genome diversity of human cytomegalovirus (HCMV) strains, often falls short to assess co-linearity of non-adjacent polymorphic sites in mixed HCMV populations. In the present study, we established a long amplicon sequencing workflow to identify number and relative quantities of unique HCMV haplotypes in mixtures. Accordingly, long read PacBio sequencing was applied to amplicons spanning over multiple polymorphic sites. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-free viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections.

Our data show that artificial HCMV DNA mixtures were correctly determined upon long amplicon sequencing down to 1% abundance of the minor DNA source. Total error rate of mapped reads ranged from 0.17 to 0.43 depending on the stringency of quality trimming. PCR products of up to 7.7 kb and a GC content <55% were efficiently generated when DNA was directly isolated from bronchoalveolar lavage samples, yet long range PCR may display a slightly lower sensitivity compared to short amplicons. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Intra-patient haplotype diversity is unevenly distributed across the target site and often interspersed by long identical stretches, thus unable to be linked by short reads. Moreover, diversity at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed populations.

Quantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.

Impact statementHuman cytomegalovirus (HCMV), a large enveloped DNA virus, displays the highest inter-host genome variability among all human herpesviruses. Primary infection, reinfection and reactivation are mostly asymptomatic but may cause devastating harm in congenitally infected newborns and in immunosuppressed individuals. Multiple distinct strains circulate in humans, each characterised by a unique assembly of well-defined polymorphic genes, most of which are linked to cell entry, persistence and immune evasion. Mixed HCMV strain infections are common and may pose a high pathogenic potential for patients at risk for symptomatic infections. To better understand the biological behaviour and dynamics of individual viral genomes it is inevitable to assess the co-linearity of polymorphic sites in a genetically heterogeneous population. In this study, we established and successfully applied a long read sequencing technique to long amplicons and identified co-linear genome stretches (haplotypes) in patient samples with mixed HCMV populations. This strategy for haplotype determination allows linkage analysis of multiple non-adjacent polymorphic sites along up to 7.7 kb. This allows a better approximation to the true strain diversity in mixed samples, which short read sequencing approaches failed to do. Thereby, improving our knowledge on mixed HCMV infections important for the clinical outcome, diagnostics, treatment and vaccine development.

Data SummarySequence data generated in this study were deposited in GenBank with the accession numbers MW560357-MW560373. Raw data of Illumina and PacBio sequencing were submitted to the NCBI Sequence Read Archive (SRA) under project number SUB8972240. BioSample accession numbers are provided in Supplementary Table 3 and 4.

Additional sequence data for reference purposes were accessed from GenBank. Accession numbers are listed in Supplementary Table 6 and 7.

category: genomics


title: A new platform for high-throughput therapy testing on iPSC-derived, immature airway from Cystic Fibrosis Patients

abstract:
Induced pluripotent, stem cell (iPSC)-derived models of airway tissue have successfully modeled the primary defect in regulated chloride conductance caused by the major Cystic Fibrosis causing mutation, F508del. However, it remains unclear if iPSC-derived airway cultures can be used in high-throughput therapy development for F508del and rarer mutations. There is an urgent need for airway tissue models that reflect the variability of patient-specific responses and are scalable for therapy development. In the current work, we describe a robust, high-throughput fluorescence assay of mutant CFTR function in iPSCs differentiated to immature airway epithelium. This assay measures reproducible functional responses to modulators targeting either the major CF mutant F508del or the nonsense mutant: W1282X-CFTR. We show that the ranking of patient-specific responses to interventions in this stem-cell based model recapitulates the ranking observed in primary nasal epithelial cultures obtained from the same individuals. In summary, these proof-of-concept studies show that this novel platform has the potential to support therapy development and precision medicine for Cystic Fibrosis patients.

One Sentence SummaryWe describe a fluorescence-based platform that enables high-throughput Cystic Fibrosis therapy testing using iPSCs differentiated to immature lung.


title: Identification of the Novel Pyroptosis-Related Gene Signature in Patients with Esophageal Adenocarcinoma

abstract: Esophageal adenocarcinoma (EAC) is a highly malignant type of digestive tract cancers with a poor prognosis despite therapeutic advances. Pyroptosis is an inflammatory form of programmed cell death, whereas the role of pyroptosis in EAC remains largely unknown. Herein, we identified a pyroptosis-related five-gene signature that was significantly correlated with the survival of EAC patients in The Cancer Genome Atlas (TCGA) cohort and an independent validation dataset. In addition, a nomogram based on the five-gene signature was constructed with novel prognostic values. Moreover, the genes in the pyroptosis-related signature, CASP1, GSDMB, IL1B, PYCARD, and ZBP1, might be involved in immune response and regulation of the tumor microenvironment. Our findings indicate that the five-gene signature provides insights into the involvement of pyroptosis in EAC progression, and is promising in the risk assessment as well as prognosis for EAC patients in clinical practice.


title: In vivo modelling of patient genetic heterogeneity identifies concurrent Wnt and PI3K activity as a potent driver of invasive cholangiocarcinoma growth.

abstract: Intrahepatic cholangiocarcinoma (ICC) is an aggressive and lethal malignancy of the bile ducts within the liver characterised by high levels of genetic heterogeneity. In the context of such genetic variability, determining which oncogenic mutations drive ICC growth has been difficult and developing modes of patient stratification and targeted therapies remains challenging. As a result, survival rates following a diagnosis with ICC have remained static since the late 1970s, whilst incidence of ICC has increased. Here, we performed the first functional in vivo study into the role that genetic heterogeneity plays in drivinga ICC via modelling of interactions between rare mutations with more common driver genes. By leveraging human ICC sequencing data to stratify and then model genetic heterogeneity in the mouse, we uncovered numerous novel tumour suppressors which, when lost, cooperate with the RAS oncoprotein to drive ICC growth. In this study, we specifically focus on a set of driver mutations that interact with KRAS to initiate aggressive, sarcomatoid-type ICC. We show that tumour growth of this cancer relies on both Wnt and PI3K signalling to drive proliferation and suppress apoptosis. Finally, we demonstrate that pharmacological co-inhibition of Wnt and PI3K in vivo substantially impedes the growth of ICC, regardless of mutational profile. As such, Wnt and PI3K activity should be considered as a signature by which patients can be stratified for treatment and inhibitors of these pathways should be levied as a treatment for patients diagnosed with ICC.


title: Synaptic Encoding of Vestibular Sensation Regulates Movement Timing and Coordination

abstract: Vertebrate vestibular circuits use sensory signals derived from the inner ear to guide both corrective and volitional movements. A major challenge in the neuroscience of balance is to link the synaptic and cellular substrates that encode body tilts to specific behaviors that stabilize posture and enable efficient locomotion. Here we address this problem by measuring the development, synaptic architecture, and behavioral contributions of vestibulospinal neurons in the larval zebrafish. First, we find that vestibulospinal neurons are born and are functionally mature before larvae swim freely, allowing them to act as a substrate for postural regulation. Next, we map the synaptic inputs to vestibulospinal neurons that allow them to encode posture. Further, we find that this synaptic architecture allows them to respond to linear acceleration in a directionally-tuned and utricle-dependent manner; they are thus poised to guide corrective movements. After loss of vestibulospinal neurons, larvae adopted eccentric postures with disrupted movement timing and weaker corrective kinematics. We used a generative model of swimming to demonstrate that together these disruptions can account for the increased postural variability. Finally, we observed that lesions disrupt vestibular-dependent coordination between the fins and trunk during vertical swimming, linking vestibulospinal neurons to navigation. We conclude that vestibulospinal neurons turn synaptic representations of body tilt into defined corrective behaviors and coordinated movements. As the need for stable locomotion is common and the vestibulospinal circuit is highly conserved our findings reveal general mechanisms for neuronal control of balance.


title: Tracing the evolution of human gene regulation and its association with shifts in environment

abstract: As humans spread throughout the world, they adapted to variation in many environmental factors, including climate, diet, and pathogens. Because many of these adaptations were likely mediated by multiple non-coding variants with small effects on gene regulation, it has been difficult to link genomic signals of selection to specific genes, and to describe the regulatory response to selection. To overcome this challenge, we adapted PrediXcan, a machine learning method for imputing gene regulation from genotype data, to analyze low-coverage ancient human DNA (aDNA). First, we used simulated genomes to benchmark strategies for adapting gene regulatory prediction to increase robustness to incomplete aDNA data. Applying the resulting models to 490 ancient Eurasians, we found that genes with the strongest divergent regulation among ancient populations with hunter-gatherer, pastoralist, and agricultural lifestyles are enriched for metabolic and immune functions. Next, we explored the contribution of divergent gene regulation to two traits with strong evidence of recent adaptation: dietary metabolism and skin pigmentation. We found enrichment for divergent regulation among genes previously proposed to be involved in diet-related local adaptation, and in many cases, the predicted effects on regulation provide explanations for previously observed signals of selection, e.g., at FADS1, GPX1, and LEPR. For skin pigmentation, we applied new models trained in melanocytes to a time series of 2999 ancient Europeans spanning ~38,000 years BP. In contrast to diet, skin pigmentation genes show little regulatory change over time, suggesting that adaptation mainly involved large-effect coding variants. This work demonstrates how aDNA can be combined with present-day genomes to shed light on the biological differences among ancient populations, the role of gene regulation in adaptation, and the relationship between ancient genetic diversity and the present-day distribution of complex traits.


title: Sexually distinct song cultures in a songbird metapopulation

abstract: Songbirds learn their songs culturally, through imitating tutors. The vocal culture of a songbird population changes as new song units (syllables) are introduced through immigration, copying errors, and innovation, while other syllables fall out of use. This leads to a diversification of the syllable pool across the species, much like the diversification and spatial patterns of human language. Vocal cultures have been well studied in male songbirds but have been largely overlooked in females. In particular, few studies compare spatial variation of male and female song cultures. Here we undertake one of the first comparisons of male and female song culture in birds, analysing song data from a metapopulation of New Zealand bellbirds Anthornis melanura, spanning an archipelago of six islands. Having classified 20,700 syllables, we compare population syllable repertoire sizes and overlap between sites and sexes. We show that males and females--both with complex songs--have distinct song cultures, sharing only 6-26% of syllable types within each site. Furthermore, male and female syllable types can be statistically discriminated based on acoustic properties. Despite diverse syllable repertoires within sites, few syllable types were shared between sites (both sexes had highly distinct site-specific dialects). For the few types shared between sites, sharing decreased with distance only for males. Overall, there was no significant difference between sexes in degree of site-site repertoire overlap. These results suggest different cultural processes at play for the two sexes, underlining the inadequacy of male-centric song research and calling for comparisons of male and female song cultures in many more species.

category: animal behavior and cognition


title: A cGAMP-containing hydrogel for prolonged SARS-CoV-2 RBD subunit vaccine exposure induces a broad and potent humoral response

abstract: The SARS-CoV-2 virus spike protein, specifically its receptor binding domain (RBD), has emerged as a promising target for generation of neutralizing antibodies. Although the RBD peptide subunit is easily manufactured and highly stable, RBD-based subunit vaccines have been hampered by its poor inherent immunogenicity. We hypothesize that this limitation can be overcome by sustained co-administration alongside a potent and optimized adjuvant. The innate immune second messenger, cGAMP, holds promise as it activates the potent anti-viral STING pathway, but has exhibited poor performance as a therapeutic due to its nonspecific pharmacodynamic profiles when administered systemically and its poor pharmacokinetics arising from rapid excretion and degradation by its hydrolase ENPP1. To overcome these limitations, we sought to mimic the natural scenario of viral infections by creating an artificial immunological niche that enables slow release of cGAMP and the RBD antigen. Specifically, we co-encapsulated cGAMP and RBD in an injectable polymer-nanoparticle (PNP) hydrogel system. This cGAMP-adjuvanted hydrogel vaccine elicited more potent, durable, and broad antibody responses and improved neutralization than both dose-matched bolus controls and a hydrogel-based vaccine lacking cGAMP. The cGAMP-adjuvanted hydrogel platform developed is suitable for delivery of other antigens and may provide enhanced immunity against a broad range of pathogens.

category: bioengineering


title: A SARS-CoV-2 nucleocapsid protein TR-FRET assay amenable to high-throughput screening

abstract: Drug development for specific antiviral agents against coronavirus disease 2019 (COVID-19) is still an unmet medical need as the pandemic continues to spread globally. Although huge efforts for drug repurposing and compound screens have put forth, only few compounds remain in late stage clinical trials. New approaches and assays are needed to accelerate COVID-19 drug discovery and development. Here we report a time-resolved fluorescence resonance energy transfer-based assay that detects the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) produced in infected cells. It uses two specific anti-NP monoclonal antibodies (MAbs) conjugated to donor and acceptor fluorophores that produces a robust ratiometric signal for high throughput screening of large compound collections. Using this assay, we measured a half maximal inhibitory concentration (IC50) for Remdesivir of 9.3 M against infection with SARS-CoV-2 USA/WA1/2020 (WA-1). The assay also detected SARS-CoV-2 South African (Beta, {beta}), Brazilian/Japanese variant P.1 (Gamma, {gamma}), and Californian (Epsilon, {varepsilon}), variants of concern or interest (VoC). Therefore, this homogeneous SARS-CoV-2 NP detection assay can be used for accelerating lead compound discovery for drug development and for evaluating drug efficacy against emerging SARS-CoV-2 VoC.


title: Dynamics of SARS-CoV-2 host cell interactions inferred from transcriptome analyses

abstract: The worldwide spread of severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) caused an urgent need for an in-depth understanding of interactions between the virus and its host. Here, we dissected the dynamics of virus replication and the host cell transcriptional response to SARS-CoV-2 infection at a systems level by combining time-resolved RNA sequencing with mathematical modeling. We observed an immediate transcriptional activation of inflammatory pathways linked to the anti-viral response followed by increased expression of genes involved in ribosome and mitochondria function, thus hinting at rapid alterations in protein production and cellular energy supply. At later stages, metabolic processes, in particular those depending on cytochrome P450 enzymes, were downregulated. To gain a deeper understanding of the underlying transcriptional dynamics, we developed an ODE model of SARS-CoV-2 infection and replication. Iterative model reduction and refinement revealed that a negative feedback from virus proteins on the expression of anti-viral response genes was essential to explain our experimental dataset. Our study provides insights into SARS-CoV-2 virus-host interaction dynamics and facilitates the identification of druggable host pathways supporting virus replication.

category: systems biology


title: Linguistic modulation of the neural encoding of phonemes

abstract: Speech comprehension entails the neural mapping of the acoustic speech signal onto learned linguistic units. This acousto-linguistic transformation is bi-directional, whereby higher-level linguistic processes (e.g., semantics) modulate the acoustic analysis of individual linguistic units. Here, we investigated the cortical topography and linguistic modulation of the most fundamental linguistic unit, the phoneme. We presented natural speech and  phoneme quilts (pseudo-randomly shuffled phonemes) in either a familiar (English) or unfamiliar (Korean) language to native English speakers while recording fMRI. This design dissociates the contribution of acoustic and linguistic processes towards phoneme analysis. We show that (1) the four main phoneme classes (vowels, nasals, plosives, fricatives) are differentially and topographically encoded in human auditory cortex, and that (2) their acoustic analysis is modulated by linguistic analysis. These results suggest that the linguistic modulation of cortical sensitivity to phoneme classes minimizes prediction error during natural speech perception, thereby aiding speech comprehension in challenging listening situations.


title: Predicted Impact of the Viral Mutational Landscape on the Cytotoxic Response against SARS-CoV-2

abstract:
The massive assessment of immune evasion due to viral mutations that potentially increase COVID-19 susceptibility can be computationally facilitated. The adaptive cytotoxic T response is critical during primary infection and the generation of long-term protection. Potential epitopes in the SARS-CoV-2 proteome were predicted for 2,915 human alleles of 71 HLA class I families. Allele families showed extreme differences in number of recognized epitopes, underscoring genetic variability of protective capacity between humans. Up to 1,222 epitopes were associated with any of the twelve supertypes, that is, allele clusters covering 90% population. Among them, the B27 supertype showed the lowest number of epitopes. Epitope escape mutations identified in ~118,000 NCBI isolates mainly involved non-conservative substitutions at the second and C-terminal position of the ligand core, or total ligand removal by large recurrent deletions. Escape mutations affected 47% of supertype epitopes, which in 21% of cases concerned isolates from two or more sub-continental areas. Some of these changes were coupled, but never surpassed 15% evaded epitopes for the same supertype in the same isolate, except for B27, which reached up to 33%. In contrast to most supertypes, eight particular allele families mostly contained alleles with few SARS-CoV-2 ligands. Isolates harboring cytotoxic escape mutations for these families co-existed geographically within sub-Saharan and Asian populations enriched in these alleles. Collectively, these data indicate that independent escape mutation events have already occurred for half of HLA class I supertype epitopes. However, it is presently unlikely that, overall, it poses a threat to the global population. In contrast, single and double mutations for susceptible alleles may be associated with viral selective pressure and alarming local outbreaks. This study highlights the automated integration of genomic, geographical and immunoinformatic information for surveillance of SARS-CoV-2 variants potentially affecting the population as a whole, as well as minority subpopulations.

AUTHOR SUMMARYThe cytotoxic T response, a type of immune response dependent upon an individual's genetics that does not require antibodies, is critical for neutralizing SARS-CoV-2 infection. The potential bypass of the cytotoxic T response by mutations acquired by the virus after one year of the pandemic is therefore of maximal concern. We have approached the complexity of human variability and more than 100.000 viral genomes in this respect using a computational strategy. We have detected numerous mutations in these genomes that mask some viral regions involved in the cytotoxic response. However, the accumulation of these changes in independent isolates is still too low to threaten the global human population. In contrast, our protocol has identified mutations that may be relevant for specific populations and minorities with cytotoxic genetic backgrounds susceptible to SARS-CoV-2 infection. Some viral variants co-existed in the same country with these human communities which warrants deeper surveillance in these cases to prevent local outbreaks. Our study support the integration of massive data of different natures in the surveillance of viral pandemics.

category: immunology


title: Microglia and border-associated mouse macrophages maintain their embryonic origin during Alzheimers disease

abstract:
Brain microglia and border-associated macrophages (BAMs) display distinct spatial, developmental, and phenotypic features. Although at steady-state, the origins of distinct brain macrophages are well-documented, the dynamics of their replenishment in neurodegenerative disorders remain elusive, particularly for disease-associated microglia (DAMs) and BAMs. In this study, we conducted a comprehensive fate-mapping analysis of murine microglia and BAMs and their turnover kinetics during Alzheimers disease (AD) progression. We used a novel inducible AD mouse model to investigate the contribution of bone marrow cells to the pool of foetal-derived brain macrophages during the development of AD. We demonstrated that microglia and DAMs remain a remarkably stable embryonic-derived population even during the progression of AD pathology, indicating that neither parenchymal macrophage subpopulation originates from, nor are replenished by, monocytes. At the border-associated brain regions, bona fide CD206+ BAMs are minimally replaced by monocytes, and their turnover rates are not accelerated by AD. In contrast, all other myeloid cells are swiftly replenished by bone marrow progenitors. This information further elucidates the turnover kinetics of these cells not only at steady-state, but also in neurodegenerative diseases, which is crucial for identifying potential novel therapeutic targets.

Impact statementInducible fate-mapping analysis demonstrates that neither microglia, disease-associated microglia nor border-associated macrophages are replenished by bone marrow-derived cells in Alzheimers disease.


title: Immunisation of ferrets and mice with recombinant SARS-CoV-2 spike protein formulated with Advax-SM adjuvant protects against COVID-19 infection

abstract: The development of a safe and effective vaccine is a key requirement to overcoming the COVID-19 pandemic. Recombinant proteins represent the most reliable and safe vaccine approach but generally require a suitable adjuvant for robust and durable immunity. We used the SARS-CoV-2 genomic sequence and in silico structural modelling to design a recombinant spike protein vaccine (Covax-19). A synthetic gene encoding the spike extracellular domain (ECD) was inserted into a baculovirus backbone to express the protein in insect cell cultures. The spike ECD was formulated with Advax-SM adjuvant and first tested for immunogenicity in C57BL/6 and BALB/c mice. The Advax-SM adjuvanted vaccine induced high titers of binding antibody against spike protein that were able to neutralise the original wildtype virus on which the vaccine was based as well as the variant B.1.1.7 lineage virus. The Covax-19 vaccine also induced potent spike-specific CD4+ and CD8+ memory T-cells with a dominant Th1 phenotype, and this was shown to be associated with cytotoxic T lymphocyte killing of spike labelled target cells in vivo. Ferrets immunised with Covax-19 vaccine intramuscularly twice 2 weeks apart made spike receptor binding domain (RBD) IgG and were protected against an intranasal challenge with SARS-CoV-2 virus 2 weeks after the second immunisation. Notably, ferrets that received two 25 or 50g doses of Covax-19 vaccine had no detectable virus in their lungs or in nasal washes at day 3 post-challenge, suggesting the possibility that Covax-19 vaccine may in addition to protection against lung infection also have the potential to block virus transmission. This data supports advancement of Covax-19 vaccine into human clinical trials.


title: U2 snRNA structure is influenced by SF3A and SF3B proteins but not by SF3B inhibitors

abstract: U2 snRNP is an essential component of the spliceosome. It is responsible for branch point recognition in the spliceosome A-complex via base-pairing of U2 snRNA with an intron to form the branch helix. Small molecule inhibitors target the SF3B component of the U2 snRNP and interfere with A-complex formation during spliceosome assembly. We previously found that the first SF3B inhibited-complex is less stable than A-complex and hypothesized that SF3B inhibitors interfere with U2 snRNA secondary structure changes required to form the branch helix. Using RNA chemical modifiers, we probed U2 snRNA structure in A-complex and SF3B inhibited splicing complexes. The reactivity pattern for U2 snRNA in the SF3B inhibited-complex is indistinguishable from that of A-complex, suggesting that they have the same secondary structure conformation, including the branch helix. This observation suggests SF3B inhibited-complex instability does not stem from an alternate RNA conformation and instead points to the inhibitors interfering with protein component interactions that normally stabilize U2 snRNPs association with an intron. In addition, we probed U2 snRNA in the free U2 snRNP in the presence of SF3B inhibitor and again saw no differences. However, increased protection of nucleotides upstream of Stem I in the absence of SF3A and SF3B proteins suggests a change of secondary structure at the very 5' end of U2 snRNA. Chemical probing of synthetic U2 snRNA in the absence of proteins results in similar protections and predicts a previously uncharacterized extension of Stem I. Because this stem must be disrupted for SF3A and SF3B proteins to stably join the snRNP, the structure has the potential to influence snRNP assembly and recycling after spliceosome disassembly.

category: molecular biology


title: Identification of the receptor-binding protein of Clostridium difficile phage CDHS-1 reveals a new class of receptor-binding domains.

abstract: As natural bacterial predators, bacteriophages have the potential to be developed to tackle antimicrobial resistance, but our exploitation of them is limited by understanding their vast uncharacterised genetic diversity1,2. Fascinatingly, this genetic diversity reflects many ways that phages can make proteins, performing similar functions that together form the familiar phage particle. Critical to infection are phage receptor-binding proteins (RBPs) that bind bacterial  receptors and initiate bacterial entry3. Here we identified and characterised Gp22, a novel RBP for phage CDHS-1 that infects pathogenic C. difficile, but that had no recognisable RBPs. We showed that Gp22 antibodies neutralised CDHS-1 infection and used immunogold-labelling and transmission electron microscopy to identify their location on the capsid. The Gp22 three-dimensional structure was resolved by X-ray crystallography revealing a new RBP class with an N-terminal L-shaped -helical superhelix domain and a C-terminal Mg2+-binding domain. The findings provide novel insights into C. difficile phage biology and phage-host interactions. This will facilitate optimal phage development and future engineering strategies4,5. Furthermore, the AlphaFold2-predicted Gp22 structure, which was strikingly accurate, paves the way for a structurome based transformation and guidance of future phage studies where many proteins lack sequence homology but have recognisable protein structures.


title: The structurome of a Clostridium difficile phage and the remarkable accurate prediction of its novel phage receptor-binding protein


title: Mitochondrial dysfunction and oxidative stress contribute to cognitive and motor impairment in FOXP1 syndrome

abstract: There is increasing evidence that mitochondrial homeostasis - influenced by both genetic and environmental factors - is crucial in neurodevelopment. FOXP1 syndrome is a neurodevelopmental disorder that manifests motor dysfunction, intellectual disability, autism and language impairment. In this study, we used a Foxp1+/- mouse model to address whether cognitive and motor deficits in FOXP1 syndrome are associated with mitochondrial dysfunction and oxidative stress. Here we show that genes with a role in mitochondrial biogenesis and dynamics (e.g. Foxo1, Pgc-1, Tfam, Opa1, and Drp1) were dysregulated in the striatum of Foxp1+/- mice at different postnatal stages. Furthermore, in the striatum of Foxp1+/- animals, mitochondrial membrane potential was disrupted, and reactive oxygen species, lipid peroxidation and cytochrome c release were significantly elevated. These features can explain the reduced neurite branching, learning and memory, endurance, and motor coordination that we observed in these animals. Taken together, we provide strong evidence of mitochondrial dysfunction in Foxp1+/- mice, suggesting that insufficient energy supply and excessive oxidative stress underlies the cognitive and motor impairment in FOXP1 deficiency.


title: Exocyst Inactivation in Urothelial Cells Disrupts Autophagy and Activates non-canonical NF-κB

abstract: Ureter obstruction is a highly prevalent event during embryonic development and is a major cause of pediatric kidney disease. We have reported that ureteric bud-specific ablation of the exocyst Exoc5 subunit in late-murine gestation results in failure of urothelial stratification, cell death, and complete ureter obstruction. However, the mechanistic connection between disrupted exocyst activity, urothelial cell death, and subsequent ureter obstruction was unclear. Here, we report that inhibited urothelial stratification does not drive cell death during ureter development. Instead, we demonstrate that the exocyst plays a critical role in autophagy in urothelial cells, and that disruption of autophagy activates a urothelial NF-{kappa}B stress response. Impaired autophagy first provokes canonical NF-{kappa}B activity which is progressively followed by increasing non-canonical NF-{kappa}B activity and cell death if the stress remains unresolved. Furthermore, we demonstrate that ureter obstructions can be completely rescued in Exoc5 conditional knockout mice by administering a single dose of pan-caspase inhibitor z-VAD-FMK at E16.5 prior to urothelial cell death. Taken together, ablation of Exoc5 disrupts autophagic stress response and activates progressive NF-{kappa}B signaling which promotes obstructive uropathy.


title: Increased frequency of recurrent in-frame deletions in new expanding lineages of SARS CoV-2 reflects immune selective pressure

abstract:
Most of the attention in the surveillance of evolution of SARS-CoV-2 has been centered on single nucleotide substitutions in the spike glycoprotein. We show that in-frame deletions (IFDs) also play a significant role in the evolution of viral genome. The percentage of genomes and lineages with IFDs is growing rapidly and they co-occur independently in multiple lineages, including emerging variants of concerns. IFDs distribution is correlated with spike mutations associated with immune escape and concentrated in proteins involved in interactions with the host immune system. Structural analysis suggests that IFDs remodel viral proteins surfaces at common epitopes and interaction interfaces, affecting the virus interactions with the immune system. We hypothesize that the increased frequency of IFDs is an adaptive response to elevated global population immunity.

SummaryMonitoring of SARS-CoV-2 genome evolution uncovers increased frequency and non-random distribution of in-frame deletions in recently emerged lineages.

category: evolutionary biology


title: Mutation signatures inform the natural host of SARS-CoV-2

abstract: The before-outbreak evolutionary history of SARS-CoV-2 is enigmatic because it shares only [~]96% genomic similarity with RaTG13, the closest relative so far found in wild animals (horseshoe bats). Since mutations on single-stranded viral RNA are heavily shaped by host factors, the viral mutation signatures can in turn inform the host. By comparing publically available viral genomes we here inferred the mutations SARS-CoV-2 accumulated before the outbreak and after the split from RaTG13. We found the mutation spectrum of SARS-CoV-2, which measures the relative rates of 12 mutation types, is 99.9% identical to that of RaTG13. It is also similar to that of two other bat coronaviruses but distinct from that evolved in non-bat hosts. The viral mutation spectrum informed the activities of a variety of mutation-associated host factors, which were found almost identical between SARS-CoV-2 and RaTG13, a pattern difficult to create in laboratory. All the findings are robust after replacing RaTG13 with RshSTT182, another coronavirus found in horseshoe bats with [~]93% similarity to SARS-CoV-2. Our analyses suggest SARS-CoV-2 shared almost the same host environment with RaTG13 and RshSTT182 before the outbreak.


title: Construction of a new chromosome-scale, long-read reference genome assembly of the Syrian hamster, Mesocricetus auratus

abstract:
BackgroundThe Syrian hamster (Mesocricetus auratus) has been suggested as a useful mammalian model for a variety of diseases and infections, including infection with respiratory viruses such as SARS-CoV-2. The MesAur1.0 genome assembly was generated in 2013 using whole-genome shotgun sequencing with short-read sequence data. Current more advanced sequencing technologies and assembly methods now permit the generation of near-complete genome assemblies with higher quality and greater continuity.

FindingsHere, we report an improved assembly of the M. auratus genome (BCM_Maur_2.0) using Oxford Nanopore Technologies long-read sequencing to produce a chromosome-scale assembly. The total length of the new assembly is 2.46 Gbp, similar to the 2.50 Gbp length of a previous assembly of this genome, MesAur1.0. BCM_Maur_2.0 exhibits significantly improved continuity with a scaffold N50 that is 6.7 times greater than MesAur1.0. Furthermore, 21,616 protein coding genes and 10,459 noncoding genes are annotated in BCM_Maur_2.0 compared to 20,495 protein coding genes and 4,168 noncoding genes in MesAur1.0. This new assembly also improves the unresolved regions as measured by nucleotide ambiguities, where approximately 17.11% of bases in MesAur1.0 were unresolved compared to BCM_Maur_2.0 in which the number of unresolved bases is reduced to 3.00%.

ConclusionsAccess to a more complete reference genome with improved accuracy and continuity will facilitate more detailed, comprehensive, and meaningful research results for a wide variety of future studies using Syrian hamsters as models.


title: Construction of a new chromosome-scale, long-read reference genome assembly for the Syrian hamster, Mesocricetus auratus


title: A PKA Inhibitor Motif within Smoothened Controls Hedgehog Signal Transduction

abstract: The Hedgehog (Hh) cascade is central to development, tissue homeostasis, and cancer. A pivotal step in Hh signal transduction is the activation of GLI transcription factors by the atypical G protein-coupled receptor (GPCR) Smoothened (SMO). How SMO activates GLI has remained unclear for decades. Here we show that SMO employs a decoy substrate sequence to physically block the active site of the PKA catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular, and organismal models, we demonstrate that interfering with SMO / PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism we uncovered echoes one utilized by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a new mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.


title: Protease Resistance of ex vivo Amyloid Fibrils implies the proteolytic Selection of disease-associated Fibril Morphologies

abstract: Several studies recently showed that ex vivo fibrils from patient or animal tissue were structurally different from in vitro formed fibrils from the same polypeptide chain. Analysis of serum amyloid A (SAA) and A{beta}-derived amyloid fibrils additionally revealed that ex vivo fibrils were more protease stable than in vitro fibrils. These observations gave rise to the proteolytic selection hypothesis that suggested that disease-associated amyloid fibrils were selected inside the body by their ability to resist endogenous clearance mechanisms. We here show, for more than twenty different fibril samples, that ex vivo fibrils are more protease stable than in vitro fibrils. These data support the idea of a proteolytic selection of pathogenic amyloid fibril morphologies and help to explain why only few amino acid sequences lead to amyloid diseases, although many, if not all, polypeptide chains can form amyloid fibrils in vitro.


title: Feeding effects on liver mitochondrial bioenergetics of Boa constrictor (Serpentes: Boidae)

abstract: Snakes are interesting examples of overcoming energy metabolism challenges as many species can endure long periods without feeding, and their eventual meals are of reasonably large sizes, thus exhibiting dual extreme adaptations. Consequently, metabolic rate increases considerably to attend to the energetic demand of digestion, absorption and, protein synthesis. These animals should be adapted to transition from these two opposite states of energy fairly quickly, and therefore we investigated mitochondrial function plasticity in these states. Herein we compared liver mitochondrial bioenergetics of the boid snake Boa constrictor during fasting and after meal intake. We fasted the snakes for 60 days, then we fed a subgroup with 30% of their body size and evaluated their maximum postprandial response. We measured liver respiration rates from permeabilized tissue and isolated mitochondria, and from isolated mitochondria, we also measured Ca2+ retention capacity, the release of H2O2, and NAD(P) redox state. Mitochondrial respiration rates were maximized after feeding, reaching until 60% increase from fasting levels when energized with complex I-linked substrates. Interestingly, fasting and fed snakes exhibited similar respiratory control ratios and citrate synthase activity. Furthermore, we found no differences in Ca2+ retention capacity, indicating no increase in susceptibility to mitochondrial permeability transition pore (PTP), or redox state of NAD(P), although fed animals exhibited increases in the release of H2O2. Thus, we conclude that liver mitochondria from B. constrictor snakes increase the maintenance costs during the postprandial period and quickly improve the mitochondrial bioenergetics capacity without compromising the redox balance.


title: Increased Histone-DNA Complexes and Endothelial-Dependent Thrombin Generation in Severe COVID-19

abstract:
ObjectiveCoagulopathy in severe COVID-19 is common but poorly understood. The purpose of this study was to determine how SARS-CoV-2 infection impacts histone levels, fibrin structure, and endogenous thrombin potential in the presence and absence of endothelial cells.

ApproachWe studied individuals with SARS-CoV-2 infection and acute respiratory distress syndrome at the time of initiation of mechanical ventilation compared to healthy controls. Blood samples were assayed for levels of histone-DNA complexes. Confocal microscopy was used to evaluate fibrin structure in clots formed from recalcified plasma samples using fluorescently-labeled fibrinogen. Endogenous thrombin potential was measured by calibrated automated thrombin assays in the presence of tissue factor and phospholipid (PCPS) or cultured human endothelial cells.

ResultsCirculating nucleosomes were elevated in the plasma of COVID-19 patients relative to healthy controls (n=6, each group). COVID-19 patient plasma thrombin generation was also altered. Despite having an increased endogenous thrombin potential, patient plasma samples exhibited prolonged lag times and times to peak thrombin in the presence of added tissue factor and PCPS. Strikingly different results were observed when endothelial cells were used in place of tissue factor and PCPS. Control plasma samples did not generate measurable thrombin (lag time >60 min); in contrast, plasma samples from COVID-19+ patients generated thrombin (mean lag time [~]20 min). Consistent with the observed alterations in thrombin generation, clots from COVID-19 subjects exhibited a denser fibrin network, thinner fibers and lower fibrin resolvability.

ConclusionsElevated histones, aberrant fibrin formation, and increased endothelial-dependent thrombin generation in COVID-19 may contribute to coagulopathy.

HIGHLIGHTSO_LIHistone-DNA complexes are significantly elevated in the plasma of patients with severe SARS-CoV-2 infection.
C_LIO_LIMeasures of thrombin generation by calibrated automated thrombography and fibrin clots formed in situ are altered in severe COVID-19.
C_LIO_LIPlasma from COVID-19 patients promotes thrombin generation on cultured endothelial cells in the absence of added tissue factor or phospholipids.
C_LIO_LIThe additive effects of histones on thrombin generation and endothelial cell function may play a major role in the thrombotic complications observed in severe SARS-CoV-2 infection.
C_LI


title: Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel

abstract: Ligand binding to membrane proteins is critical for many biological signaling processes. However, individual binding events are rarely directly observed, and their asynchronous dynamics are occluded in ensemble-averaged measures. For membrane proteins, single-molecule approaches that resolve these dynamics are challenged by dysfunction in nonnative lipid environments, lack of access to intracellular sites, and costly sample preparation. Here, we introduce an approach combining cell-derived nanovesicles, microfluidics, and single-molecule fluorescence colocalization microscopy to track individual binding events at a cyclic nucleotide-gated TAX-4 ion channel critical for sensory transduction. Our observations reveal dynamics of both nucleotide binding and a subsequent conformational change likely preceding pore opening. We further show that binding of the second ligand in the tetrameric channel is less cooperative than previously estimated from ensemble-averaged binding measures. This approach is broadly applicable to studies of binding dynamics for proteins with extracellular or intracellular domains in native cell membrane.


title: CALR frameshift mutations in MPN patient-derived iPS cells accelerate maturation of megakaryocytes

abstract: Calreticulin (CALR) mutations are driver mutations in myeloproliferative neoplasms (MPNs), leading to activation of the thrombopoietin receptor, and causing abnormal megakaryopoiesis. Here, we generated patient-derived CALRins5- or CALRdel52-positive induced pluripotent stem (iPS) cells to establish a MPN disease model for molecular and mechanistic studies. We demonstrated myeloperoxidase deficiency in CD15+ granulocytic cells derived from homozygous CALR-mutant iPS cells, rescued by repairing the mutation using CRISPR/Cas9. iPS cell-derived megakaryocytes showed characteristics of primary megakaryocytes such as formation of demarcation membrane system and cytoplasmic pro-platelets protrusions. Importantly, CALR mutations led to enhanced megakaryopoiesis and accelerated megakaryocytic development in a thrombopoietin-independent manner. Mechanistically, our study identified differentially regulated pathways in mutated vs. unmutated megakaryocytes, such as hypoxia signaling, which represents a potential target for therapeutic intervention. Altogether, we demonstrate key aspects of mutated CALR-driven pathogenesis, dependent on its zygosity and found known and novel therapeutic targets, making our model a valuable tool for clinical drug screening in MPNs.


title: p53-mediated neurodegeneration in the absence of the nuclear protein Akirin2

abstract: Proper gene regulation is critical for both neuronal development and maintenance as the brain matures. We previously demonstrated that Akirin2, an essential nuclear protein that interacts with transcription factors and chromatin remodeling complexes, is required for the embryonic formation of the cerebral cortex. Here we show that Akirin2 plays a mechanistically distinct role in maintaining healthy neurons during cortical maturation. Restricting Akirin2 loss to excitatory cortical neurons resulted in progressive neurodegeneration via necroptosis and severe cortical atrophy with age. Comparing transcriptomes from Akirin2-null postnatal neurons and cortical progenitors revealed that targets of the tumor suppressor p53, a regulator of both proliferation and cell death encoded by Trp53, were consistently upregulated. Heterozygous deletion of Trp53 rescued neurodegeneration in Akirin2-null neurons. These data: 1) implicate Akirin2 as a critical neuronal maintenance protein; 2) identify p53 pathways as mediators of Akirin2 functions; and 3) suggest Akirin2 dysfunction may be relevant to neurodegenerative diseases.


title: Polybasic patches in both C2 domains of Synaptotagmin-1 are required for evoked neurotransmitter release

abstract:
Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release. It is composed of a single-pass transmembrane domain linked to two tandem C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is not well understood. Calcium binding to C2B, but not to C2A, is critical for synchronous release and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here we asked what critical feature of C2A may be responsible for its functional role, and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1K189-192A and Syt1K326-327A) side-by-side to determine their relative contributions to Syt1 function in cultured cortical mouse neurons and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The readily releasable vesicle pool or spontaneous release were not affected, so both patches are specifically required for synchronization of release. We suggest these patches contribute to cooperative binding to membranes, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release.

Significance StatementSynaptotagmin-1 is a vesicular calcium sensor required for synchronous neurotransmitter release. Its tandem cytosolic C2 domains (C2A and C2B) bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. How calcium-binding to Synaptotagmin-1 leads to release and the relative contributions of the two C2 domains is not clear: unlike C2B, calcium-binding to C2A is not critical for evoked release, yet both domains are needed for Syt1 function. Combining electrophysiological recordings from cultured neurons and optical tweezers measurements that probe single C2 domain-membrane interactions, we show that conserved polybasic regions in both domains contribute to membrane binding cooperatively, and both are required for evoked release, likely by increasing the overall affinity of Synaptotagmin-1 for negatively charged membranes.


title: D-cysteine is an endogenous regulator of neural progenitor cell dynamics in the mammalian brain

abstract: D-amino acids are increasingly recognized as important signaling molecules in the mammalian central nervous system. However, the D-stereoisomer of the amino acid with the fastest in vitro spontaneous racemization rate, cysteine, has not been examined in mammals. Using chiral high-performance liquid chromatography and an stereospecific luciferase assay, we identify endogenous D-cysteine in the mammalian brain. We identify serine racemase (SR), which generates the NMDA glutamate receptor co-agonist D-serine, as a candidate biosynthetic enzyme for D-cysteine. Levels of D-cysteine are enriched over twentyfold in the embryonic mouse brain compared to the adult. D-cysteine reduces the proliferation of cultured mouse embryonic neural progenitor cells (NPCs) by approximately 50%, effects not shared with D-serine or L-cysteine. The antiproliferative effect of D-cysteine is mediated by the transcription factors FoxO1 and FoxO3a. The selective influence of D-cysteine on NPC proliferation is reflected in overgrowth and aberrant lamination of the cerebral cortex in neonatal SR knockout mice. Finally, we perform an unbiased screen for D-cysteine-binding proteins in NPCs by immunoprecipitation with a D-cysteine-specific antibody followed by mass spectrometry. This approach identifies myristoylated alanine-rich C-kinase substrate (MARCKS) as a putative D-cysteine-binding protein. Together, these results establish endogenous mammalian D-cysteine and implicate it as a physiologic regulator of NPC homeostasis in the developing brain.


title: Members of the CUGBP Elav-Like Family of RNA-Binding Proteins are Expressed in Distinct Populations of Primary Sensory Neurons

abstract: Primary sensory Dorsal Root Ganglia (DRG) neurons are diverse, with distinct populations that respond to specific stimuli. Previously, we observed that functionally distinct populations of DRG neurons express mRNA transcript variants with different 3 untranslated regions (3UTRs). 3UTRs harbor binding sites for interaction with RNA-binding proteins (RBPs) critical for targeting mRNAs to subcellular domains, modulating transcript stability and regulating the rate of translation. In the current study we sought to determine if 3UTR-binding proteins are restricted to specific DRG neuron populations. Analysis of publicly available single-cell RNA-Sequencing (scRNA-Seq) data generated from adult mice revealed that 17 3UTR-binding RBPs were enriched in specific populations of DRG neurons. This included 4 members of the CUGBP Elav-Like Family (CELF). CELF2 and CELF4 were enriched in peptidergic, CELF6 in both peptidergic and nonpeptidergic and CELF3 in tyrosine hydroxylase-expressing neurons. CELF4 is a known regulator of neural excitability, likely through modulation of protein synthesis via binding to interaction sites within the 3UTRs of mRNAs. Immunofluorescence studies showed 60% of CELF4+ neurons are small diameter C fibers and 33% medium diameter myelinated (likely Ad) fibers. Co-expression analyses using transcriptomic data and quantitative immunofluorescence revealed that CELF4 is enriched in nociceptive neurons that express GFRA3, CGRP and the capsaicin receptor TRPV1. Finally, genes with CELF4 binding motifs expressed in CELF4+ neurons are significantly associated with gene ontology (GO) terms such as "RNA-binding" and "translation". We propose that CELF4 may therefore control a novel regulon that coordinates the translation of mRNAs encoding components of the protein translation apparatus in nociceptors.


title: The relationship between mucosal microbiota, colitis and systemic inflammation in Chronic Granulomatous Disorder

abstract:
BackgroundChronic granulomatous disorder (CGD) is a primary immunodeficiency which is frequently complicated by an inflammatory colitis and is associated with systemic inflammation.

ObjectiveTo investigate the role of the microbiome in the pathogenesis of colitis and systemic inflammation.

MethodsWe performed 16S rDNA sequencing on mucosal biopsy samples from each segment of 10 CGD patients colons, and conducted compositional and functional pathway prediction analyses.

ResultsThe microbiota in samples from colitis patients demonstrated reduced taxonomic alpha diversity compared to unaffected patients, even in apparently normal bowel segments. Functional pathway richness was similar between the colitic and non-colitic mucosa, although metabolic pathways involved in butyrate biosynthesis or utilisation were enriched in patients with colitis and correlated positively with faecal calprotectin levels. One patient with very severe colitis was dominated by Enterococcus spp., while among other patients Bacteroides spp. abundance correlated with colitis severity measured by faecal calprotectin and an endoscopic severity score. In contrast, Blautia abundance associated with low severity scores and mucosal health. Several taxa and functional pathways correlated with concentrations of inflammatory cytokines in blood but not with colitis severity. Notably, dividing patients into  High and  Low systemic inflammation groups demonstrated clearer separation than on the basis of colitis status in beta diversity analyses.

ConclusionThe microbiome is abnormal in CGD-associated colitis and altered functional characteristics probably contribute to pathogenesis. Furthermore, the relationship between the mucosal microbiome and systemic inflammation, independent of colitis status, implies that the microbiome in CGD can influence the inflammatory phenotype of the condition.

Key MessagesThe colonic mucosal microbiome and bacterial metabolic pathways in patients with CGD colitis differ from patients without colitis, even in macroscopically normal bowel segments.

The mucosal microbiome and bacterial metabolic pathways in patients with CGD also differ according to the extent of systemic inflammation, independently from the presence of colitis, suggesting a role for the gut microbiota in the inflammatory phenotype of this condition.

Capsule summaryThe pathogenesis of chronic granulomatous disorder (CGD)-associated colitis and other inflammatory complications is unclear. We demonstrate potentially treatable alterations in the mucosa-associated microbiome in CGD colitis and microbial differences which associate with systemic inflammation independently of colitis status.