title: Sloppiness: fundamental study, new formalism and quantification

abstract: Precise estimation of parameters in a complex dynamical system is often challenging, even if provided with adequate quality and quantity of data. A major challenge is the possible presence of large regions in the parameter space over which model predictions are nearly identical. This property, known as sloppiness, has been reasonably well-addressed in the past decade, studying its possible impacts and remedies. However, certain critical unanswered questions concerning sloppiness, its quantification and practical implications in identification still prevail. In this work, we systematically examine sloppiness at a fundamental level and formalise a new theoretical definition of sloppiness. Further, we propose a method to quantify sloppiness for non-linear predictors. The proposed method aids in the characterisation of a model structure around a point of interest in the parameter space and detecting local structural unidentifiability. Further, we establish a mathematical relationship between practical identifiability and sloppiness in linear predictors. Finally, we demonstrate the proposed formalism and methods on standard models.


title: Nuclear inhibitor of protein phosphatase 1 (NIPP1) regulates CNS tau phosphorylation and myelination during development

abstract: Nuclear inhibitor of protein phosphatase 1 (NIPP1) is a known regulator of gene expression and has been shown to play roles in many physiological or pathological processes such as stem cell proliferation and skin inflammation. While NIPP1 has many regulatory roles in proliferating cells, its function in the central nervous system (CNS) has not been directly investigated. In the present study, we examined NIPP1 CNS function using a conditional knockout (cKO) mouse model, in which the nipp1 gene is excised from neural precursor cells. These mice demonstrate severe developmental impairments that lead to premature lethality within the first few postnatal weeks. To delineate some of the neurological changes occurring in these animals, we first assessed microtubule associated protein tau, a known target of NIPP1 activity. Furthermore, observed tremors prompted exploration of myelin integrity, an integral structure for CNS function, whose disruption is associated with various neurological disorders and neurodegenerative diseases. First, immunoblotting demonstrated increased phospho-tau and altered AKT and PP1 activity in NIPP1 cKO mice, suggesting increased tau phosphorylation likely results from a shift in kinase/phosphatase activity. Second, immunoblots, electron microscopy, and electrophysiology demonstrated a myelin deficit within the brain and optic nerve. Our study suggests that NIPP1 in neural precursors regulates phosphorylation of tau and CNS myelination and may represent a novel therapeutic target for neurodegenerative diseases.

category: neuroscience


title: Perivascular SPP1 Mediates Microglial Engulfment of Synapses in Alzheimers Disease Models

abstract: Microglia are phagocytes of the brain parenchyma, where they interact with neurons to engulf synapses in a context-dependent manner. Genetic studies in Alzheimers disease (AD) highlight dysfunctional phagocytic signaling in myeloid cells as disease-associated pathway. In AD models, there is a region-specific reactivation of microglia-synapse phagocytosis involving complement; however, what drives microglia-synapse engulfment remains unknown. Here, we show that SPP1 (Osteopontin), a glycoprotein associated with inflammation, is regionally upregulated and modulates microglial synaptic engulfment in AD mouse models. Ultrastructural examination revealed SPP1 expression predominantly by perivascular macrophages, a subtype of border-associated macrophages, in the hippocampus of mice and patient tissues. Cell-cell interaction networks of single-cell transcriptomics data suggested that perivascular SPP1 drives microglial functional states in the hippocampal microenvironment of AD mice. Absence of Spp1 expression resulted in failure of microglia to mediate synaptic phagocytosis. This study suggests a critical role for perivascular SPP1 in neuroimmune crosstalk in AD-relevant context.


title: Effect of LSD and music on the time-varying brain dynamics

abstract: Psychedelics are getting closer to being widely used in clinical treatment. Music is known as a key element of psychedelic-assisted therapy due to its psychological effects, specifically on the emotion, meaning-making and sensory processing. However, there is still a lack of understanding in how psychedelics influence brain activity in experimental settings involving music listening. The main goal of our research is to investigate the effect of music, as a part of "setting", on the brain states dynamics after lysergic acid diethylamide (LSD) intake. We used an open dataset, where a group of 15 participants underwent two scanning sessions under LSD influence and under placebo. Every scanning session contained three runs: two resting-state runs separated by one run with music listening. We applied K-Means clustering to identify the repetitive patterns of brain activity, so-called brain states. For further analysis, we calculated states dwell time, fractional occupancy and transition probability. We found that the brain states dynamics during the resting-state and listening to music on both LSD and placebo does not differ significantly. Furthermore, we found that the music itself could potentially have a long-term influence on the resting-state, in particular on the states involving task-positive networks. Collectively, these findings suggest that the whole-brain states dynamics during psychedelic experience and placebo is relatively stable, however, music, as a crucial element of "setting", can potentially have an influence on the subjects resting-state. Further studies should replicate these results on a larger sample size.


title: Delay-related activity in marmoset prefrontal cortex

abstract: Persistent delay-period activity in prefrontal cortex (PFC) has long been regarded as a neural signature of working memory (WM). Electrophysiological investigations in macaque PFC have provided much insight into WM mechanisms, however a barrier to understanding is the fact that a portion of PFC lies buried within the principal sulcus in this species and is inaccessible for laminar electrophysiology or optical imaging. The relatively lissencephalic cortex of the New World common marmoset (Callithrix jacchus) circumvents such limitations. It remains unknown however, whether marmoset PFC neurons exhibit persistent activity. Here, we addressed this gap by conducting wireless electrophysiological recordings in PFC of marmosets performing a delayed-match-to-location task on a home cage-based touchscreen system. As in macaques, marmoset PFC neurons exhibited sample, delay, and response-related activity that was directionally tuned and linked to correct task performance. Models constructed from population activity consistently and accurately predicted stimulus location throughout the delay period, supporting a framework of delay activity in which mnemonic representations are relatively stable in time. Taken together, our findings support the existence of common neural mechanisms underlying WM performance in PFC of macaques and marmosets, and thus validate the marmoset as a suitable model animal for investigating the microcircuitry underlying WM.


title: Dichotomic Regulation of Striatal Plasticity by Dynorphin

abstract: Modulation of corticostriatal plasticity alters the information flow throughout basal ganglia circuits and represents a fundamental mechanism for motor learning, action selection, and reward. Synaptic plasticity in the striatal direct- and indirect-pathway spiny projection neurons (dSPNs and iSPNs) are dichotomically regulated by two distinct networks of GPCR signaling cascades. While it is well-known that dopamine D2 and adenosine A2a receptors bidirectionally regulate iSPN plasticity, it remains unclear how D1 signaling modulation of synaptic plasticity is counteracted by a dSPN-specific Gi signaling. Here, we show that striatal dynorphin selectively suppresses long-term potentiation (LTP) through Kappa Opioid Receptor (KOR) in dSPNs. Both KOR antagonism and conditional deletion of dynorphin in dSPNs enhance LTP counterbalancing with different levels of D1 receptor activation. Behaviorally, mice lacking dynorphin specifically in dSPNs show normal motor behavior and reward-based learning, but enhanced flexibility during reversal learning. These findings support a model in which D1R and KOR signaling bidirectionally modulate synaptic plasticity in striatal direct pathways and behavior.


title: Overactivation of a sleep-active neuron decouples survival from the need to sleep

abstract:
Sleep is a state of behavioral quiescence that is closely associated with survival. Sleep-active neurons promote sleep and survival. It is not known, however, whether sleep-active neurons need to cause sleep to promote survival. Here, we tested how depolarization of the sleep-active RIS neuron in Caenorhabditis elegans controls sleep and survival during larval starvation. Survival always increased with raised RIS depolarization. RIS depolarization promoted sleep over a long range. Unexpectedly, however, high levels of RIS depolarization caused a nearly complete loss of sleep. Similarly, overexpression of sleep-inducing FLP-11 neuropeptides in RIS inhibited sleep, indicating that overactive transmission from RIS inhibits sleep. Despite the loss of sleep, survival was normal following FLP-11 overexpression. Thus, RIS overactivation abolishes sleep yet supports survival. These results indicate that regulation of sleep and survival are separable functions of a sleep-active neuron that are normally coupled but can be uncoupled by sleep-neuron over activation.

Author SummarySleep behavior and survival benefits are independently regulated functions of the sleep neuron RIS in Caenorhabditis elegans.


title: All-optical visualization of specific molecules in the ultrastructural context of brain tissue

abstract:
Understanding the molecular anatomy and neural connectivity of the brain requires imaging technologies that can map the 3D nanoscale distribution of specific proteins in the context of brain ultrastructure. Light and electron microscopy (EM) enable visualization of either specific labels or anatomical ultrastructure, but combining molecular specificity with anatomical context is challenging. Here, we present pan-Expansion Microscopy of tissue (pan-ExM-t), an all-optical mouse brain imaging method that combines [~]24-fold linear expansion of biological samples with fluorescent pan-staining of protein densities (providing EM-like ultrastructural context), and immunolabeling of protein targets (for molecular imaging). We demonstrate the versatility of this approach by imaging the established synaptic markers Homer1, Bassoon, PSD-95, Synaptophysin, the astrocytic protein GFAP, myelin basic protein (MBP), and anti-GFP antibodies in dissociated neuron cultures and mouse brain tissue sections. pan-ExM-t reveals these markers in the context of ultrastructural features such as pre and postsynaptic densities, 3D nanoarchitecture of neuropil, and the fine structures of cellular organelles. pan-ExM-t is adoptable in any neurobiological laboratory with access to a confocal microscope and has therefore broad applicability in the research community.

HighlightsO_LIpan-ExM-t visualizes proteins in the context of synaptic ultrastructure
C_LIO_LILipid labeling in pan-ExM-t reveals organellar and cellular membranes
C_LIO_LIAll-optical, easily accessible alternative to correlative light/electron microscopy
C_LIO_LIHigh potential for high throughput connectomics studies
C_LI


title: Neuronal nuclear calcium signaling suppression of microglial reactivity is mediated by osteoprotegerin after traumatic brain injury

abstract:
BackgroundTraumatic Brain Injury (TBI) is characterized by massive changes in neuronal excitation, from acute excitotoxicity to chronic hyper- or hypoexcitability. Nuclear calcium signaling pathways are involved in translating changes in synaptic inputs and neuronal activity into discrete transcriptional programs which not only affect neuronal survival and synaptic integrity, but also the crosstalk between neurons and glial cells. Here we report the effects of blunting neuronal nuclear calcium signals in the context of TBI.

MethodsWe used AAV vectors to express the genetically-encoded and nuclear-targeted calcium buffer parvalbumin (PV.NLS.mCherry) or the calcium/calmodulin buffer CaMBP4.mCherry in neurons only. Upon TBI, the extent of neuroinflammation, neuronal death and synaptic loss were assessed by immunohistochemistry and targeted transcriptome analysis. Modulation of the overall level of neuronal activity was achieved by PSAM/PSEM chemogenetics targeted to parvalbumin interneurons. The functional impact of neuronal nuclear Calcium buffering in TBI was assessed by quantification of spontaneous whisking.

ResultsBuffering neuronal nuclear calcium unexpectedly resulted in a massive and long-lasting increase in the recruitment of reactive microglia to the injury site, which was characterised by a disease-associated and phagocytic phenotypes. This effect was accompanied by a substantial surge in synaptic loss and significantly reduced whisking activity. Transcriptome analysis revealed a complex effect of TBI in the context of neuronal nuclear calcium buffering, with upregulation of complement factors, chemokines and interferon-response genes, as well as the downregulation of synaptic genes and epigenetic regulators compared to control conditions. Notably, nuclear calcium buffering led to a substantial loss in neuronal osteoprotegerin (OPG). Whereas stimulation of neuronal firing induced OPG expression. Viral re-expression of OPG resulted in decreased microglial recruitment and synaptic loss. OPG upregulation was also observed in the CSF of human TBI patients, underscoring its translational value.

ConclusionNeuronal nuclear calcium signals regulate the degree of microglial recruitment and reactivity upon TBI via, among others, osteoprotegerin signals. Our findings support a model whereby neuronal activity altered after TBI exerts a powerful impact on the neuroinflammatory cascade, which in turn contributes to the overall loss of synapses and functional impairment.


title: Single-cell transcriptomics reveal hyperacute cytokine and immune checkpoint axis in patients with poor neurological outcomes after cardiac arrest

abstract: Neurological injury is a major driver of mortality among patients hospitalized after cardiac arrest (CA). The early systemic inflammatory response after CA is associated with neurological injury and mortality but remains poorly defined. We determine the innate immune network induced by clinical CA at single-cell resolution. Immune cell states diverge as early as 6h post-CA between patients with good or poor neurological outcomes at hospital discharge. Nectin-2+ monocyte and Tim-3+ natural killer (NK) cell subpopulations associate with poor outcomes, and interactome analysis highlights their crosstalk via cytokines and immune checkpoints. Ex vivo studies on peripheral blood cells from CA patients demonstrate that immune checkpoints are a compensatory mechanism against inflammation after CA. IFN{gamma}/IL-10 induce Nectin-2 on monocytes; in a negative feedback loop, Nectin-2 suppresses IFN{gamma} production by NK cells. The initial hours after CA may represent a window for therapeutic intervention in the resolution of inflammation via immune checkpoints.

category: immunology


title: A one-step CRISPR-based strategy for endogenous gene tagging in Drosophila melanogaster

abstract: The ability to tag endogenous genes to create fluorescent fusion proteins has revolutionized the studies of protein subcellular localization and dynamics and regulation in live cells. Here, we report a precise, rapid, and one-step endogenous gene tagging system in Drosophila melanogaster, which allows us to screen for engineered lines without the visible eye marker. Specifically, we developed an efficient screening strategy for the identification of the homologous integration events by employing a PCR-based method to characterize individual flies using only a small segment of their middle leg. Here, we detail design and construction of gRNA plasmids and donor plasmid, and methods for screening, and confirmation of engineered lines. Together, these protocols make tagging any endogenous protein in Drosophila more efficient and faster, enabling studies of a wide range of cellular processes in live cells within intact organisms.

category: molecular biology


title: Detection and molecular characterization of a phytoplasma associated with Cucumber (Cucumis sativus) and its first report from India

abstract: Cucumber (Cucumis sativus) plants exhibiting typical phyllody symptoms were collected from farmers field of Chintamani, Chickballapur districts of Karnataka (India). Disease incidence of phyllody was 2-3%. The etiology of the cucumber phyllody phytoplasma (CuPP) was confirmed by amplifying 16S rRNA gene from symptomatic plants using PCR followed by nested PCR using universal primers pairs P1/P7 and R16F2n/R16R2. After general detection the non-ribosomal SecY and rp (ribosomal protein) genes was amplified using specific primers. The PCR amplified products 16SrRNA (1.2 kb), SecY (1.6 kb) rp gene (1.2 kb) was cloned and sequenced. Sequence and phylogenetic analyses of 16S rRNA, secY and rp genes revealed that the detected phytoplasma is a member of the 16SrI group (Candidatus Phytoplasma asteris). Further on the basis of computer-simulated RFLP (= in silico RFLP) analysis of amplified F2n/R2 region of 16S rRNA gene indicates that, the detected phytoplasma was belongs to the subgroup X (16SrII-X). This is the first report on phytoplasma associated with phyllody disease of cucumber in India.


title: Transcriptional profiling from whole embryos to single neuroblast lineages in Drosophila

abstract: Embryonic development results in the production of distinct tissue types, and different cell types within each tissue. A major goal of developmental biology is to uncover the "parts list" of cell types that comprise each organ. Here we perform single cell RNA sequencing (scRNA-seq) of the Drosophila embryo to identify the genes that characterize different cell and tissue types during development. We assay three different timepoints, revealing a coordinated change in gene expression within each tissue. Interestingly, we find that the elav and mhc genes, whose protein products are widely used as markers for neurons and muscles, respectively, show broad pan-embryonic expression, indicating the importance of post-transcriptional regulation. We next focus on the central nervous system (CNS), where we identify genes characterizing each stage of neuronal differentiation: from neural progenitors, called neuroblasts, to their immediate progeny ganglion mother cells (GMCs), followed by new-born neurons, young neurons, and the most mature neurons. Finally, we ask whether the clonal progeny of a single neuroblast (NB7-1) share a similar transcriptional identity. Surprisingly, we find that clonal identity does not lead to transcriptional clustering, showing that neurons within a lineage are diverse, and that neurons with a similar transcriptional profile (e.g. motor neurons, glia) are distributed among multiple neuroblast lineages. Although each lineage consists of diverse progeny, we were able to identify a previously uncharacterized gene, Fer3, as an excellent marker for the NB7-1 lineage. Within the NB7-1 lineage, transcriptional clusters are identifiable in neuroblasts and neurons, and each cluster is composed of current temporal transcription factor (e.g. Hunchback, Kruppel, Pdm, and Castor), novel temporal factors, and/or targets of the temporal transcription factors. In conclusion, we have characterized the embryonic transcriptome for all major tissue types and for three stages of development, as well as the first transcriptomic analysis of a single, identified neuroblast lineage, finding a lineage-enriched transcription factor.

category: developmental biology


title: Colouring coral larvae for tracking dispersal

abstract:
O_LIEcologists have long sought to understand larval dispersal characteristics of benthic marine invertebrates. Direct quantification of coral larvae dispersal has proven challenging, in part due to their complex life-history, minute size, and widespread dispersal at the scale of kilometres. Instead, indirect methods such as particle modelling, chemical signatures, and genetic correlation are often used in dispersal studies.
C_LIO_LIHere, we develop a direct method of quantifying larval dispersal by applying vital stains to coral larvae, allowing differentiation and direct tracking of millions of larvae from the pelagic dispersal stage through to the sedentary stages of attachment and metamorphosis on coral reefs.
C_LIO_LINeutral red and Nile blue stains were extremely effective at staining coral larvae, while alizarin red and calcein blue showed no visible results. Differences in toxicity to vital stains was noted among species, with Acropora spp. exhibiting decreased larval survival and settlement, while Merulinidae spp. were unaffected. By experimenting with different incubation times and concentrations, our results indicate that neutral red can be effectively applied for short periods (<20 minutes) at low concentrations (1-100 mg l-1), whereas Nile blue requires longer stain times (>60 minutes) at higher concentrations (100-1000 mg l-1).
C_LIO_LIThe strong colour of both neutral red and Nile blue stains was retained by newly settled larvae in lab settings upwards of five days following settlement, providing a direct method of differentiating between newly settled larvae on reefs. Field-validation of Nile blue applied to coral larvae from wild-captured coral slicks demonstrates the efficacy of staining across a diverse range of coral taxa.
C_LIO_LIVital staining provides a simple, rapid (<60 mins), and low cost (<AUD$0.00001 per larva) method of colouring coral larvae that allows for direct tracking of dispersal and recruitment in studies of reef connectivity and restoration.
C_LI

category: ecology


title: Community adaptation to temperature explains abrupt soil bacterial community shift along a geothermal gradient on Iceland

abstract: Understanding how and why soil microbial communities respond to temperature changes is important for understanding the drivers of microbial distribution and abundance. A unique area in Iceland, where soil temperatures have increased due to geothermic activity four years prior to sampling, creating a stable gradient of ambient to +40{degrees}C, allowed us to investigate the shape of the response to warming of soil bacterial communities, and their associated community temperature adaptation. We used 16S rRNA amplicon sequencing to profile bacterial communities, and bacterial growth based assays (3H Leu-incorporation) to characterize community adaptation using a temperature sensitivity index (SI, log (growth at 40{degrees}C/4{degrees}C)). Samples were taken from [≥]9 levels of warming (covering almost up to +40{degrees}C above ambient soil temperature), under both grassland (Agrostis capillaris) and forest (Picea sitchensis) vegetation. The soils had very different community composition, but temperature adaptation was the same. Both diversity and community composition as well SI showed similar threshold dynamics along the soil temperature gradient. There were no significant changes up to soil warming of approx. 6-9 {degrees}C, beyond which all indices shifted in parallel. The consistency of these responses gives strong support for a decisive role for direct temperature effects in driving bacterial community shifts along soil temperature gradients.


title: NUMT PARSER: automated identification and removal of nuclear mitochondrial pseudogenes (numts) for accurate mitochondrial genome reconstruction in Panthera

abstract: Nuclear mitochondrial pseudogenes (numts) may hinder the reconstruction of mtDNA genomes and affect the reliability of mtDNA datasets for phylogenetic and population genetic comparisons. Here, we present the program NO_SCPLOWUMTC_SCPLOW PO_SCPLOWARSERC_SCPLOW, which allows for the identification of DNA sequences that likely originate from numt pseudogene DNA. Sequencing reads are classified as originating from either numt or true cytoplasmic mitochondrial (cymt) DNA by direct comparison against cymt and numt reference sequences. Classified reads can then be parsed into cymt or numt datasets. We tested this program using whole genome shotgun-sequenced data from two ancient Cape lions (Panthera leo), because mtDNA is often the marker of choice for ancient DNA studies and the genus Panthera is known to have numt pseudogenes. NO_SCPLOWUMTC_SCPLOW PO_SCPLOWARSERC_SCPLOW decreased sequence disagreements that were likely due to numt pseudogene contamination and equalized read coverage across the mitogenome by removing reads that likely originated from numts. We compared the efficacy of NO_SCPLOWUMTC_SCPLOW PO_SCPLOWARSERC_SCPLOW to two other bioinformatic approaches that can be used to account for numt contamination. We find that NO_SCPLOWUMTC_SCPLOW PO_SCPLOWARSERC_SCPLOW outperformed approaches that rely only on read alignment or Basic Local Alignment Search Tool (BLAST) properties, and was effective at identifying sequences that likely originated from numts while having minimal impacts on the recovery of cymt reads. NO_SCPLOWUMTC_SCPLOW PO_SCPLOWARSERC_SCPLOW therefore improves the reconstruction of true mitogenomes, allowing for more accurate and robust biological inferences.

category: evolutionary biology


title: Full-length dystrophin is expressed across human tissues and DMD downregulation commonly occurring in tumours coincides with Duchenne-like molecular alterations

abstract: Mutations in the DMD gene, encoding dystrophins, cause Duchenne muscular dystrophy (DMD). However, loss of DMD expression impacts a broader spectrum of cells than those affected in DMD. Tumours were used as a model to investigate whether DMD loss across dissimilar tissues evokes related outcomes. Transcriptomic, proteomic, and mutation datasets from forty-nine healthy tissues and corresponding primary tumours (10,542 samples), and 180 related tumour cell lines were analysed. DMD expression was widespread across healthy tissues at levels comparable to common housekeeping genes, with corresponding full-length dystrophin identified by mass spectrometry. DMD expression was reduced in 80% of analysed tumours due to transcriptional downregulation not somatic mutations. The full-length transcript encoding Dp427m was decreased significantly in 68% of tumours, while Dp71 showed variability of expression. Hierarchical clustering analysis of the most highly expressed DMD transcripts revealed six clusters distinguishing malignant from healthy tissues. Transcriptomes of primary tumours and corresponding tumour cell lines with low DMD expression showed enrichment of specific pathways in the downregulated genes, compared to tumours and cell lines with higher DMD expression. Pathways consistently identified, such as ECM-receptor interaction, calcium signalling and PI3K-Akt, are also altered in DMD. Thus, DMD transcription occurs across a spectrum of healthy tissues and the molecular signature associated with its downregulation, which frequently occurs in malignancies, is concordant with changes found in Duchenne muscles, even though these malignancies originate from tissues not commonly associated with dystrophin expression or function. Therefore, the importance of the DMD gene extends beyond its roles identified in DMD.

category: genetics


title: Active vision during prey-capture in wild marmoset monkeys

abstract: Here, we examined prey-capture in wild common marmosets (Callithrix jacchus) to explicate the active role of primate vision for high-precision, goal-directed motor actions in challenging natural environments. We observed distinct marmoset hunting strategies that each relied on the close integration of visual processes and dynamic biomechanical motor movements, but differed based on several factors including prey size/speed, substrate, and their relative distance to the prey. Marmoset positional behavior in these complex arboreal settings often involves extending the body, including inversions, while relying on vision to guide prey capture. Applying markerless pose estimation technology, analyses revealed details of how primate vision both guides and corrects rapid motor actions in real-time during prey-capture in the wild. These findings illustrate the significance of active vision during primate ethological behaviors in response to the very challenges the visual system evolved to solve.

category: animal behavior and cognition


title: Microenvironmental engineering improves the self-organization of patterned microvascular networks

abstract: The construction of three-dimensional (3D) microvascular networks with defined structures remains challenging. Emerging bioprinting strategies provide a means of patterning endothelial cells (ECs) into the geometry of 3D microvascular networks, but the microenvironmental cues necessary to promote their self-organization into cohesive and perfusable microvessels are unknown. To this end, we reconstituted microvessel formation in vitro by patterning thin lines of closely packed ECs fully embedded within a 3D extracellular matrix (ECM) and observed how different microenvironmental parameters influenced EC behaviors and their self-organization into microvessels. We found that the inclusion of fibrillar matrices, such as collagen I, into the ECM positively influenced cell condensation into extended geometries such as cords. We also identified the presence of a high molecular weight protein(s) in fetal bovine serum (FBS) that negatively influenced EC condensation. This component destabilized cord structure by promoting cell protrusions and destabilizing cell-cell adhesions. Endothelial cords cultured in the presence of fibrillar collagen and the absence of this protein activity were able to polarize, lumenize, incorporate mural cells, and support fluid flow. These optimized conditions allowed for the construction of branched and perfusable microvascular networks directly from patterned cells in as little as three days. These findings reveal important design principles for future microvascular engineering efforts based on bioprinting techniques.

category: bioengineering


title: Systematic classification error profoundly impacts inference in high-depth Whole Genome Shotgun Sequencing datasets

abstract: There is little consensus in the literature as to which approach for classification of Whole Genome Shotgun (WGS) sequences is best. In this paper, we examine two of the most popular algorithms, Kraken2 and Metaphlan2 utilizing four publicly available datasets. As expected from previous literature, we found that Kraken2 reports more overall taxa while Metaphlan2 reports fewer taxa while classifying fewer overall reads. To our surprise, however, Kraken 2 reported not only more taxa but many more taxa that were significantly associated with metadata. This implies that either Kraken2 is more sensitive to taxa that are biologically relevant and are simply missed by Metaphlan2, or that Kraken2s classification errors are generated in such a way to impact inference. To discriminate between these two possibilities, we compared Spearman correlations coefficients of each taxa against each taxa with higher abundance from the same dataset. We found that Kraken2, but not Metaphlan2, showed a consistent pattern of classifying low abundance taxa that generated high correlation coefficients with higher abundance taxa. Neither Metaphlan2, nor 16S sequences that were available for two of our four datasets, showed this pattern. Simple simulations based on a variable Poisson error rate sampled from the uniform distribution with an average error rate of 0.0005 showed strikingly strong concordance with the observed correlation patterns from Kraken2. Our results suggest that Kraken2 consistently misclassifies high abundance taxa into the same erroneous low abundance taxa creating "phantom" taxa have a similar pattern of inference as the high abundance source. Because of the large sequencing depths of modern WGS cohorts, these "phantom" taxa will appear statistically significant in statistical models even with a low overall rate of classification error from Kraken. Our simulations suggest that this can occur with average error rates as low as 1 in 2,000 reads. These data suggest a novel metric for evaluating classifier accuracy and suggest that the pattern of classification errors should be considered in addition to overall classification error rate since consistent classification errors have a more profound impact on inference compared to classification errors that do not always result in assignment to the same erroneous taxa. This work highlights fundamental questions on how classifiers function and interact with large sequencing depth and statistical models that still need to be resolved for WGS, especially if correlation coefficients between taxa are to be used to build covariance networks. Our work also suggests that despite its limitations, 16S rRNA sequencing may still be useful as neither of the two most popular 16S classifiers showed these patterns of inflated correlation coefficients between taxa.

category: bioinformatics


title: recolorize: improved color segmentation of digital images (for people with other things to do)

abstract:
O_LIColor patterns are easy to observe, but difficult to quantify. A necessary first step for most digital color pattern analysis methods is generating color maps, where every pixel in an image is assigned to one of a set of discrete color pattern elements (color segmentation).
C_LIO_LIMost automatic methods for color segmentation rely on color value thresholds or k-means clustering. While these work in specialized cases, these methods often fail due to biological variation in pattern distribution and intensity, technical variation in images, and poor scalability for batch clustering. As a result, color segmentation is the bottleneck step in a wide variety of analyses.
C_LIO_LIHere we present recolorize, an R package for color segmentation that succeeds in many cases where currently used methods fail. Recolorize has three major components: (1) an effective two-part algorithm for color clustering where color distributions are binned and combined according to perceived similarity; (2) a toolkit for making minor manual adjustments to automatic output where needed; and (3) easy export options to a variety of methods that require accurate color maps.
C_LIO_LIThis paper illustrates how to use recolorize, includes examples where we effectively address many of the problems that fail using k-means clustering, and combines the package with two of the most popular R packages for quantifying color pattern variation (patternize and pavo).
C_LI


title: A Deep Learning Method for MiRNA/IsomiR Target Detection

abstract:
MotivationAccurate identification of microRNA (miRNA) targets at base-pair resolution has been an open problem for over a decade. The recent discovery of miRNA isoforms (isomiRs) adds more complexity to this problem. Despite the existence of many methods, none considers isomiRs, and their performance is still suboptimal. We hypothesize that by taking the isomiR-mRNA interaction into account and applying a deep learning model to study miRNA-mRNA interaction features, we may improve the accuracy of miRNA target predictions.

ResultsWe developed a deep learning tool called DMISO to capture the intricate features of miRNA/isomiR-mRNA interactions. Based on 10-fold cross-validation, DMISO showed high precision (95%) and recall (90%). Evaluated on three independent datasets, DMISO had superior performance to five tools, including three popular conventional tools and two recently developed deep learning-based tools. By applying two popular feature interpretation strategies, we demonstrated the importance of the miRNA regions other than their seeds and the potential contribution of the RNA-binding motifs within miRNAs/isomiRs and mRNAs to the miRNA/isomiR-mRNA interactions.

AvailabilityThe source code and tool are available at http://hulab.ucf.edu/research/projects/DMISO.

Contacthaihu@cs.ucf.edu, xiaoman@mail.ucf.edu

Supplementary informationSupplementary data are available online.


title: DeepVelo: Deep Learning extends RNA velocity to multi-lineage systems with cell-specific kinetics

abstract: 1The introduction of RNA velocity in single-cell studies has opened new ways of examining cell differentiation and tissue development. Existing RNA velocity estimation methods are based on strong assumptions of either complete observation of cells in steady states or a predefined dynamics pattern parameterized by constant coefficients. These assumptions are violated in complex and heterogenous single-cell sequencing datasets and thus limit the application of these techniques. Here we present DeepVelo, a novel method that predicts the cell-specific dynamics of splicing kinetics using Graph Convolution Networks (GCNs). DeepVelo generalizes RNA velocity to cell populations containing time-dependent kinetics and multiple lineages, which are common in developmental and pathological systems. We applied DeepVelo to disentangle multifaceted kinetics in the processes of dentate gyrus neurogenesis, pancreatic endocrinogenesis, and hindbrain development. DeepVelo infers time-varying cellular rates of transcription, splicing and degradation, recovers each cells stage in the underlying differentiation process and detects putative driver genes regulating these processes. DeepVelo relaxes the constraints of previous techniques and facilitates the study of more complex differentiation and lineage decision events in heterogeneous single-cell RNA sequencing data.


title: Molecular-Dynamics Simulation Methods for Macromolecular Crystallography

abstract: To assess the potential benefits of molecular-dynamics (MD) simulations for macromolecular crystallography (MX), we performed room-temperature X-ray diffraction studies of the catalytic subunit of mouse protein kinase A (PKA-C). We then performed crystalline MD simulations of PKA-C, computed simulated electron densities from the water, protein, and ion components of the MD simulations, and carefully compared them to the initial crystal structure. The results led to the development of an MD-MX analysis procedure and several associated methods: 1) density comparison to evaluate consistency between the MD and the initial crystal structure model; 2) water building to generate alternative solvent models; and 3) protein remodeling to improve the crystal structure where interpretation of density is unclear. This procedure produced a revised structure of PKA with a new ordered water model and a modified protein structure. The revisions yield new insights into PKA mechanisms, including: a sensitivity of the His294 conformation to protonation state, with potential consequences for regulation of substrate binding; a remodeling of the Lys217 side chain along with a bound phosphate; an alternative conformation for Lys213 associated with binding to the regulatory subunit; and an alternative conformation for catalytic base Asp166 and nearby waters, suggesting a mechanism of progression of the phosphotransfer reaction via changes in Mg2+ coordination. Based on the benefits seen applying these methods to PKA, we recommend incorporating our MD-MX procedure into MX studies, to decide among ambiguous interpretations of electron density that occur, inevitably, as part of standard model refinement.

category: biophysics


title: Characterizing the extracellular matrix transcriptome of cervical, endometrial, anduterine cancers

abstract: Increasingly, the matrisome, a set of proteins that form the core of the extracellular matrix (ECM) or are closely associated with it, has been demonstrated to play a key role in tumor progression. However, in the context of gynecological cancers, the matrisome has not been well characterized. A holistic, yet targeted, exploration of the tumor microenvironment is critical for better understanding the progression of gynecological cancers, identifying key biomarkers for cancer progression, establishing the role of gene expression in patient survival, and for assisting in the development of new targeted therapies. In this work, we explored the matrisome gene expression profiles of cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), uterine corpus endometrial carcinoma (UCEC), and uterine carcinosarcoma (UCS) using publicly available RNA-seq data from The Cancer Genome Atlas (TCGA) and The Genotype-Tissue Expression (GTEx) portal. We hypothesized that the matrisomal expression patterns of CESC, UCEC, and UCS would be highly distinct with respect to genes which are differentially expressed and hold inferential significance with respect to tumor progression, patient survival, or both. Through a combination of statistical and machine learning analysis techniques, we identified sets of genes and gene networks which characterized each of the gynecological cancer cohorts. Our findings demonstrate that the matrisome is critical for characterizing gynecological cancers and transcriptomic mechanisms of cancer progression and outcome. Furthermore, while the goal of pan-cancer transcriptional analyses is often to highlight the shared attributes of these cancer types, we demonstrate that they are highly distinct diseases which require separate analysis, modeling, and treatment approaches. In future studies, matrisome genes and gene ontology terms that were identified as critical for predicting patient survival or cancer stage can be evaluated as potential drug targets and incorporated into in vitro models of disease.

category: cancer biology


title: 3'UTR-dependent dynamic changes in TP53 mRNA localization regulate p53 tumor suppressor activity

abstract: The tumor suppressor p53 triggers senescence in response to oncogenic stress in primary cells. However, the mechanisms by which tumor cells retaining p53 bypass senescence are not fully understood. Here we report that p53 cytostatic activity is inhibited in tumor cells by the 3 untranslated region (3UTR) of its mRNA, without altering p53 levels. 3UTR inhibition requires a long U-rich element (URE) and its binding protein, HuR. The 3UTR excluded TP53 mRNAs from a perinuclear compartment containing the CK2 kinase, suppressing p53 phosphorylation on an activating CK2 site, Ser392. In primary cells undergoing oncogene-induced senescence and tumor cells treated with genotoxic agents, TP53 mRNAs became concentrated in the perinuclear cytoplasm, coinciding with p53 phosphorylation and activation by CK2. In both cases, perinuclear re-localization of TP53 transcripts required AMPK2-dependent HuR nuclear translocation. ATM kinase activity was essential for DNA damage-induced spatial reprogramming of TP53 mRNAs, likely through phosphorylation and inactivation of MDM2. MDM2 was required for peripheral localization of TP53 transcripts and negatively regulated levels of the AMPK2 activating kinase, CaMKK{beta}. Our findings reveal a critical role for 3UTR sequences in suppressing p53 protein activity and provide a new mechanistic framework for p53 activation by DNA damaging agents.


title: Increased adipose tissue indices of androgen catabolism and aromatization in women with metabolic dysfunction

abstract:
BackgroundBody fat distribution is a risk factor for obesity-associated comorbidities, and adipose tissue dysfunction plays a role in this association. In humans, there is a sex difference in body fat distribution, and steroid hormones are known to regulate several cellular processes within adipose tissue. Our aim was to investigate if intra-adipose steroid concentration and expression or activity of steroidogenic enzymes were associated with features of adipose tissue dysfunction in individuals with severe obesity.

MethodsSamples from 40 bariatric candidates (31 women, 9 men) were included in the study. Visceral (VAT) and subcutaneous adipose tissue (SAT) were collected during surgery. Adipose tissue morphology was measured by a combination of histological staining and semi-automated quantification. Following extraction, intra-adipose and plasma steroid concentrations were determined by liquid chromatography, electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Aromatase activity was estimated using product-over-substrate ratio, while AKR1C2 activity was measured directly by fluorogenic probe. Gene expression was measured by quantitative PCR.

ResultsVAT aromatase activity was positively associated with VAT adipocyte hypertrophy (p-valueadj < 0.01) and negatively with plasma HDL-cholesterol (p-valueadj < 0.01), while SAT aromatase activity predicted dyslipidemia in women even after adjustment for waist circumference, age and hormonal contraceptive use. We additionally compared women with high and low visceral adiposity index (VAI) and found that VAT excess is characterized by adipose tissue dysfunction, increased androgen catabolism mirrored by increased AKR1C2 activity and higher aromatase expression and activity indices.

ConclusionIn women, increased androgen catabolism or aromatization is associated with visceral adiposity and adipose tissue dysfunction.

DISCLOSURE SUMMARYAT obtained consulting fees form Bausch Health, Novo Nordisk and research funding from Johnson & Johnson Medical Companies as well as Medtronic and GI Windows for studies unrelated to this manuscript. The other authors have nothing to disclose.

category: physiology


title: Electron tomography analysis of the prolamellar body and its transformation into grana thylakoids in the cryofixed Arabidopsis cotyledon

abstract: The para-crystalline structure of prolamellar bodies (PLBs) and light-induced etioplasts-to-chloroplasts transformation have been investigated with electron microscopy methods. However, these studies suffer from chemical fixation artifacts and limited volumes of three-dimensional reconstruction. We have examined Arabidopsis thaliana cotyledon cells with electron tomography (ET) to visualize etioplasts and their conversion into chloroplasts. We employed the scanning mode of ET for imaging large volumes and high-pressure freezing to improve sample preservation. PLB tubules were arranged in a zinc blende-type lattice like carbon atoms in diamonds. Within 2 hours after illumination, the lattice collapsed from the PLB exterior and the disorganized tubules merged to form thylakoid sheets, a.k.a. pre-granal thylakoids. These pre-granal thylakoids in PLBs vicinity folded and overlapped with each other to create grana stacks. Since the nascent pre-granal thylakoids had curved membranes in their tips, we examined the expression and localization of CURT1 proteins. CURT1A transcript was most abundant in de-etiolating cotyledon samples, and CURT1A concentrated to the PLB periphery. In curt1a etioplasts, PLB-associated thylakoids were swollen and failed to form grana stacks. By contrast, PLBs had cracks in their lattices in curt1c etioplasts. Our data provide evidence that CURT1A is required for pre-granal thylakoid assembly from PLB tubules during de-etiolation, while CURT1C contributes to the cubic crystal growth under darkness.

category: plant biology


title: Ralstonia solanacearum effector RipAF1 ADP-ribosylates host FBN1 to induce resistance against bacterial wilt

abstract: Ralstonia solanacearum (Rso) causes destructive bacterial wilt across a broad range of host plants by inducing jasmonic acid (JA) signaling while suppressing salicylic acid (SA) signaling pathways during disease development. In the present study, we show that Rso type III effector RipAF1 exerts a negative effect on bacterial virulence by subverting disease signaling in association with bacterial wilt. The ADP-ribosylation activity of RipAF1 was verified both in vivo and in vitro. Host fibrillin FBN1 was identified as a RipAF1-interacting protein that acted as a susceptible factor for bacterial wilt. In particular, RipAF1 directly ADP-ribosylates FBN1 at the E175/K207 residues, thus interfering with the mediation of disease signaling by FBN1. Together, these results suggest that RipAF1 exerts a role in defense induction by ADP-ribosylation of the susceptible factor FBN1 in the host plant.


title: ASTF1, an AP2/ERF-family transcription factor and ortholog of cultivated tomato LEAFLESS, is required for acylsugar biosynthesis

abstract: Acylsugars, specialized metabolites produced by solanaceous trichomes, provide protection against biotic and abiotic stresses. Here, we report ACYLSUGAR TRANSCRIPTION FACTOR1 (ASTF1/Sopen05g008450; AP2/ERF-family member) positively regulates acylsugar biosynthesis. Virus-induced gene silencing (VIGS) of ASTF1 in Solanum pennellii reduced acylsugar production by 65%. Most acylsugar (and several flavonoid) metabolic genes were downregulated in ASTF1-silenced plants, and these genes showed strong co-expression with ASTF1. In promoters of potential ASTF1-targets, we identified three enriched motifs, and one motif showed similarity with binding sites of other AP2/ERFs. Phylogenetic analysis and data mining indicated trichome-enriched expression of ASTF1 orthologs in several acylsugar-producing solanaceous species, suggesting a conserved role in acylsugar biosynthesis. This was supported by VIGS of ASTF1 orthologs in Nicotiana benthamiana. Broader phylogenetic analysis revealed relationships among specialized metabolic AP2/ERFs in several asterid species and provided clues about evolutionary emergence of acylsugar phenotype. Cultivated tomato ortholog (LEAFLESS/Solyc05g013540) has been reported to coordinate leaf initiation with transient expression at incipient primordia, and data mining revealed downregulation of trichome-preferentially-expressed genes, including acylsugar (and flavonoid) metabolic genes, in leafless mutants shoot apices, indicating remarkable spatiotemporal functional diversity. Our work will pave a way to disentangle acylsugar regulatory network and holds promise for future metabolic engineering of acylsugar production.


title: Gut microbiome-linked metabolites in the pathobiology of depression and anxiety - a role for bile acids

abstract:
BackgroundThe gut microbiome may play a role in the pathogenesis of neuropsychiatric diseases including major depressive disorder (MDD). Bile acids (BAs) are steroid acids that are synthesized in the liver from cholesterol and further processed by gut-bacterial enzymes, thus requiring both human and gut microbiome enzymatic processes in their metabolism. BAs participate in a range of important host functions such as lipid transport and metabolism, cellular signaling and regulation of energy homeostasis. BAs have recently been implicated in the pathophysiology of Alzheimers and several other neuropsychiatric diseases, but the biochemical underpinnings of these gut microbiome-linked metabolites in the pathophysiology of depression and anxiety remains largely unknown.

MethodUsing targeted metabolomics, we profiled primary and secondary BAs in the baseline serum samples of 208 untreated outpatients with MDD. We assessed the relationship of BA concentrations and the severity of depressive and anxiety symptoms as defined by the 17-item Hamilton Depression Rating Scale (HRSD17) and the 14-item Hamilton Anxiety Rating Scale (HRSA-Total), respectively. We also evaluated whether the baseline metabolic profile of BA informs about treatment outcomes.

ResultsThe concentration of the primary BA chenodeoxycholic acid (CDCA) was significantly lower at baseline in both severely depressed (log2 fold difference (LFD)= -0.48; p=0.021) and highly anxious (LFD= -0.43; p=0.021) participants compared to participants with less severe symptoms. The gut bacteria-derived secondary BAs produced from CDCA such as lithocholic acid (LCA) and several of its metabolites, and their ratios to primary BAs, were significantly higher in the more anxious participants (LFDs range=[0.23,1.36]; ps range=[6.85E-6,1.86E-2]). The interaction analysis of HRSD17 and HRSA-Total suggested that the BA concentration differences were more strongly correlated to the symptoms of anxiety than depression. Significant differences in baseline CDCA (LFD= -0.87, p=0.0009), isoLCA (LFD= -1.08, p=0.016) and several BA ratios (LFDs range [0.46, 1.66], ps range [0.0003, 0.049]) differentiated treatment failures from remitters.

ConclusionIn patients with MDD, BA profiles representing changes in gut microbiome compositions are associated with higher levels of anxiety and increased probability of first-line treatment failure. If confirmed, these findings suggest the possibility of developing gut microbiome-directed therapies for MDD characterized by gut dysbiosis.


title: Cognitive dysfunction and anxiety resulted from synaptic downscaling, hippocampal atrophy and ventricular enlargement in i.c.v. streptozotocin injected male Wistar rats

abstract:
Intracerebroventricular streptozotocin (ICV-STZ) injection is among the best animal models to simulate sporadic Alzheimers disease (AD) (Hoyer, 2002, 2000, 1998; Salkovic-Petrisic and Hoyer, 2007). Brain insulin signaling abnormality (Grunblatt et al., 2007), neurodegeneration (Kamat, 2015), neuroinflammation (Liu et al., 2022), cholinergic damage (Blokland and Jolles, 1993; Roy et al., 2021), mitochondrial dysfunction (C. Correia et al., 2013), genetic abnormality (Chen et al., 2012), respiratory problem (Ebel et al., 2017), oxidative stress (Sharma and Gupta, 2001), gliosis (Rostami et al., 2017), sleep disturbances (Song et al., 2018) associated with cognitive abnormalities seen in ICV-STZ injected rats. Available experimental evidences has used varying doses of STZ (<1 to 3mg/kg) and studied its effect for different study durations, ranging from 14-21 (short), 30-42 (mild), 90-105 (moderate) and 250-270 (long) days (Chen et al., 2013, 2012; Grunblatt et al., 2007; Knezovic et al., 2015; Kraska et al., 2012; Mehla et al., 2013; Rostami et al., 2017; Salkovic-Petrisic et al., 2006). These studies indicated that 3mg/kg of body weight is the optimum dose for inducing sporadic AD in the rodents. However studies on the pathological process with relating the morphological and functional abnormalities reported were illusive.

Therefore in the present study, we have investigated the effect of 3mg/kg ICV-STZ on three cognitive domains viz. spatial, episodic and avoidance and even neuropsychiatric abnormality i.e. anxiety with extensive morphological examination in the layers associated with hippocampus proper. Further, we have observed the onset of diffuse amyloid plaques, in association with enlargement of lateral ventricles and down scaling of spines in granular cells of dentate gyrus. Finally, to dissect the functional changes in spatial and anxiety variables we had examined the probability of fraction of time spent in Morris water maze and changes in ethological patterns of STZ injected rats in elevated plus maze.


title: Application of light-sheet mesoscopy to image host-pathogen interactions in intact organs

abstract: Human African Trypanosomiasis (HAT) is a disease caused by the extracellular parasite Trypanosoma brucei that affects the central nervous system (CNS) during the chronic stage of the infection, inducing neuroinflammation, coma, and death if left untreated. However, little is known about the structural change happening in the brain as result of the infection. So far, infection-induced neuroinflammation has been observed with conventional methods, such as immunohistochemistry, electron microscopy, and 2-photon microscopy only in small portions of the brain, which may not be representative of the disease. In this paper, we have used a newly-developed light-sheet illuminator to image the level of neuroinflammation in chronically infected mice and we use analyse these data to compare the infection in naive controls. This system was developed for imaging in combination with the Mesolens objective lens, providing sub-cellular resolution for imaging volumes exceeding 39 mm3 in an acquisition time of only 8 hours. The mouse brain specimens were cleared using CUBIC+, followed by antibody staining to locate Glial Fibrillary Acid Protein (GFAP) expressing cells, primarily astrocytes and ependymocytes, used here as a proxy for cell reactivity and gliosis. The large capture volume allowed us to detect GFAP+ cells and spatially resolve the innate responses to T. brucei infection. Based on morphometric analyses and spatial distribution of GFAP+ cells, our data demonstrates a significant increase in cell dendrite branching around the lateral ventricle, as well as dorsal and ventral third ventricles, that are negatively correlated with the branch extension in distal sites from the circumventricular spaces. To our knowledge, this is the first report highlighting the potential of light-sheet mesoscopy to characterise the inflammatory responses of the mouse brain to parasitic infection at the cellular level in intact cleared organs, opening new avenues for the development of new mesoscale imaging techniques for the study of host-pathogen interactions.


title: Small silencing RNAs expressed from W-linked retrocopies of Masculinizer target the male-determining gene PxyMasc during female sex determination in the Diamondback moth Plutella xylostella

abstract:
The Lepidoptera are an insect order of cultural, economic and environmental importance, representing c. 10% of all described living species. Yet, for all but one of these species (silkmoth, Bombyx mori) the molecular genetics of how sexual fate is determined remains unknown. We investigated this in the diamondback moth (DBM - Plutella xylostella), a globally important, highly invasive and economically damaging pest of cruciferous crops. Our previous work uncovered a regulator of male sex determination in DBM - PxyMasc, a homologue of B. mori Masculinizer - which although initially expressed in embryos of both sexes, is then reduced in female embryos, leading to female-specific splicing of doublesex. Here, through sequencing small RNA libraries generated from early embryos and sexed larval pools, we identified a variety of small silencing RNAs (predominantly piRNAs) complementary to PxyMasc, whose temporal expression correlated with the reduction in PxyMasc transcript observed previously in females. Analysis of these small RNAs showed that they are expressed from tandemly-arranged, multi-copy arrays found exclusively on the W (female-specific) chromosome, which we term  Pxyfem. Analysis of the Pxyfem sequences showed that they are partial cDNAs of PxyMasc mRNA transcripts, likely integrated into transposable element graveyards by the non-canonical action of retrotransposons (retrocopies), and that their apparent similarity to B. mori feminizer more probably represents convergent evolution. Our study helps elucidate the sex determination cascade in this globally important pest and highlights the  shortcuts which retrotransposition events can facilitate in the evolution of complex molecular cascades, including sex determination.

Significance statementUncovering the mechanisms which species have evolved to determine sex is of fundamental interest and provides avenues for pest management through genetic manipulation of these pathways. In insects, much of what is known regarding sex determination is concentrated within the Diptera and Hymenoptera, despite other orders (e.g. Lepidoptera) being of great ecological and economic importance. Here, using small RNA sequencing of embryonic and early larval samples, we uncover an RNAi-based sex determination system which silences the male determining gene PxyMasc in the Diamondback moth (Plutella xylostella) - a global pest of cruciferous crops. We track production of these silencing RNAs back to the W-chromosome where they are expressed from partial cDNA copies of PxyMasc. Our analysis suggests these are PxyMasc  retrocopies, integrated via the non-canonical action of LTR retrotransposons and that similarities between this system and the feminizer system in Bombyx mori likely represent convergent evolution.


title: mRNAid, an Open-Source Platform for Therapeutic mRNA Design and Optimization Strategies

abstract: Recent COVID-19 vaccines unleashed the potential of mRNA-based therapeutics. mRNA optimization is indispensable for reducing immunogenicity, ensuring stability, and maximizing protein output. We present mRNAid, an experimentally validated software for mRNA optimization and visualization that generates mRNA sequences with comparable if not superior characteristics to commercially optimized sequences. To encompass all aspects of mRNA design, we also interrogated the impact of uridine content, nucleoside analogs and UTRs on expression and immunogenicity.


title: Reduced stroke outcome in old-aged female mice maintained on a dietary vitamin B12 deficiency

abstract: The global population is aging and the prevalence of age-related diseases, such as stroke, is predicted to increase. A vitamin B12 deficiency (vit. B12 def.) is common in the elderly, because of changes in metabolism. Clinical studies have reported that a vit. B12 def results in worse outcome after stroke, the mechanisms through which a vit. B12 def. changes the brain requires further investigation. This study investigated the role of vit. B12 def. on stroke outcome and mechanisms using aged female mice. Eighteen month old females were put on a control or vit. B12 def. diet for four weeks, after which an ischemic stroke was induced in the sensorimotor cortex. After damage, motor function was measured and animals were euthanized and tissues were collected for analysis. Vit. B12 def. animals had increased levels of total homocysteine in plasma and liver, choline levels were also increased in the liver. Vit. B12 def. had larger damage volume in brain tissue and more apoptosis. In the cecum, changes in creatinine and methylmalonic acid were observed in vit. B12 def animals, pathway analysis showed dysfunction in B12 transport. Analysis of mitochondrial metabolomics in brain tissue showed reduced levels of metabolites involved in the TCA cycle in vit. B12 def animals. Meanwhile, pathway analysis showed significant, widespread dysfunction in phenylalanine, tyrosine, and tryptophan biosynthesis. Motor function after stroke was impaired in vit. B12 def. animals. A dietary vitamin B12 deficiency impairs motor function through increased apoptosis and changes in mitochondrial metabolism in brain tissue.


title: Microglia Detect Externalized Phosphatidylserine on Synapses for Elimination via TREM2 in Alzheimer's Disease Models

abstract:
Genetic studies implicate phagocytosis pathways in microglia to be a major Alzheimers disease (AD)-associated process. Microglia phagocytose synapses in AD mouse models, suggesting a role for microglia in region-specific synapse loss, a pathological hallmark of AD. However, whether specific synapses are targeted for elimination, and if so, how, remains to be elucidated. Here, we show that synapses externalize phosphatidylserine (PtdSer) upon challenge by {beta}-amyloid oligomers, which are then selectively engulfed by microglia. Mechanistically, we find that Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) is critical for microglia to sense and preferentially engulf AD synapses. In brains of mice and humans, TREM2 dysfunction leads to exacerbation of apoptotic synapses. Our work altogether suggests a fundamental role for microglia as brain-resident macrophages to remove damaged synapses in AD. We provide mechanistic insight into how TREM2 variants associated with increased risk of developing AD may contribute to defective microglia-synapse function.

One-Sentence summaryMicroglia selectively engulf synapses in Alzheimer-like mouse brains via PtdSer-TREM2 signaling.


title: Tissue-wide Genetic and Cellular Landscape Shapes the Execution of Sequential PRC2 Functions in Neural Stem Cell Lineage Progression

abstract: The generation of a correctly-sized cerebral cortex with all-embracing neuronal and glial cell-type diversity critically depends on faithful radial glial progenitor (RGP) cell proliferation/differentiation programs. Temporal RGP lineage progression is regulated by Polycomb Repressive Complex 2 (PRC2) and loss of PRC2 activity results in severe neurogenesis defects and microcephaly. How PRC2-dependent gene expression instructs RGP lineage progression is unknown. Here we utilize Mosaic Analysis with Double Markers (MADM)-based single cell technology and demonstrate that PRC2 is not cell-autonomously required in neurogenic RGPs but rather acts at the global tissue-wide level. Conversely, cortical astrocyte production and maturation is cell-autonomously controlled by PRC2-dependent transcriptional regulation. We thus reveal highly distinct and sequential PRC2 functions in RGP lineage progression that are dependent on complex interplays between intrinsic and tissue-wide properties. In a broader context our results imply a critical role for the genetic and cellular niche environment in neural stem cell behavior.


title: Mice expressing P301S mutant human tau have deficits in interval timing

abstract:
Interval timing is a key executive process that involves estimating the duration of an interval over several seconds or minutes. Patients with Alzheimers disease (AD) have deficits in interval timing. Since temporal control of action is highly conserved across mammalian species, studying interval timing tasks in animal AD models may be relevant to human disease. Amyloid plaques and tau neurofibrillary tangles are hallmark features of AD. While rodent models of amyloid pathology are known to have interval timing impairments, to our knowledge, interval timing has not been studied in models of tauopathy. Here, we evaluate interval timing performance of P301S transgenic mice, a widely studied model of tauopathy that overexpresses human tau with the P301S mutation. We employed the switch interval timing task, and found that P301S mice consistently underestimated temporal intervals compared to wild-type controls, responding early in anticipation of the target interval. Our study indicating timing deficits in a mouse tauopathy model could have relevance to human tauopathies such as AD.

HIGHLIGHTSO_LIWe examined interval timing behavior in mice expressing P301S mutant tau
C_LIO_LIP301S mice responded earler than littermate controls
C_LIO_LIThese data provide insight into animal models of tauopathy
C_LI


title: High-level motor planning allows flexible walking at different gait patterns in a neuromechanical model

abstract: Humans are able to adopt almost any desired gait pattern on the fly when walking. We postulate that this flexibility in humans is partially due to the ability to control the whole body during walking as a volitional, goal-directed movement that can be planned and changed, rather than having to rely on habitual, reflexive control that is adapted over long time-scales. Here we present a neuromechanical model that accounts for this flexibility by combining movement goals and motor plans on a kinematic task level with low-level spinal feedback loops. We show that the model is able to walk at a wide range of different gait patterns by choosing a small number of high-level control parameters representing a movement goal. A larger number of parameters governing the low-level reflex loops in the spinal cord, on the other hand, remains fixed. We also show that the model is able to generalize the learned behavior by re-combining the high-level control parameters and walk with gait patterns it had not encountered before. Furthermore, the model can transition between different gaits without loss of balance by switching to a new set of control parameters in real time.


title: Neural systems underlying RDoC social constructs: An activation likelihood estimation meta-analysis

abstract: ObjectiveNeuroscientists have sought to identify the underlying neural systems supporting mental processes involved in social cognition. These processes allow us to interact and communicate with others, form social relationships, and navigate the social world. Through the use of NIMHs Research Domain Criteria (RDoC) framework, we evaluated consensus among hundreds of studies that examined brain activity during social and social cognition tasks to elucidate, at a large scale, regions comprising the "social brain". In addition, we examined convergence across tasks corresponding to the four RDoC social constructs, including Affiliation and Attachment, Social Communication, Perception and Understanding of Self, and Perception and Understanding of Others. We assessed activation patterns by performing a series of coordinate-based meta-analyses using the activation likelihood estimate (ALE) method. One meta-analysis was performed on whole-brain coordinates reported from 864 fMRI contrasts of healthy social processing (239 publications; 6,232 participants) using the NiMARE Python package. The meta-analysis of all social contrasts revealed consensus in the "social brain", including the medial prefrontal cortex (mPFC), anterior cingulate cortex (ACC), posterior cingulate cortex (PCC), temporoparietal junction (TPJ), bilateral insula, amygdala, fusiform gyrus, precuneus, and thalamus. Additionally, four separate RDoC-based meta-analyses revealed differential convergence associated with the four social constructs. These outcomes highlight the neural support underlying these social constructs and inform future research on alterations among neurotypical and atypical populations.


title: Substance P and adenosine signaling pathways regulate exosomal sorting of miR-21 in colonic epithelial cells

abstract:
Background & AimsMicroRNAs (miRNAs) are transported in body fluids within exosomes, and exosomal-miRNA sorting is highly selective. In colonic epithelial cells (CECs), Substance P-neurokinin-1-receptor (SP/NK1R) signaling regulates miR-21 sorting into secreted exosomes. Here, we studied the molecular mechanisms driving miR-21 sorting into colonic epithelial exosomes (CEEs) under SP-stimulation.

MethodsWe performed studies with human colonic epithelial NCM460 cells overexpressing neurokinin-1-receptor (NCM460-NK1R) and in intestinal-epithelial-specific NK1R knockout mice. SP-regulated gene targets were validated by real-time polymerase chain reaction (RT-PCR) and immunoblotting. Small non-coding RNAs (sncRNAs) were isolated from NCM460-NK1R cells and secreted exosomes, and 3'-end adenylated and 3'-end uridylated fractions were separated. Cellular and exosomal sncRNA fractions were processed for miR-21 and miR-1307-3p expression and 3'-end adenylation to uridylation (A/U) ratios using RT-PCR. Pharmacological inhibition studies in NCM460-NK1R cells were performed in the presence of the Adenosine A2B receptor (ADORA2B) antagonist, PSB-1115, followed by RT-PCR and immunoblotting. Mass spectrometry validated in silico interacting partners of miR-21 in CECs.

ResultsIn CECs, miR-21 is predominantly polyuridylated under SP-stimulation and preferentially sorted to CEEs. SP/NK1R signaling activation upregulates extracellular adenosine (ADO) signaling via ADORA2B concomitant with reduced ADO uptake via epithelial-specific equilibrative nucleoside transporter-2 (ENT-2). Mass spectrometry and immunoblotting revealed upregulation of TUT7 in SP-stimulated CEEs. Knockdown of TUT7 decreased both TUT7 and miR-21 content in these exosomes. Pharmacological inhibition of ADORA2B in CECs resulted in decreased TUT7 and miR-21 in CEEs, regardless of SP stimulation.

ConclusionsSP/NK1R coupling in CECs activates ADORA2B and its downstream signaling cascade which mediates TUT7/miR-21 interaction and subsequent miR-21 polyuridylation and exosomal export.

SynopsisSubstance P-neurokinin-1 receptor signaling regulates terminal uridyl transferase7-mediated predominant 3-end polyuridylation and exosomal recruitment of miR-21 in human colonic epithelial NCM460 cells overexpressing NK-1R via activation of Adenosine A2B receptor and its downstream signaling cascade. Because Adenosine A2B receptor signaling is involved in IBD pathogenesis, it could therefore be exploited as a pharmacological target for the therapeutic benefits in IBD.

category: cell biology


title: Methacrylic acid-based biomaterials promote peripheral innervation in the subcutaneous space of mice

abstract:
Peripheral nerve innervation is essential for regulating tissue repair and regeneration. MAA-based biomaterials have been previously shown to promote angiogenesis. Here we show a new role for MAA-based biomaterials in promoting terminal axon nerve growth. Our results demonstrate that MAA-based biomaterials promote peripheral nerve growth in an Igf-1 and Shh dependent manner. The resulting nerves increased the sensitivity of treated mice paws to nociception. iDISCO clearing showed that MAA increased the presence of peripheral nerve structures in whole explants. MAA was also able to increase the expression of key neuronal markers and growth factors in a peripheral neuropathy model, the diabetic db/db mouse, suggesting that MAA-based biomaterials may be relevant to treatment of peripheral neuropathy. Moreover, in a peripheral neuropathy model, MAA was able to up-regulate the expression of growth factors for an extended duration suggesting MAA may prevent degeneration through an effect on factors that promote survival. As all tissues are innervated, MAA-based biomaterials could have broad applications in the promoting regeneration and preventing degeneration of peripheral nerves.

HIGHLIGHTSO_LIMethacrylic acid-based biomaterials promote axon growth in-situ without exogenous growth factors or cells
C_LIO_LIMethacrylic acid-based biomaterial induced terminal axon growth displays nociception, an indicator of functional outgrowth
C_LIO_LIMethacrylic acid-based biomaterials terminal axon growth is Igf-1 and Shh driven
C_LI


title: A Python Package for the Localization of Protein Modifications in Mass Spectrometry Data

abstract: Determining the correct localization of post-translational modifications (PTMs) on peptides aids in interpreting their effect on protein function. While most algorithms for this task are available as standalone applications or incorporated into software suites, improving their versatility through access from popular scripting languages facilitates experimentation and incorporation into novel workflows. Here we describe pyAscore, an efficient and versatile implementation of the Ascore algorithm in Python for scoring the localization of user defined PTMs in data dependent mass spectrometry. pyAscore can be used from the command line or imported into Python scripts and accepts standard file formats from popular software tools used in bottom-up proteomics. Access to internal objects for scoring and working with modified peptides adds to the toolbox for working with PTMs in Python. pyAscore and is available as an open source package for Python 3.6+ on all major operating systems and can be found at pyascore.readthedocs.io.


title: Immunopharmacological evaluation of adjuvant efficacy of Monophosphoryl lipid-A and CpG ODN with SARS-CoV-2 RBD antigen

abstract: SARS-CoV-2 infection has made the mankind to witness most sever and serious pandemic situation in the history. Millions of people have suffered and are still suffering with this infection which has caused a mass mortality in the past three years. Development of an effective vaccine to control the spread of infection and to prevent this viral infection is need of the hour. Adjuvanted vaccines have proven their efficacy in controlling many other viral infections like flu, keeping this context in view we have evaluated the immunopharmacological efficacy of two adjuvants MPL-A and CpG ODN in combination with MF59 emulsion against SARS-CoV-2 antigen. From the data obtained we can infer that both the adjuvants were capable of eliciting a potent antibody response against antigen alone and MF59 groups. Comparatively MPL-A was eliciting a Th1 polarized response in terms of IgG2a and cytokine production. Both the adjuvants were capable of enhancing the CD 4, 8 and 19 cell populations. Overall the pre clinical evaluation has given a clue of the effectiveness of MPL-A and CpG adjuvants against SARS-CoV-2 antigen.


title: Impact of prior infection on SARS-CoV-2 antibody responses in vaccinated long-term care facility staff

abstract:
SARS-CoV-2 emerged in 2019 and has resulted in millions of deaths worldwide. Certain populations are at higher risk for infection, especially staff and residents at long term care facilities (LTCF), due to the congregant living setting, and residents with many comorbidities. Prior to vaccine availability, these populations represented a large fraction of total COVID-19 cases and deaths in the U.S. Due to the high-risk setting and outbreak potential, staff and residents were among the first groups to be vaccinated. To define the impact of prior infection on response to vaccination, we measured antibody responses in a cohort of staff members at a LTCF, many of whom were previously infected by SARS-CoV-2. We found that neutralizing, receptor-binding-domain (RBD) and nucleoprotein (NP) binding antibody levels were significantly higher post-full vaccination course in individuals that were previously infected, and NP antibody levels could discriminate individuals with prior infection from vaccinated individuals. While an anticipated antibody titer increase was observed after vaccine booster dose in naive individuals, boost response was not observed in individuals with previous COVID-19 infection. We observed a strong relationship between neutralizing antibodies and RBD-binding antibodies post-vaccination across all groups, suggesting RBD-binding antibodies may be used as a correlate of neutralization. One individual with high levels of neutralizing and binding antibodies experienced a breakthrough infection (prior to the introduction of Omicron), demonstrating that the presence of antibodies is not always sufficient for complete protection against infection. These results highlight that history of COVID-19 exposure significantly increases SARS-CoV-2 antibody responses following vaccination.

ImportanceLong-term care facilities (LTCFs) have been disproportionately impacted by COVID-19, due to their communal nature, high-risk profile of residents and vulnerability to respiratory pathogens. In this study, we analyzed the role of prior natural immunity to SARS-CoV-2 on post-vaccination antibody responses. The LTCF in our cohort experienced a large outbreak with almost 40% of staff becoming infected. We found that individuals that were infected prior to vaccination, had higher levels of neutralizing and binding antibodies post-vaccination. Importantly, the second vaccine dose significantly boosted antibody levels in those that were immunologically naive prior to vaccination, but not those that had prior immunity. Regardless of pre-vaccination immune status, levels of binding and neutralizing antibodies were highly correlated. The presence of NP-binding antibodies can be used to identify individuals that were previously infected when pre-vaccination immune status is not known. Our results reveal that vaccination antibody responses differ depending on prior natural immunity.


title: Screening of SARS-CoV-2 Antivirals Through a Cell-Based RNA-Dependent RNA Polymerase (RdRp) Reporter Assay

abstract:
COVID-19 (Coronavirus Disease 2019) caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus-2) continues to pose international public health threat and thus far, has resulted in greater than 5.6 million deaths worldwide. Vaccines are critical tools to limit COVID-19 spread, but antiviral drug development is an ongoing global priority due to fast spreading COVID-19 variants that may elude vaccines efficacies. The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is an essential enzyme of viral replication and transcription machinery complex. Therefore, the RdRp is an attractive target for the development of effective anti-COVID-19 therapeutics. In this study, we developed a cell-based assay to determine the enzymatic activity of SARS-CoV-2 RdRp through luciferase reporter system. The SARS-CoV-2 RdRp reporter assay was validated using a known inhibitors of RdRp polymerase, remdesivir along with other anti-virals including ribavirin, penciclovir, rhoifolin, 5CT, and dasabuvir. Among these inhibitors, dasabuvir (FDA-approved drug) exhibited promising RdRp inhibitory activity. Anti-viral activity of dasabuvir was also tested on the replication of SARS-CoV-2 through infection of Vero E6 cells. Dasabuvir inhibited the replication of SARS-CoV-2, USA-WA1/2020 as well as B.1.617.2 (delta variant) in Vero E6 cells in a dose-dependent manner with IC50 values 9.47 M and 10.48 M, for USA-WA1/2020 and B.1.617.2 variants, respectively). Our results suggests that dasabuvir can be further evaluated as a therapeutic drug for COVID-19. In addition, our assays provide robust, target-specific, and high-throughput screening compatible (z- and z-factors of > 0.5) platforms that will be a valuable tool for the screening SARS-CoV-2 RdRp inhibitors.

SignificanceSARS-CoV-2 has caused a major public crisis world has seen in recent history. Development of vaccines and emergency use authorization of anti-virals are helping in reducing the burden of SARS-CoV-2 caused hospitalization and deaths. However, there is still need for optimal anti-viral(s) that can efficiently block viral propagation, and targeting viral polymerase (RdRp) is an among the most suitable targets for clamping viral replication. In this study, we developed a cell-based assay to screen potential compounds capable of blocking RdRp activity. The efficacy of our assay was validated by using already approved anti-virals, which reduced RdRp activity and slowed the replication of two SARS-CoV-2 variants (WA1 USA-WA1/2020 and B.1.617.2) in a cell culture model. This confirmed that our system can be used for identifying potential anti-SARS-CoV-2 anti-virals.

category: microbiology


title: Targeted protein S-nitrosylation of ACE2 as potential treatment to prevent spread of SARS-CoV-2 infection

abstract: Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and thus spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E-protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model, and thus provide a novel avenue for therapy.


title: Evolution of Delta variant by non-Spike signature co-appearing mutations: trailblazer of COVID-19 disease outcome

abstract: The high transmissibility and infectivity of a SARS-CoV-2 variant is usually ascribed to the Spike mutations, while emerging non-spike mutations might be a serious threat to the current Spike-recombinant vaccines. In addition to mutations in structural Spike glycoprotein, rapid accumulation of mutations across non-structural genes is leading to continuous virus evolution, altering its pathogenicity. We performed whole genome sequencing of SARS-CoV-2 positive samples collected from different clinical groups from eastern India, during the second pandemic wave (April-May, 2021). In addition to the several common spike mutations in Delta variant, two mutually explicit signature constellations of non-spike co-appearing mutations were identified, driving symptomatic and asymptomatic infections. We attempted to correlate these unique signatures of non-Spike co-appearing mutations to COVID-19 disease outcome. Results revealed that the Delta strains harboring a unique constellation of 9 non-spike co-appearing mutations could be the wheeler and dealer of symptomatic infection, even post vaccination. The strains predominantly driving asymptomatic infection possessed 7 non-spike co-appearing mutations, which were mutually exclusive in contrast to the set of mutations causing symptomatic disease. Phylodynamic analysis depicted high probability of emergence of these unique sub-clusters within India, with subsequent spread worldwide. Interestingly, some mutations of this signature were selected in Omicron and IHU variants, which suggest that gradual accumulation of such co-existing mutations may lead to emergence of more "vaccine-evading variants" in future. Hence, unfaltering genome sequencing and tracking of non-Spike mutations might be significant in formulation of any future vaccines against emerging SARS-CoV-2 variants that might evade the current vaccine-induced immunity.

category: genomics


title: Time series analysis of SARS-CoV-2 genomes and correlations among highly prevalent mutations

abstract: The efforts of the scientific community to tame the recent SARS-CoV-2 pandemic seems to have been diluted by the emergence of new viral strains. Therefore, it becomes imperative to study and understand the effect of mutations on viral evolution, fitness and pathogenesis. In this regard, we performed a time-series analysis on 59541 SARS-CoV-2 genomic sequences from around the world. These 59541 genomes were grouped according to the months (January 2020-March 2021) based on the collection date. Meta-analysis of this data led us to identify highly significant mutations in viral genomes. Correlation and Hierarchical Clustering of the highly significant mutations led us to the identification of sixteen mutation pairs that were correlated with each other and were present in >30% of the genomes under study. Among these mutation pairs, some of the mutations have been shown to contribute towards the viral replication and fitness suggesting the possible role of other unexplored mutations in viral evolution and pathogenesis. Additionally, we employed various computational tools to investigate the effects of T85I, P323L, and Q57H mutations in Non-structural protein 2 (Nsp2), RNA-dependent RNA polymerase (RdRp) and Open reading frame 3a (ORF3a) respectively. Results show that T85I in Nsp2 and Q57H in ORF3a mutations are deleterious and destabilize the parent protein whereas P323L in RdRp is neutral and has a stabilizing effect. The normalized linear mutual information (nLMI) calculations revealed the significant residue correlation in Nsp2 and ORF3a in contrast to reduce correlation in RdRp protein.


title: The network of SARS-CoV-2-cancer molecular interactions and pathways

abstract:
BackgroundRelatively little is known about the long-term impacts of SARS-CoV-2 biology, including whether it increases the risk of cancer. This study aims to identify the molecular interactions between COVID-19 infections and cancer processes.

Materials and MethodsWe integrated recent data on SARS-CoV-2 - host protein interactions, risk factors for critical illness, known oncogenes, tumor suppressor genes and cancer drivers in EpiGraphDB, a database of disease biology and epidemiology. We used these data to reconstruct the network of molecular links between SARS-CoV-2 infections and cancer processes in various tissues expressing the angiotensin-converting enzyme 2 (ACE2) receptor. We applied community detection algorithms and Gene Set Enrichment Analysis (GSEA) to identify cancer-relevant pathways that may be perturbed by SARS-CoV-2 infection.

ResultsIn lung tissue, the results showed that 4 oncogenes are potentially targeted by SARS-CoV-2, and 92 oncogenes interact with other human genes targeted by SARS-CoV-2. We found evidence of potential SARS-CoV-2 interactions with Wnt and hippo signaling pathways, telomere maintenance, DNA replication, protein ubiquitination and mRNA splicing. Some of these pathways were potentially affected in multiple tissues.

ConclusionsThe long-term implications of SARS-CoV-2 infection are still unknown, but our results point to the potential impact of infection on pathways relevant to cancer affecting cell proliferation, development and survival, favoring DNA degradation, preventing the repair of damaging events and impeding the translation of RNA into working proteins. This highlights the need for further research to investigate whether such effects are transient or longer lasting. Our results are openly available in the EpiGraphDB platform at https://epigraphdb.org/covid-cancer and the repository https://github.com/MRCIEU/covid-cancer (https://doi.org/10.5281/zenodo.6391588).


title: The E2 glycoprotein holds key residues for Mayaro virus adaptation to the urban Aedes aegypti mosquito

abstract:
Adaptation to mosquito vectors suited for transmission in urban settings is a major driver in the emergence of arboviruses. To better anticipate future emergence events, it is crucial to assess their potential to adapt to new vector hosts. In this work, we used two different experimental evolution approaches to study the adaptation process of an emerging alphavirus, Mayaro virus (MAYV), to Aedes aegypti, an urban mosquito vector of many arboviruses. We identified E2-T179N as a key mutation increasing MAYV replication in insect cells and enhancing transmission by live Aedes aegypti. In contrast, this mutation decreased viral replication and binding in human fibroblasts, a primary cellular target of MAYV in humans. We also showed that MAYV E2-T179N was attenuated in vivo in a mouse model. We then used structural and experimental analyses to show that MAYV E2-T179N bound less efficiently to human cells, though the decrease in replication or binding was not mediated through interaction with one of the host receptors, Mxra8. When this mutation was introduced in the closely related chikungunya virus, which has caused major outbreaks globally in the past two decades, we observed increased replication in both human and insect cells, suggesting E2 position 179 is an important determinant of alphavirus host-adaptation, although in a virus-specific manner. Collectively, these results indicate that adaptation at the T179 residue in MAYV E2 may result in increased vector competence - but coming at the cost of optimal replication in humans - and may represent a first step towards a future emergence event.

Author SummaryMosquito-borne viruses must replicate in both mosquito and vertebrate hosts to be maintained in nature successfully. When viruses that are typically transmitted by forest dwelling mosquitoes enter urban environments due to deforestation or travel, they must adapt to urban mosquito vectors to transmit effectively. For mosquito-borne viruses, the need to also replicate in a vertebrate host like humans constrains this adaptation process. Towards understanding how the emerging alphavirus, Mayaro virus, might adapt to transmission by the urban mosquito vector, Aedes aegypti, we used natural evolution approaches to identify several viral mutations that impacted replication in both mosquito and vertebrate hosts. We show that a single mutation in the receptor binding protein increased transmission by Aedes aegypti but simultaneously reduced replication and disease in a mouse model, suggesting that this mutation alone is unlikely to be maintained in a natural transmission cycle between mosquitoes and humans. Understanding the adaptive potential of emerging viruses is critical to preventing future pandemics.


title: Tracing carbon metabolism with stable isotope metabolomics reveals the legacy of diverse carbon sources in soil

abstract:
Tracking the metabolic activity of whole soil communities can improve our understanding of the transformation and fate of carbon in soils. We used stable isotope metabolomics to trace 13C from nine labeled carbon sources into the water-soluble metabolite pool of an agricultural soil over time. Soil was amended with a mixture of all nine sources, with one source isotopically labeled in each treatment. We compared changes in the 13C-enrichment of metabolites with respects to carbon source and time over a 48-day incubation and contrasted differences between soluble versus insoluble sources. Whole soil metabolite profiles varied singularly by time, while the composition of 13C-labeled metabolites differed primarily by carbon source (R2 = 0.68) rather than time (R2 = 0.07) with source-specific differences persisting throughout incubations. The 13C-labeling of metabolites from insoluble carbon sources occurred at a slower rate than soluble sources but yielded a higher average atom % 13C in metabolite markers of biomass (amino acids and nucleic acids). The 13C-enrichment of metabolite markers of biomass stabilized at between 5 - 15 atom % 13C by the end of incubations. Temporal patterns in the 13C-enrichment of TCA cycle intermediates, nucleobases (uracil and thymine), and by-products of DNA salvage (allantoin) closely tracked microbial activity. Our results demonstrate that metabolite production in soils is driven by the carbon source supplied to the community, and that the fate of carbon in metabolite profiles do not tend to converge over time as a result of ongoing microbial processing and recycling.

ImportanceCarbon metabolism in soil remains poorly described due to the inherent difficulty of obtaining information on the microbial metabolites produced by complex soil communities. Our study demonstrates the use of stable isotope probing (SIP) to study carbon metabolism in soil by tracking 13C from supplied carbon sources into metabolite pools and biomass. We show that differences in the metabolism of sources influences the fate of carbon in soils. Heterogeneity in 13C-metabolite profiles corresponded with compositional differences in the metabolically active populations, providing a basis for how microbial community composition is correlated with the quality of soil carbon. Our study demonstrates the application of SIP-metabolomics in studying soils and identifies several metabolite markers of growth, activity, and other aspects of microbial function.


title: Divergent LD-transpeptidase-independent effects of peptidoglycan carboxypeptidases on intrinsic ss-lactam and vancomycin resistance

abstract:
Vancomycin and {beta}-lactams are clinically important antibiotics that inhibit the formation of peptidoglycan cross-links, but their binding targets are different. The binding target of vancomycin is D-alanine-D-alanine (D-Ala-D-Ala), whereas that of {beta}-lactam is penicillin-binding proteins (PBPs). In this study, we revealed the divergent effects of peptidoglycan (PG) carboxypeptidases on vancomycin and {beta}-lactam resistance in Escherichia coli and Bacillus subtilis. The deletion of PG carboxypeptidases induced sensitivity to most {beta}-lactams, whereas it induced strong resistance toward vancomycin. Notably, both of two phenotypes did not have strong association with LD-transpeptidases, which are necessary for the formation of PG 3-3 cross-links and covalent bonds between PG and an Lpp outer membrane (OM) lipoprotein. Vancomycin resistance was induced by increased amount of decoy D-Ala-D-Ala residues within PG, whereas {beta}-lactam sensitivity was associated with physical interactions between PG carboxypeptidase and PBPs. The presence of OM permeability barrier strongly strengthened vancomycin resistance, but it significantly weakened {beta}-lactam sensitivity. Collectively, our results revealed two distinct LD-transpeptidase-independent functions of PG carboxypeptidases, which involved inverse modulation of bacterial resistance to clinically important antibiotics, {beta}-lactams and vancomycin, and presented evidence for a link between PG carboxypeptidase and PBPs.

IMPORTANCEBacterial peptidoglycan (PG) hydrolases play important roles in various aspects of bacterial physiology, including cytokinesis, PG synthesis, quality control of PG, PG recycling, and stress adaptation. Of all the PG hydrolases, the role of PG carboxypeptidases is poorly understood, especially regarding their impacts on antibiotic resistance. To date, most studies on PG carboxypeptidases are focused on LD-transpeptidase-related roles. We have revealed two distinct LD-transpeptidase-independent functions of PG carboxypeptidases with respect to antibiotic resistance. The deletion of PG carboxypeptidases led to sensitivity to most {beta}-lactams, while it caused strong resistance to vancomycin. The underlying molecular mechanisms of two phenotypes were not associated with LD-transpeptidases. Therefore, our study provides novel insights into the roles of PG carboxypeptidases in the regulation of antibiotic resistance and a potential clue for the development of a drug to improve the clinical efficacy of {beta}-lactam antibiotics.

One sentence summaryEffect of peptidoglycan carboxypeptidase on antibiotic


title: The impact of PrsA over-expression on the Bacillus subtilis transcriptome during fed-batch fermentation of alpha-amylase production

abstract: The production of the alpha-amylase (AMY) enzyme in Bacillus subtilis at a high rate leads to the accumulation of unfolded AMY, which causes secretion stress. The over-expression of the PrsA chaperone aids the enzyme folding and reduces stress. To identify affected pathways and potential mechanisms involved in the reduced growth, we analyzed the transcriptomic differences during fed-batch fermentation between a PrsA over-expressing strain and a control in a time-series RNA-seq experiment. We observe transcription in 542 previously un-annotated regions, of which 234 had significant changes in expression levels between the samples. Moreover, 1,791 protein-coding sequences, 80 non-coding genes, and 20 riboswitches overlapping UTR regions of coding genes had significant changes in expression. Via gene-set over-representation analysis of the differentially expressed genes, we identified putatively regulated biological processes; overall the analysis suggests that the PrsA over-expression affects ATP biosynthesis activity, amino acid metabolism, and cell wall stability. The investigation of the protein interaction network points to a potential impact on cell motility signaling. We discuss the impact of these highlighted mechanisms for reducing secretion stress or detrimental aspects of PrsA over-expression during AMY production.