title: BlotIt - Optimal alignment of western blot and qPCR experiments

abstract: Biological systems are frequently analyzed by means of mechanistic mathematical models. In order to infer model parameters and provide a useful model that can be employed for systems understanding and hypothesis testing, the model is often calibrated on quantitative, time-resolved data. To do so, it is typically important to compare experimental measurements over broad time ranges and various experimental conditions, e.g. perturbations of the biological system. However, most of the established experimental techniques such as Western blot, or quantitative real-time polymerase chain reaction only provide measurements on a relative scale, since different sample volumes, experimental adjustments or varying development times of a gel lead to systematic shifts in the data. In turn, the number of measurements corresponding to the same scale enabling comparability is limited. Here, we present a new flexible method to align measurement data that obeys different scaling factors. We propose an alignment model to estimate these scaling factors and provide the possibility to adapt this model depending on the measurement technique of interest. In addition, an error model can be specified to adequately weight the different data points and obtain scaling-model based confidence intervals of the finally scaled data points. Our approach is applicable to all sorts of relative measurements and does not need a particular experimental condition that has been measured over all available scales. An implementation of the method is provided with the R package blotIt including refined ways of visualization.


category: systems biology


title: Azole resistance associated regulatory motifs within the promoter of cyp51A in Aspergillus fumigatus

abstract: Aspergillus fumigatus is one of the deadliest fungal species causing hundreds of thousands of deaths each year. As azoles provide the preferred first-line option for treatment of Aspergillosis, the increase in rates of resistance and the poor therapeutic outcomes for those infected with a resistant isolate constitutes a serious global health threat. Azole resistance is frequently associated with specific tandem repeat duplications of a promoter element upstream of cyp51A, the gene which encodes the target for this drug class in A. fumigatus. This promoter element is recognized by the activating transcription factors SrbA and AtrR. This region also provides a docking platform for the CCAAT-binding-complex (CBC) and HapX that cooperate in the regulation of genes involved in iron-consuming pathways including cyp51A. Here, we studied the regulatory contribution of SrbA, AtrR, CBC and HapX binding sites on cyp51A expression and azole resistance during different iron availability employing promoter mutational analysis and protein/DNA interaction analysis. This strategy revealed iron status-dependent and -independent roles of these regulatory elements. We show that promoter occupation by both AtrR and SrbA is required for iron-independent steady-state transcriptional activation of cyp51A and its induction during short-term iron exposure relies on HapX binding. We further uncover the HapX binding site as repressor element the disruption of which elevates cyp51A expression and azole resistance regardless of iron availability.

category: molecular biology


title: The evolution of novel biotic interactions at ecological margins in response to climate change involves alleles from across the geographical range of the UK Brown Argus butterfly

abstract: Understanding the rate and extent to which populations can adapt to novel environments at their ecological margins is fundamental to predicting the persistence of biological communities during ongoing and rapid global change. Recent range expansion in response to climate change in the UK butterfly Aricia agestis is associated with the evolution of novel interactions with a larval food plant, and the loss of its ability to use its ancestral larval host species. Using ddRAD analysis of 61210 variable SNPs from 261 females from throughout the UK range of this species, we identify genomic regions at multiple chromosomes that are associated with these evolutionary responses, and their association with demographic history and ecological variation. Gene flow appears widespread throughout the range, despite the apparently fragmented nature of the habitats used by this species. Patterns of haplotype variation between selected and neutral genomic regions suggest that evolution associated with climate adaptation is polygenic, resulting from the independent spread of existing alleles throughout the established range of this species, rather than the colonisation of pre-adapted genotypes from coastal populations. These data suggest that rapid responses to climate change do not depend on the availability of pre-adapted genotypes. Instead, the evolution of novel forms of biotic interaction in Aricia agestis has occurred during range expansion, through the assembly of novel genotypes from alleles from multiple localities.

category: evolutionary biology


title: A Microtubule Mechanostat Enables Cells to Navigate Confined Environments

abstract: Cells migrating through complex 3D environments face considerable mechanical challenges. Cortical contractility must be highly coordinated to drive both cell movement and squeeze the large nucleus through confined regions. How cells protect themselves against mechanical forces and achieve nuclear transmigration in this context is largely unknown. Here, we demonstrate that cells experiencing confinement form a microtubule-dependent mechanostat in response to compressive forces. The mechanostat is an adaptive feedback mechanism whereby compressive loading of microtubules recruits CLASPs (cytoplasmic linker-associated proteins) to dynamically tune and repair the lattice. These reinforced microtubules allow the cell to withstand force and spatiotemporally organize contractility signaling pathways. Disruption of the mechanostat imbalances cortical contractility, stalling migration and ultimately resulting in catastrophic cell rupture.

category: cell biology


title: Relative enrichment - a density-based colocalization measure for single-molecule localization microscopy

abstract: Dual-color single-molecule localization microscopy (SMLM) provides unprecedented possibilities for detailed studies of colocalization of different molecular species in a cell. However, the informational richness of the data is not fully exploited by current analysis tools that often reduce colocalization to a single value. Here, we describe a new tool specifically designed for determination of co-localization in both 2D and 3D from SMLM data. The approach uses a novel function that describes the relative enrichment of one molecular species on the density distribution of a reference species. The function reframes the question of colocalization by providing a density-context relevant to multiple biological questions. Moreover, the function visualize enrichment (i.e. colocalization) directly in the images for easy interpretation. We demonstrate the approachs functionality on both simulated data and cultured neurons, and compare it to current alternative measures. The method is available in a Python function for easy and parameter-free implementation.


title: Partitioning variance in reproductive success, within years and across lifetimes

abstract: Variance in reproductive success ([Formula], with k = number of offspring)--is the stuff of evolution. Evolutionary change by natural selection requires variation in offspring number, and the ratio [Formula] is the most important factor that reduces effective size (Ne) compared to census size (N). Various frameworks have been proposed to parse factors that contribute to [Formula], but none has focused on age-specific values of{phi} , which indicate the degree to which reproductive skew is overdispersed among individuals of the same age and sex. Here I show how an ANOVA framework can be used to partition [Formula] into between-age and within-age components. For seasonal reproduction, the between-age effect depends on mean age-specific fecundity (bx, available in many published life tables), but few empirical data are available on the within-age effect, which depends on{phi} x. Analysis of variance in lifetime reproductive success [Formula] is more complicated; however, by using age at death rather than age as the treatment, the same ANOVA framework can be used to partition overall [Formula] into three components: between-treatment, within-treatment, and longevity. For both seasonal and lifetime reproduction, I also show how to quantify the expected contribution of random demographic stochasticity to these variance components. Analyses of empirical and simulated data illustrate the new methods and show that the largely-neglected within-age effect a) typically represents a substantial component of the overall variance in offspring number (even under a null model of random reproductive success), and b) can dominate the overall variance when{phi} x>l.


title: A paternal bias in germline mutation is widespread across amniotes and can arise independently of cell divisions

abstract: In humans and other mammals, germline mutations are more likely to arise in fathers than in mothers. Although this sex bias has long been attributed to DNA replication errors in spermatogenesis, recent evidence from humans points to the importance of mutagenic processes that do not depend on cell division, calling into question our understanding of this basic phenomenon. Here, we infer the ratio of paternal-to-maternal mutations, , in 42 species of amniotes, from putatively neutral substitution rates of sex chromosomes and autosomes. Despite marked differences in gametogenesis, physiologies and environments across species, fathers consistently contribute more mutations than mothers in all the species examined, including mammals, birds and reptiles. In mammals,  is as high as 4 and correlates with generation times; in birds and snakes,  appears more stable around 2. These observations can be explained by a simple model, in which mutations accrue at equal rates in both sexes during early development and at a higher rate in the male germline after sexual differentiation, with a conserved paternal-to-maternal ratio across species. Thus,  may reflect the relative contributions of two or more developmental phases to total germline mutations, and is expected to depend on generation time even if mutations do not track cell divisions.


title: Perception of RNA Nanotechnology among students and recommendations towards improved educational outreach

abstract: Translating new technologies to industrial and biomedical applications requires a highly skilled workforce. In the past, colleges and graduate schools played a primary role in preparing students for various areas of industry and medicine. The learning process and introduction of new concepts have recently extended beyond college education. High schools saw the rise of specialized career programs, while both high and middle school curricula got infused with challenging concepts. Nucleic acids, such as DNA and RNA, are broadly known for their role in the foundation of life. However, nucleic acid nanotechnology, an area of material science manipulating DNA and RNA to create complex structures with controlled properties, is less known. Herein, I report the results of a study investigating the perception of RNA nanotechnology among school students and suggest educational resources to improve understanding of RNA nanotechnology.

category: scientific communication and education


title: Myosin 10 supports mitotic spindle bipolarity by promoting PCM integrity and supernumerary centrosome clustering

abstract: Myosin 10 (Myo10) is a member of the MyTH4/FERM domain family of unconventional, actin-based motor proteins. Studies have implicated Myo10 in supporting cell adhesion via its integrin-binding FERM domain, and spindle positioning and spindle pole integrity via its microtubule-binding MyTH4 domain. Here we characterized Myo10s contribution to mitosis using Myo10 knockout HeLa cells and MEFs isolated from a Myo10 knockout mouse. Most notably, both of these knockout cells exhibit a pronounced increase in the frequency of multipolar spindles. Staining of unsynchronized metaphase cells showed that the primary driver of spindle multipolarity in knockout MEFs and knockout HeLa cells lacking supernumerary centrosomes is PCM fragmentation, which creates y-tubulin-positive, centriole-negative microtubule asters that serve as additional spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing centrosome clustering. These results argue, therefore, that Myo10 supports spindle bipolarity by maintaining PCM integrity in both normal and cancer cells, and by promoting supernumerary centrosome clustering in cancer cells. Finally, we present evidence that the defect in spindle pole integrity in Myo10 knockout cells is likely due to a defect in pole stability rather than pole maturation, and that Myo10 promotes supernumerary centrosome clustering at least in part by promoting cell adhesion during mitosis.


title: Live cell biosensors based on the fluorescence lifetime of environment-sensing dyes

abstract: Most biosensors used with fluorescence lifetime imaging (FLIM) have been based on fluorescence resonance energy transfer (FRET). Here we examined the capabilities of FLIM biosensors based on environment-sensing dyes. We screened merocyanine dyes to find an optimal combination of environment-induced FLIM changes, photostability and brightness at wavelengths suitable for live cell imaging. A biosensor reporting conformational changes of endogenous Cdc42 protein was tested in vitro and in live cells. In addition to the known advantages of FLIM (quantitation independent of photobleaching, intracellular biosensor distribution, and excitation intensity), the spectral properties of the merocyanine-FLIM biosensor (mcFLIM) provided enhanced sensitivity at low activation levels (<10%) where FRET is typically least sensitive. We leveraged these properties and the phasor representation of FLIM to determine the specific concentration of activated Cdc42 across the cell.


title: Differentiated kidney tubular cell-derived extracellular vesicles enhance maturation of tubuloids

abstract: Advanced kidney in vitro models such as organoids or tubuloids still lack the intrinsic expression of various transport proteins needed for active secretory function. Extracellular vesicles (EVs), cell-derived structures that constitute the organs microenvironment, are known to regulate various cellular processes, including kidney development and regeneration across the nephron. In this study, we propose a new application of renal tubular epithelial cell EVs as modulators for tubuloid functional maturation by increasing the levels of various differentiation markers such as organic anion transport 1 (OAT1), a protein involved in endogenous waste excretion. First, we show that EVs from engineered proximal tubule cells increased the expression of several transcription factors and epithelial transporters in tubuloids that resulted in improved cellular transport capacity. Next, a more in-depth proteomic data analysis demonstrated that EVs can trigger various biological pathways, including mesenchymal-to-epithelial transition, which is crucial in the tubular epithelial maturation process. Moreover, we demonstrated that EV-treated tubuloid-derived cells in a 3D tubular conformation as part of a bioartificial kidney can generate a tight polarized epithelial monolayer with formation of dense cilia structures. In conclusion, EVs from renal tubular epithelial cells can phenotypically improve tubuloid maturation, thereby enhancing their potential as preclinical models and functional units in regenerative therapies.


title: Impact of cell culture on the transcriptomic programs of primary and iPSC-derived human alveolar type 2 cells

abstract: The alveolar epithelial type 2 cell (AEC2) is the facultative progenitor of lung alveoli tasked to maintain distal lung homeostasis. AEC2 dysfunction has been implicated in the pathogenesis of a number of pulmonary diseases, including idiopathic pulmonary fibrosis (IPF), highlighting the importance of human in vitro models of the alveolar epithelium. However, AEC2-like cells captured in cell culture have yet to be directly compared to their in vivo counterparts at single cell resolution. Here, we apply single cell RNA sequencing to perform head-to-head comparisons between the global transcriptomes of freshly isolated primary (1{degrees}) adult human AEC2s, their isogenic cultured progeny, and human iPSC-derived AEC2s (iAEC2s) cultured in identical conditions. We find each population occupies a distinct transcriptomic space with both types of cultured AEC2s (1{degrees} and iAEC2s) exhibiting similarities to and differences from freshly purified 1{degrees} cells. Across each cell type, we find an inverse relationship between proliferative states and AEC2 maturation states, with uncultured 1{degrees} AEC2s being most quiescent and mature, their cultured progeny being more proliferative/less mature, and cultured iAEC2s being most proliferative/least mature. iAEC2s also express significantly lower levels of major histocompatibility complex (MHC) genes compared to 1{degrees} cells, suggesting immunological immaturity. Cultures of either type of human AEC2 (1{degrees} or iAEC2) do not generate detectable type 1 alveolar cells in these defined conditions; however, iAEC2s after co-culture with fibroblasts can give rise to a subset of cells expressing "transitional cell markers" recently described in fibrotic lung tissue of patients with pulmonary fibrosis or in mouse models of pulmonary fibrosis. Hence, we provide direct comparisons of the transcriptomic programs of 1{degrees} and engineered AEC2s, two in vitro model systems that can be harnessed for studies of human lung health and disease.


title: Very small spontaneous structural mutation rate in green algae

abstract: Structural mutations are very important in evolution and led to major innovations, but our knowledge of the spontaneous structural mutation rate is very limited. We so cannot have a complete view of the adaptive potential of species and new variant in a population without addressing this lack. Here, we used Illumina genomic data of mutation accumulation experiments from five green algae species and measured very low structural mutation rate, between 5x10-12 to 2x10-11 mutations per site, at least ten times lower than the nucleotide mutation rate in these species. Such low rates of structural mutations are typical of species with large effective population sizes, and suggests a very strong selection toward a small structural mutation rate. Last, we show that the rate of true positive detection of de novo structural mutations is variant types dependent and globally poor, highlighting the necessity of long read sequencing in future mutation accumulation studies.


title: Foldseek: fast and accurate protein structure search

abstract:
Highly accurate structure prediction methods are generating an avalanche of publicly available protein structures. Searching through these structures is becoming the main bottleneck in their analysis. Foldseek enables fast and sensitive comparisons of large structure sets. It reaches sensitivities similar to state-of-the-art structural aligners while being four orders of magnitude faster. Foldseek is free open-source software available at foldseek.com and as a webserver at search.foldseek.com.

Contactsoeding@mpinat.mpg.de, martin.steinegger@snu.ac.kr

category: bioinformatics


title: Binding of cardiolipin to the KcsA channel at the membrane outer leaflet allosterically opens the inner gate

abstract: Membrane proteins embedded in the membrane undergo changes in their actions under the influence of membrane lipids. Here, we present a novel type of lipid action on the potassium channel KcsA from Streptomyces lividans. Cardiolipin is present in various cellular membranes, including the host membrane of KcsA. Although the M0 domain, a nontransmembrane helix, is known to sense anionic lipids in the inner leaflet, we found that divalent anionic cardiolipin in the outer leaflet of the membrane interacts with positively charged residues, Arg64 and Arg89, on the extracellular side of the transmembrane domain. This binding propagates its action across the membrane toward the intracellular region of KcsA, thus, opening the inner gate. Such a long-range allosteric effect has not been found for channel-lipid interactions.

category: biophysics


title: Sub-10 nm fluorescence imaging

abstract: Advances in superresolution microscopy demonstrated single-molecule localization precisions of a few nanometers. However, translation of such high localization precisions into sub-10 nm spatial resolution in biological samples remains challenging. Here, we show that resonance energy transfer between fluorophores separated by less than 10 nm results in accelerated fluorescence blinking and consequently lower localization probabilities impeding sub-10 nm fluorescence imaging. We demonstrate that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used advantageously to determine the number and distance even of spatially unresolvable fluorophores in the sub-10 nm range. In combination with genetic code expansion (GCE) with unnatural amino acids and bioorthogonal click-labeling with small fluorophores photoswitching fingerprint analysis enables sub-10 nm resolution fluorescence imaging in cells.


title: Aging clocks, entropy, and the limits of age-reversal

abstract: We analyze aging signatures in the DNA-methylation and Electronic Medical Records from the UK Biobank datasets and observe that aging is driven by a large number of individually rare and independent transitions between metastable states in a vast configuration space. The compound effect of the configuration changes can be captured by a single stochastic variable, the thermodynamic biological age (tBA), tracking the entropy produced and hence the information lost in the aging process. We show that tBA increases with age, causes the linear aging drift of physiological state variables, reduces resilience, and drives the exponential acceleration of the risks of chronic diseases and death. The entropic character of the aging drift sets limits on possibilities of age-reversal. However, the universal features of configuration transitions suggest practical ways of controlling the rate of aging and thus promising the strongest possible life-extension effects.


title: Coupling comprehensive pesticide-wide association study to iPSC dopaminergic screening identifies and classifies Parkinson-relevant pesticides

abstract: Parkinsons disease (PD) is a complex, multi-factorial neurodegenerative disease, known to involve genetic, aging-related components, but also to be highly sensitive to environmental factors. In particular, ample evidence links pesticides to PD etiology. Here, establishing a field-to-bench paradigm, we have combined record-based exposure assessment in a population-based epidemiologic study of PD with testing in dopaminergic neurons produced from iPSCs to further identify and classify PD-relevant pesticides. First, agricultural pesticide-application records in California enabled us to investigate exposure to nearly 300 specific pesticides and PD risk in a comprehensive, pesticide-wide association study (PWAS). We implicated long-term exposure to 53 pesticide active ingredients in PD risk and identified their relevant co-exposure profiles. Second, to identify which of these pesticides might contribute to PD through direct effects on dopaminergic neurons, we employed a live-cell imaging screening paradigm in which neurons, definitively identified with a tyrosine hydroxylase reporter, were exposed to 43 of the high-risk pesticides. Using detailed morphometric measures, we found 10 pesticides were directly toxic to these neurons. Further, we analyzed pesticides typically used in combinations in cotton farming. Among these "cotton cluster" pesticides, co-exposures resulted in markedly greater toxicity than any single pesticide. Trifluralin was a pivotal driver of toxicity to dopaminergic neurons and led to marked mitochondrial dysfunction. Our field-to-bench paradigm may prove useful to mechanistically dissect pesticide exposure implicated in PD risk, and guide agricultural policy in the future.

category: neuroscience


title: Influence of earlier human activity on gut microbiota and immune responses of Darwin's finches in the Galapagos Islands

abstract: Urbanization can influence animal traits, including immunity and gut microbiota. This idea has been studied in well-established urban areas, but few studies have determined the effect of early-stage urbanization on the gut microbiota and its relationship with immunity. Over the past several decades, the Galapagos Islands have seen rapid resident human population growth and tourist activity, leading to varying levels of human activity of four of thirteen Islands. Consequently, diet, gut microbiota, and immunity of endemic animals, such as Darwins finches, may have changed. The goal of this study was to determine the effect of land use on the immune response, gut microbiota, and body measurements of Darwins finches early-on in the rapid increase in human activity in the Islands. Specifically, we compared proxies of the immune response (haptoglobin levels, lysozyme activities, complement antibody levels, and natural antibody levels), gut microbiota (bacterial diversity, community structure and membership, and relative abundance of bacterial taxa), and body measurements (body mass, tarsus length, and scaled mass index) across undeveloped, agricultural, and urban areas in 2008 for medium ground finches (Geospiza fortis) and small ground finches (G. fuliginosa). We found that lysozyme activity was lower and bacterial diversity was higher in urban areas compared to non-urban areas across both finch species. For small ground finches, relative abundances of bacterial genera (Pseudoxanthomonas, Cloacibacterium, and Dietzia spp.) were higher in urban areas compared to non-urban areas, but this pattern was not observed in medium ground finches. Medium ground finches were smaller in undeveloped areas compared to the other two areas, but body measurements of small ground finches did not differ across areas. Our historical data provides an interesting comparison to more recent data and demonstrates that even early human activity can influence traits of organisms.

category: ecology


title: psntools - a Python package for protein structure network analysis

abstract: The application of network theory to investigate protein structures and conformational ensembles through Protein Structure Networks (PSNs) has proven particularly insightful to study protein dynamics, the potentially disruptive effects of disease-related mutations, and allosteric mechanisms. Here, we present psntools, a novel Python package for downstream analysis of PSNs. psntools is completely PSN-agnostic, in contrast with several available tools in the community. psntools relies only on a few Python dependencies, most notably MDAnalysis and NetworkX, works without external software, and can be incorporated into Python-based analysis pipelines. We also present an example of the usage of psntools on a case of study of biological interest, which helped produce novel insights on the structural details of the interaction between BCL-xL and the BH3 motif of BECLIN-1. The psntools package and the data associated with the case study are available at https://github.com/ELELAB/psntools.


title: De novo mass spectrometry peptide sequencing with a transformer model

abstract: Tandem mass spectrometry is the only high-throughput method for analyzing the protein content of complex biological samples and is thus the primary technology driving the growth of the field of proteomics. A key outstanding challenge in this field involves identifying the sequence of amino acids--the peptide--responsible for generating each observed spectrum, without making use of prior knowledge in the form of a peptide sequence database. Although various machine learning methods have been developed to address this de novo sequencing problem, challenges that arise when modeling tandem mass spectra have led to complex models that combine multiple neural networks and post-processing steps. We propose a simple yet powerful method for de novo peptide sequencing, Casanovo, that uses a transformer framework to map directly from a sequence of observed peaks (a mass spectrum) to a sequence of amino acids (a peptide). Our experiments show that Casanovo achieves state-of-the-art performance on a benchmark dataset using a standard cross-species evaluation framework which involves testing with out-of-distribution samples, i.e., spectra with never-before-seen peptide labels. Casanovo not only achieves superior performance but does so at a fraction of the model complexity and inference time required by other methods.


title: Optimal pathways control fixation of multiple mutations during cancer initiation

abstract: Cancer starts after initially healthy tissue cells accumulate several specific mutations or other genetic alterations. The dynamics of tumor formation is a very complex phenomenon due to multiple involved biochemical and biophysical processes. It leads to a very large number of possible pathways on the road to final fixation of all mutations that marks the beginning of the cancer, complicating the understanding of microscopic mechanisms of tumor formation. We present a new theoretical framework of analyzing the cancer initiation dynamics by exploring the properties of effective free-energy landscape of the process. It is argued that although there are many possible pathways for the fixation of all mutations in the system, there are only few dominating pathways on the road to tumor formation. The theoretical approach is explicitly tested in the system with only two mutations using analytical calculations and Monte Carlo computer simulations. Excellent agreement with theoretical predictions is found for a large range of parameters, supporting our hypothesis and allowing us to better understand the mechanisms of cancer initiation. Our theoretical approach clarifies some important aspects of microscopic processes that lead to tumor formation.


title: Data-driven models of optimal chromatic coding in the outer retina

abstract: The functional properties of the outermost retinal circuits involved in color discrimination are not well understood. Recent experimental work on zebrafish has elucidated the in-vivo activity of photoreceptors and horizontal cells as a function of the stimulus spectrum, highlighting the appearance of chromatic-opponent signals at the first synaptic connection between cones and horizontal cells. These findings, together with the observed lack of gap junctions, suggest that the mechanism yielding early color-opponency in zebrafish is dominated by inhibitory feedback. We discuss the observed neuronal activity in the context of efficient codification of chromatic information, hypothesizing that opponent chromatic signals provide optimal codification, minimizing signal redundancy. We examine whether these functional properties are general across species by studying the dynamic properties of dichromatic and trichromatic outer retinal networks. Our findings show that dominant inhibitory feedback mechanisms provide an unambiguous codification of chromatic stimuli, whereas this property is not guaranteed in networks with strong excitatory inter-cone connections, for example via gap junctions. This provides a plausible explanation for the absence of gap junctions observed in the outermost zebrafish retinal layers. In addition, our study suggests that the simplest zebrafish-like network with dominant inhibitory feedback capable of optimally codifying chromatic information requires at least two successive inhibitory feedback layers. Finally, we contrast the chromatic codification performance of zebrafish-inspired retinal networks with networks having different opsin combinations. We find that optimal combinations lead to a chromatic codification improvement of only 13% compared with zebrafish opsins, suggesting that the zebrafish retina performs nearly optimal codification of chromatic information in its habitat.


title: What do Functional Synchronization Networks Indicate About Underlying Structure and System Dynamics? A network theory study in the islet

abstract:
Network theory provides tools for quantifying communication within heterogeneous biological systems. Calcium-based imaging is a common technique used to analyze cellular dynamics and create functional networks. However, the use of functional networks in understanding the underlying biological structure, formed by physical connections, has not been well investigated. The islet of Langerhans is a biological system with relatively simple architecture that affords the opportunity for high-fidelity experimental and computation analysis, making it an ideal system to answer biological network theory-based questions. Here, we analyze both experimental and computational models of the islet to quantify the relationship between the gap junction structural network and functional synchronization networks. We show that functional networks poorly reflect gap junction structure. Instead, they indicate intrinsic factors driving excitability, such as cellular metabolism. This surprising finding provides a new interpretation of functional networks within biological systems.

TeaserWe use experimental and computational models to provide a new interpretation of intracellular synchronization in biological networks.


title: Understanding Hybrid Lipid-Polymer Gene Delivery Nanoparticle Assembly with Fluorescence Cross Correlation Spectroscopy

abstract: Hybrid lipid-polymer-nucleic acid nanoparticles (LPNPs) provide a range of delivery strategies for nonviral gene therapy. However, due to several pairwise interactions between their components, these formulations are difficult to predict and characterize. Here, we employed, for the first time, a novel methodology based on fluorescence cross correlation spectroscopy (FCCS) to directly quantify the extent of association between polycation-DNA cores (polyplexes) and cationic liposome shells. This leads to a rapid and easy evaluation of LPNP formation. As a result, we were able to unveil two critical insights for predicting LPNP assembly. Firstly, the interaction between the polycation (polylysine) and DNA is robust, with the polycation not being displaced by liposomes. Hence, the polyplex cores and liposome shells have to be oppositely charged to associate. Secondly, and most importantly, the liposome:poliplex number ratio ({rho}N), as opposed to the more common charge ratio, is a critical parameter to predict stable LPNP formation. We find that{rho} N[≥]1 is required to ensure that every polyplex is enveloped by a liposome, avoiding the coexistence of oppositely charged species, thus inhibiting aggregation. Since the chosen LPNP components are common, these observations should be applicable to many LPNP systems. Furthermore, this FCCS-based methodology is relevant to other self-assembly composite materials, for applications beyond gene delivery.


title: Astrocytes regulate inhibition in Fragile X Syndrome

abstract: Fragile X syndrome (FXS) is a leading genetic cause of autism-like symptoms that include sensory hypersensitivity and cortical hyperexcitability. Recent observations in humans and Fmr1 knockout (KO) animal models of FXS suggest abnormal GABAergic signaling. As most studies focused on neuron-centered mechanisms, astrocytes contribution to abnormal inhibition is largely unknown. Here we propose a non-neuronal mechanism of abnormal inhibitory circuit development in FXS. Astrocyte-specific deletion of Fmr1 during postnatal period leads to increased astrocytic GABA levels, but negatively impacts synaptic GABAA receptors and parvalbumin (PV) cell development. Developmental deletion of Fmr1 from astrocytes also affects communications between excitatory neurons and PV cells, impairing sound-evoked gamma synchronization in the cortex, while enhancing baseline and on-going sound-evoked EEG power, and leading to increased locomotor activity and altered social behaviors in adult mice. These results demonstrate a profound role of astrocytic FMRP in the development of inhibitory circuits and shaping normal inhibitory responses.


title: Neutrophils degranulate GAG-containing proteoglycofili, which block Shigella growth and degrade virulence factors

abstract:
Summary paragraphNeutrophil degranulation plays a central role in their ability to kill pathogens but also to stimulate other immune cells1-3. Here we show that neutrophil degranulation, induced in hypoxia or upon Shigella infection in vitro and in vivo, leads to the release of polymers called neutrophil Proteoglycofili (PGF). PGF are mainly composed of granular proteins (myeloperoxidase, elastase, lactoferrin, cathelicidin, albumin) pre-stored in various types of granules, and chondroitin sulfate. PGF individual fibers have a diameter of 43.9 {+/-} 20.3 nm and. They secreted by viable neutrophils and do not contain DNA, as opposed to NETs which contains also granular proteins, chondroitin sulfate in addition to chromatin, released upon neutrophil disintegration and cell death.

We demonstrated that PGF block the growth of Shigella and other bacteria and degrade Shigella virulence factors. The degradation of the chondroitin sulfate polymers with testes hyaluronidases destabilizes PGF ultrastructure and abolishes its antimicrobial activity. Our results provide novel insights in the neutrophil degranulation process and open new doors for the investigation of PGF contribution to cytokines concentration gradient formation and adaptive immune cells activation. Further investigations are required to better appreciate the importance of this "sterile blaster" in infectious or inflammatory diseases.

category: immunology


title: CameraTrapDetectoR: Automatically detect, classify, and count animals in camera trap images using artificial intelligence

abstract: Motion-activated wildlife cameras, or camera traps, are widely used in biological monitoring of wildlife. Studies using camera traps amass large numbers of images and analyzing these images can be a large burden that inhibits research progress. We trained deep learning computer vision models using data for 168 species that automatically detect, count, and classify common North American domestic and wild species in camera trap images. We provide our trained models in an R package, CameraTrapDetectoR. Three types of models are available: a taxonomic class model classifies objects as mammal (human and non-human) or avian; a taxonomic family model that recognizes 31 mammal, avian, and reptile families; a species model that recognizes 75 domestic and wild species including all North American wild cat species, bear species, and Canid species. Each model also includes a category for vehicles and empty images. The models performed well on both validation datasets and out-of-distribution testing datasets as mean average precision values ranged from 0.80 to 0.96. CameraTrapDetectoR provides predictions as an R object (a data frame) and flat file and provides the option to create plots of the original camera trap image with the predicted bounding box and label. There is also the option to apply models using a Shiny Application, with a point-and-click graphical user interface. This R package has the potential to facilitate application of deep learning models by biologists using camera traps.


title: Increased H3K27 trimethylation contributes to cone survival in a mouse model of cone dystrophy

abstract: Inherited retinal diseases (IRDs) are a heterogeneous group of blinding disorders, which result in dysfunction or death of the light-sensing cone and rod photoreceptors. Despite individual IRDs being rare, collectively, they affect up to 1:2000 people worldwide, causing a significant socioeconomic burden, especially when cone-mediated central vision is affected. This study uses the Pde6ccpfl1 mouse model of achromatopsia, a cone-specific vision loss IRD, to investigate the potential gene-independent therapeutic benefits of a histone demethylase inhibitor GSK-J4 on cone cell survival. We investigated the effects of GSK-J4 treatment on cone cell survival in vivo and ex vivo and changes in cone-specific gene expression via single-cell RNA sequencing. A single intravitreal GSK-J4 injection led to transcriptional changes in pathways involved in mitochondrial dysfunction, endoplasmic reticulum stress, among other key epigenetic pathways, highlighting the complex interplay between methylation and acetylation in healthy and diseased cones. Furthermore, continuous administration of GSK-J4 in retinal explants increased cone survival. Our results suggest that IRD-affected cones respond positively to epigenetic modulation of histones, indicating the potential of this approach in the development of a broad class of novel therapies to slow cone degeneration.


title: All-optical manipulation of the Drosophila olfactory system.

abstract: Thanks to its well-known neuroanatomy, limited brain size, complex behaviour, and extensive genetic methods, Drosophila has become an indispensable model in neuroscience. A vast number of studies have focused on its olfactory system and the processing of odour information. Optogenetics is one of the recently developed genetic tools that significantly advance this field of research, allowing to replace odour stimuli by direct neuronal activation with light. This becomes a universal all-optical toolkit when spatially selective optogenetic activation is combined with calcium imaging to read out neuronal responses. Initial experiments showed a successful implementation to study the olfactory system in fish and mice, but the olfactory system of Drosophila has been so far precluded from an application. To fill this gap, we present here optogenetic tools to selectively stimulate functional units in the Drosophila olfactory system, combined with two-photon calcium imaging to read out the activity patterns elicited by these stimuli at different levels of the brain. This method allows to study the spatial and temporal features of the information flow and reveals the functional connectivity in the olfactory network.


title: Ascending neurons convey behavioral state to integrative sensory and action selection centers in the brain

abstract: Knowledge of ones own behavioral state--whether one is walking, grooming, or resting--is critical for contextualizing sensory cues including interpreting visual motion and tracking odor sources. Additionally, awareness of ones own posture is important to avoid initiating destabilizing or physically impossible actions. Ascending neurons (ANs), interneurons in the vertebrate spinal cord or insect ventral nerve cord (VNC) that project to the brain, may provide such high-fidelity behavioral state signals. However, little is known about what ANs encode and where they convey signals in any brain. To address this gap, we performed a large-scale functional screen of AN movement encoding, brain targeting, and motor system patterning in the adult fly, Drosophila melanogaster. Using a new library of AN sparse driver lines, we measured the functional properties of 247 genetically-identifiable ANs by performing two-photon microscopy recordings of neural activity in tethered, behaving flies. Quantitative, deep network-based neural and behavioral analyses revealed that ANs nearly exclusively encode high-level behaviors--primarily walking as well as resting and grooming--rather than low-level joint or limb movements. ANs that convey self-motion--resting, walking, and responses to gust-like puff stimuli--project to the brains anterior ventrolateral protocerebrum (AVLP), a multimodal, integrative sensory hub, while those that encode discrete actions--eye grooming, turning, and proboscis extension--project to the brains gnathal ganglion (GNG), a locus for action selection. The structure and polarity of AN projections within the VNC are predictive of their functional encoding and imply that ANs participate in motor computations while also relaying state signals to the brain. Illustrative of this are ANs that temporally integrate proboscis extensions over tens-of-seconds, likely through recurrent interconnectivity. Thus, in line with long-held theoretical predictions, ascending populations convey high-level behavioral state signals almost exclusively to brain regions implicated in sensory feature contextualization and action selection.


title: Medullary kappa-opioid receptor neurons inhibit pain and itch through a descending circuit

abstract: In perilous and stressful situations, the ability to suppress pain can be critical for survival. The rostral ventromedial medulla (RVM) contains neurons that robustly inhibit nociception at the level of the spinal cord through a top-down modulatory pathway. Although much is known about the role of the RVM in the inhibition of pain, the precise ability to directly manipulate pain-inhibitory neurons in the RVM has never been achieved. We now expose a cellular circuit that inhibits nociception and itch in mice. Through a combination of molecular, tracing, and behavioral approaches, we found that RVM neurons containing the kappa-opioid receptor (KOR) inhibit itch and nociception. With chemogenetic inhibition, we uncovered that these neurons are required for stress-induced analgesia. Using intersectional chemogenetic and pharmacological approaches, we determined that RVMKOR neurons inhibit nociception and itch through a descending circuit. Lastly, we identified a dynorphinergic pathway arising from the periaqueductal gray (PAG) that modulates nociception within the RVM. These discoveries highlight a distinct population of RVM neurons capable of broadly and robustly inhibiting itch and nociception.


title: Pairing-dependent plasticity in a dissected fly brain is input-specific and requires synaptic CaMKII enrichment and nighttime sleep

abstract:
In Drosophila, in vivo functional imaging studies revealed that associative memory formation is coupled to a cascade of neural plasticity events in distinct compartments of the mushroom body (MB). In-depth investigation of the circuit dynamics, however, will require an ex vivo model that faithfully mirrors these events to allow direct manipulations of circuit elements that are inaccessible in the intact fly. The current ex vivo models have been able to reproduce the fundamental plasticity of aversive short-term memory, a potentiation of the MB intrinsic neurons (Kenyon cells; KCs) responses after artificial learning ex vivo. However, this potentiation showed different localization and encoding properties from those reported in vivo and failed to generate the previously reported suppression plasticity in the mushroom body output neurons (MBONs). Here, we develop an ex vivo model using the female Drosophila brain that recapitulates behaviorally evoked plasticity in the KCs and MBONs. We demonstrate that this plasticity accurately localizes to the MB 3 compartment and is encoded by a coincidence between KCs activation and dopaminergic input. The formed plasticity is input-specific, requiring pairing of the conditioned stimulus (CS) and unconditioned stimulus (US) pathways; hence we name it pairing-dependent plasticity (PDP). PDP formation requires an intact CaMKII gene and is blocked by previous-night sleep deprivation but is rescued by rebound sleep. In conclusion, we show that our ex vivo preparation recapitulates behavioral and imaging results from intact animals and can provide new insights into mechanisms of memory formation at the level of molecules, circuits, and brain state.

Significance StatementThe mammalian ex vivo LTP model enabled in-depth investigation of the hippocampal memory circuit. We develop a parallel model to study the Drosophila mushroom body (MB) memory circuit. Pairing activation of the conditioned stimulus and unconditioned stimulus pathways in dissected brains induces a potentiation pairing-dependent plasticity (PDP) in the axons of {beta} Kenyon cells and a suppression PDP in the dendrites of their postsynaptic MB output neurons, localized in the MB 3 compartment. This PDP is input-specific and requires the 3 untranslated region of CaMKII. Interestingly, ex vivo PDP carries information about the animals experience before dissection; brains from sleep deprived animals fail to form PDP while those from animals who recovered 2 hours of their lost sleep form PDP.


title: Genotypic Sex Determination Systems could be Adaptations to Extreme Temperature Environments in Reptiles and to Endothermy in Mammals and Birds

abstract: In some vertebrates species, environmental temperatures (TSD) determine their sex determination. In others, it is controlled by genomic mechanisms (GSD). One hypothesis suggests that GSD systems could have evolved in ectothermic TSD species to escape the geographical limitation imposed by environmental temperatures. Recently, it has been found that TSD reptiles species tend to breed at warmer temperatures than GSD species, especially in habitat with four months of warm temperatures. Here we obtain the pivotal temperature (the one that generates equal ratios of male and females) in 53 reptiles species (from four orders: sphenodontia, crocodilia, testudines and squamata) and compare it with the environmental temperature in the nest during the breeding season in 100 TSD reptiles species and 78 GSD reptiles species. Our results show that GSD species statistically breed in temperatures that would cause a sex bias if they had TSD systems, whereas species with TSD breed in a similar range of temepratrues to the pivotal ones. Additionally, we also found that the body temperature of more than 1,200 endothermic species statistically exceeds the pivotal temperature suggesting that GSD is necessary for endotherms to avoid sex bias that could lead to extinction. Finally, we observed that one of the 100 most invasive species worldwide, Trachemys scripta elegans, a turtle species with TSD, has never been able to establish in countries with less than four months of warm temperatures, confirming a restriction in the geographic range in TSD species caused by extreme temperatures. Altogether, these results suggest that GSD could have evolved as an adaptation to avoid the biased sex ratios that extreme temperature may cause in species with a TSD system.


title: An atlas of gene expression variation across the Caenorhabditis elegans species

abstract: Phenotypic variation in diverse organism-level traits have been studied in Caenorhabditis elegans wild strains, but differences in gene expression and the underlying variation in regulatory mechanisms are largely unknown. Here, we use natural variation in gene expression to connect genetic variants to differences in organismal- level traits, including drug and toxicant responses. We performed transcriptomic analysis on 207 genetically distinct C. elegans wild strains to study natural regulatory variation of gene expression. Using this massive dataset, we performed genome-wide association mappings to investigate the genetic basis underlying gene expression variation and revealed complex genetic architectures. We found a large collection of hotspots enriched for expression quantitative trait loci across the genome. We further used mediation analysis to understand how gene expression variation could underlie organism-level phenotypic variation for a variety of complex traits. These results reveal the natural diversity in gene expression and possible regulatory mechanisms in this keystone model organism, highlighting the promise of gene expression variation in shaping phenotypic diversity.

category: genetics


title: Fate tracing reveals differences between Reelin+ HSCs and Desmin+ HSCs in activation, migration, and proliferation activities

abstract: The activation of hepatic stellate cells (HSCs) which comprise distinct clusters, is the main cause of liver fibrogenesis in response to different etiologies of chronic liver injuries. In this study, we constructed a novel ReelinCreERT2 transgenic mouse in which cells expressing Reelin were fully marked and demonstrated that about 50% HSCs were labeled. These ReelinCreERT2-labeled HSCs displayed distinct characteristics in migration, activation and proliferation compared to Desmin+ HSCs (total HSCs) in cholestatic (bile duct ligation; BDL) or hepatotoxic (carbon tetrachloride; CCl4) liver injuries. In BDL-induced fibrotic livers, Desmin+ HSCs were activated with increased proliferation and accumulation activities around the portal triad, but mGFP+ HSCs did not show proliferation or accumulation activity around the portal triad, and only a small part was activated. In CCl4-induced fibrotic livers, most of Desmin+ and mGFP+ HSCs were activated along with proliferation and accumulation potential around the central vein, however fewer mGFP+ HSCs were activated compared to Desmin+ HSCs. Moreover, in the regression of CCl4-induced fibrosis, mGFP+ HSCs were apoptosed whereas Desmin+ HSCs recovered to normal state. Besides, we didnt find evidence that mGFP+ HSCs transdifferentiated into hepatocytes or cholangiocytes through mesenchymal-epithelial transition (MET).

category: pathology


title: Taxonomic practice, creativity, and fashion: What's in a spider name?

abstract: Theres a secret pleasure in naming new species. Besides traditional etymologies recalling the sampling locality, habitat, or morphology of the species, etymologies may be tributes to some meaningful person (for example, the species collector, the authors husband or wife, or a celebrity), pop culture references, and even exercises of enigmatography. The possibility of choosing witty or even playful names for new species departs from the otherwise impersonal and old-fashioned writing style thats common in taxonomic papers; but, how has the descriptors choice for specific etymologies changed over the 300+ years since the introduction of the Linnaean binomial system of nomenclature? Using an unprecedented dataset of 48,464 etymologies (all valid species and subspecies of spiders described between 1757 and May 2020), we tested the hypothesis that species names given by taxonomists are deeply influenced by their cultural background. In particular, we asked whether naming practices change through space (continent in which the species was found) or have changed through time (year of description). We observed spatial and temporal differences in the way taxonomists name new species. In absolute terms, etymologies referring to morphology were the most frequently used. In relative terms, however, references to morphology peaked in 1850-1900 and then began to decline, with a parallel increase in etymologies dedicated to people and geography. Currently, these are the most widely used, with [~]38% of all etymologies of spider species described in the last ten years referring to geography, [~]25% to people, and [~]25% to morphology. Interestingly, there has been a dramatic increase in etymologies referring to pop culture and other cultural aspects in the last two decades, especially in Europe and the Americas. While such fashionable names often carry little or no biological information regarding the species itself, they help give visibility to the science of taxonomy, a discipline currently facing a profound crisis within academia. Taxonomy is among the most unchanged disciplines across the last centuries in terms of background, tools, rules, and writing style; but our analysis suggests that taxonomists remain deeply influenced by their living time and space.


title: Novel methodology for localizing and studying the insect dorsal rim area morphology in 2D and 3D

abstract: 1.Polarized light-based navigation in insects is facilitated by a specialised polarisation-sensitive part of the eye known as the dorsal rim area (DRA). Existing methods to study the anatomy of the DRA are in most cases destructive and very time consuming. Here, we present a novel method that allows for DRA localization using 3D volumetric images acquired through micro-computed tomography in combination with 2D photographs. We used the method on size polymorphic buff-tailed bumblebees, Bombus terrestris. We found that the size of the DRA can be easily acquired from photographs of the dorsal part of the eye. An allometric analysis of the size of the DRA in relation to body size in B. terrestris showed that the DRA region increases with body size but not at the same rate. Using our method, we localised the DRA in a 3D volume rendering of the eye using 2D photos localization of an individuals DRA onto the volumetric image stack of the head allowed for individual-level descriptions of the ommatidial structures (lens, crystalline cones, rhabdoms) to be performed on three different eye regions (DRA, non-DRA, proximate to DRA). The only feature distinctly unique to the DRA ommatidia was the smaller dimension of crystalline cones in comparison to other regions. Using our novel methodology we provide the first individual-level description of DRA ommatidial features and a comparison of how the DRA area varies with body size in bumblebees.

category: zoology


title: Automatic identification of intestinal parasites in reptiles using microscopic stool images and convolutional neural networks

abstract: Captive environments trigger the propagation and multiplication of parasites among different reptile species, thus weakening their immune response and causing infections and diseases. Technological advances of convolutional neural networks have opened a new field for detecting and classifying diseases which have shown great potential to overcome the shortcomings of manual detection performed by experts. Therefore, we propose an approach to identify six captive reptiles parasitic agents (Ophionyssus natricis, Blastocystis sp, Oxiurdo egg, Rhytidoides similis, Strongyloides, Taenia) from a microscope stool images dataset. Towards this end, we first use an image segmentation stage to detect the parasite within the image, which combines the Contrast Limited Adaptive Histogram Equalization (CLAHE) technique, the OTSU binarization method, and morphological operations. Then, we carry out a classification stage through Mobilenet CNN under a transfer learning scheme. This method was validated on a stool image dataset containing 3616 images data samples and 26 videos from the six parasites mentioned above. The results obtained indicate that our transfer learning-based approach can learn a helpful representation from the dataset. We obtained an average accuracy of 98.66% across the six parasitic agent classes, which statistically outperformed, at a 95% confidence level, a custom CNN trained from scratch.


title: Anatomical connectivity profile development constrains medial-lateral topography in the dorsal prefrontal cortex

abstract: The prefrontal cortex (PFC) is a highly variable, evolutionarily expanded brain region that is engaged in multiple cognitive processes. The subregions of the PFC mature relatively late compared with other brain regions, and the maturation times vary between these subregions. Among these, the dorsomedial and dorsolateral prefrontal cortex (dmPFC and dlPFC) share a parallel topographic pattern of functional connectivity, while participating in different types of complex behaviors. However, the developmental trajectories of the two areas remain obscure. In this study, we uncovered differences in the developmental trends of the dmPFC and dlPFC. These differences were mainly caused by structural and functional changes in the medial area of the superior frontal gyrus (SFG). The developmentally different arealization patterns were verified using multiple parcellation approaches with multimodal data, including structural magnetic resonance imaging (sMRI), diffusion MRI (dMRI), resting state functional MRI (rfMRI), and a publicly available transcriptomic dataset. Human brain gene expression data was also used to perform downstream analyses, which could inform us about the potential biological mechanisms underlying the developmentally different arealizations. Furthermore, behavioral analyses hinted at the effects of regionalization on ontogeny. In brief, this study revealed a tendency toward a medial-lateral prefrontal division and can provide a fuller understanding of the potential underlying genetic underpinnings as well as of the potential effects on developmental behavior.


title: Functional geometry of auditory cortical resting state networks derived from intracranial electrophysiology

abstract: Critical details remain unresolved about the organization of the human auditory cortical hierarchy and its relationship to higher order brain networks. We investigated this organization using diffusion map embedding (DME) applied to resting state intracranial electroencephalography (iEEG) obtained in neurosurgical patients. DME was applied to functional connectivity measured between regions of interest (ROIs). ROIs exhibited a hierarchical organization, symmetric between the two hemispheres and robust to the choice of iEEG frequency band, connectivity metric, and imaging modality. Tight clusters of canonical auditory and prefrontal ROIs were maximally segregated in embedding space. Clusters consistent with ventral and dorsal auditory processing streams were paralleled by a cluster suggestive of a third stream linking auditory and limbic structures. Portions of anterior temporal cortex were characterized as global hubs. This approach lays the foundation for identifying network changes during active speech and language processing and elucidating mechanisms underlying disorders of auditory processing.


title: NMNAT2 is the major NAD+ provider for vesicular glycolysis generating on-board energy for fast axonal transport cargos

abstract: Axonal transport, an ATP demanding process, plays a critical role in maintaining axonal and neuronal health. ATP in neurons is synthesized by glycolysis and mitochondrial respiration, both driven by proper NAD+/NADH redox potentials. NMNAT2 is the major neuronal NAD+ synthesizing enzyme and is a key axonal maintenance factor. Here, we show that NMNAT2 co-migrates with fast vesicular cargos in axons and is required for fast axonal transport in the distal axons of cortical neurons. Using SoNar sensor imaging to detect axonal NAD+ and NADH, we show that NMNAT2 is critical in maintaining NAD+/NADH potentials in distal axons. With Syn-ATP sensor imaging to detect synaptic vesicle ATP levels (sv-ATP), we demonstrate that glycolysis is the major provider of sv-ATP and NMNAT2 deletion significantly reduces sv-ATP levels. NAD+ supplementation of NMNAT2 KO neurons restores sv-ATP levels and fast axonal transports in a glycolysis-dependent manner. Together, these data show that NMNAT2 maintains the local NAD+/NADH redox potential and sustains "on-board" glycolysis to meet the bioenergetic demands of fast vesicular transport in distal axons. Intriguingly, mitochondrial respiration contributes significantly to sv-ATP levels in NMNAT2 KO axons. This finding suggests that NMNAT2 deletion induces metabolic plasticity by engaging mitochondria. Surprisingly, NMN, the substrate for NMNAT2 in NAD+ synthesis, supplementation restores sv-ATP and axonal transport in NMNAT2 KO axons with an efficacy similar to NAD+. NMNs restoration of NMNAT2 KO distal axonal sv-ATP levels and vesicle transport requires mitochondrial respiration, while rescue by NAD+ is independent of mitochondrial respiration. Based on these findings, we hypothesize that NMN rescues glycolysis to restore axonal transport in NMNAT2 KO axons by taking advantage of the compensatory ATP-generating metabolic pathways triggered by NMNAT2 loss.


title: Distinct sensitivities to SARS-CoV-2 variants in vaccinated humans and mice

abstract: The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has led to the development of a large number of vaccines, several of which are now approved for use in humans. Understanding vaccine-elicited antibody responses against emerging SARS-CoV-2 variants of concern (VOC) in real time is key to inform public health policies. Serum neutralizing antibody titers are the current best correlate of protection from SARS-CoV-2 challenge in non-human primates and a key metric to understand immune evasion of VOC. We report that vaccinated BALB/c mice do not recapitulate faithfully the breadth and potency of neutralizing antibody responses against VOC, as compared to non-human primates or humans, suggesting caution should be exercised when interpreting data for this animal model.


title: Neutralization Of SARS-CoV-2 Variants By A Human Polyclonal Antibody Therapeutic (COVID-HIG, NP-028) With High Neutralizing Titers To SARS-CoV-2

abstract:
Since the start of the COVID-19 outbreak the World Health Organization (WHO) has classified multiple SARS-CoV-2 Variants-of-Concern and Variants-of-Interest (VOC/VOI) with mutations in their Spike protein that increase transmissibility and/or reduce the effectiveness of vaccines and monoclonal antibody therapeutics. The emergence of these variants represents a significant health risk and highlights the need for additional COVID-19 therapeutics that maintain the ability to neutralize current, as well as future variants.

COVID-HIG (NP-028) is a polyclonal Anti-SARS-CoV-2 human Immunoglobulin purified from source human plasma screened for high antibody titers to SARS-CoV-2 antigens. COVID-HIG was previously evaluated in INSIGHT 013 clinical trial [NCT04546581] which was an international, multi-center, adaptive, randomized, double-blind, placebo-controlled trial of the safety, tolerability and efficacy of a single dose infusion (up to 400 mL) of Anti-Coronavirus Hyperimmune Intravenous Immunoglobulin (hIVIG) for the treatment of adult recently hospitalized COVID-19 patients (N=593). COVID-HIG is currently being evaluated for clinical efficacy in a Phase 3 placebo-controlled study INSIGHT 012 (NCT04910269) to compare the safety and efficacy of a single infusion of anti-COVID-19 hyperimmune immunoglobulin (hIVIG) versus placebo among adults with recently diagnosed SARS-CoV-2 infection who do not require hospitalization.

In the present study, in-vitro pseudovirus and live virus neutralization assays were used to assess the impact of SARS-CoV-2 variant spike mutations on neutralizing potency of COVID-HIG. These assays are valuable tools for monitoring the potential impact of variant mutations on efficacy of antibody therapeutics as well as vaccines/natural immunity.

To date, COVID-HIG (NP-028) has been shown to retain neutralizing potency against 20 full spike protein sequence SARS-CoV-2 pseudovirus variants including all currently classified VOC/VOI (Alpha, Beta, Gamma, Delta/Delta+, Eta, Iota, Kappa, Lambda, Mu as of Sept 2021) as well as 4 live virus variants (Alpha, Beta, Gamma, and Iota).


title: Antibody Evasion Properties of SARS-CoV-2 Omicron Sublineages

abstract: The identification of the Omicron variant (B.1.1.529.1 or BA.1) of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in Botswana in November 20211 immediately raised alarms due to the sheer number of mutations in the spike glycoprotein that could lead to striking antibody evasion. We2 and others3-6 recently reported results in this Journal confirming such a concern. Continuing surveillance of Omicron evolution has since revealed the rise in prevalence of two sublineages, BA.1 with an R346K mutation (BA.1+R346K) and B.1.1.529.2 (BA.2), with the latter containing 8 unique spike mutations while lacking 13 spike mutations found in BA.1. We therefore extended our studies to include antigenic characterization of these new sublineages. Polyclonal sera from patients infected by wild-type SARS-CoV-2 or recipients of current mRNA vaccines showed a substantial loss in neutralizing activity against both BA.1+R346K and BA.2, with drops comparable to that already reported for BA.12,3,5,6. These findings indicate that these three sublineages of Omicron are antigenically equidistant from the wild-type SARS-CoV-2 and thus similarly threaten the efficacies of current vaccines. BA.2 also exhibited marked resistance to 17 of 19 neutralizing monoclonal antibodies tested, including S309 (sotrovimab)7, which had retained appreciable activity against BA.1 and BA.1+R346K2-4,6. This new finding shows that no presently approved or authorized monoclonal antibody therapy could adequately cover all sublineages of the Omicron variant.

category: microbiology


title: Boosting with Omicron-matched or historical mRNA vaccines increases neutralizing antibody responses and protection against B.1.1.529 infection in mice

abstract: The B.1.1.529 Omicron variant jeopardizes vaccines designed with early pandemic spike antigens. Here, we evaluated in mice the protective activity of the Moderna mRNA-1273 vaccine against B.1.1.529 before or after boosting with preclinical mRNA-1273 or mRNA-1273.529, an Omicron-matched vaccine. Whereas two doses of mRNA-1273 vaccine induced high levels of serum neutralizing antibodies against historical WA1/2020 strains, levels were lower against B.1.1.529 and associated with infection and inflammation in the lung. A primary vaccination series with mRNA-1273.529 potently neutralized B.1.1.529 but showed limited inhibition of historical or other SARS-CoV-2 variants. However, boosting with mRNA-1273 or mRNA-1273.529 vaccines increased serum neutralizing titers and protection against B.1.1.529 infection. Nonetheless, the levels of inhibitory antibodies were higher, and viral burden and cytokines in the lung were slightly lower in mice given the Omicron-matched mRNA booster. Thus, in mice, boosting with mRNA-1273 or mRNA-1273.529 enhances protection against B.1.1.529 infection with limited differences in efficacy measured.


title: The mechanisms of catalysis and ligand binding for the SARS-CoV-2 NSP3 macrodomain from neutron and X-ray diffraction at room temperature

abstract: The NSP3 macrodomain of SARS CoV 2 (Mac1) removes ADP-ribosylation post-translational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the COVID-19 pandemic. Here, we determined neutron and X-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose-bound states. We characterize extensive solvation in the Mac1 active site, and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a re-evaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.

category: biochemistry


title: The mechanism of RNA capping by SARS-CoV-2

abstract: The SARS-CoV-2 RNA genome contains a 5-cap that facilitates translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made is not completely understood. Here, we reconstitute the SARS-CoV-2 7MeGpppA2-O-Me-RNA cap using virally encoded non-structural proteins (nsps). We show that the kinase-like NiRAN domain5 of nsp12 transfers RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers RNA to GDP, forming the cap core structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N-terminus of nsp9 and the kinase-like active site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.


title: Designing effective siRNAs to silence structural proteins associated genes of Indian SARS-CoV-2 strains: an in silico approach

abstract: SARS-CoV-2 is a highly transmissible and pathogenic coronavirus that first emerged in late 2019 and has since triggered a pandemic of acute respiratory disease named COVID-19 which poses a significant threat to all public health institutions in the absence of specific antiviral treatment. Since the outbreak began in March 2020, India has reported 4.77 lakh Coronavirus deaths, according to the World Health Organization (WHO). The innate RNA interference (RNAi) pathway, on the other hand, allows for the development of nucleic acid-based antiviral drugs in which complementary small interfering RNAs (siRNAs) mediate the post-transcriptional gene silencing (PTGS) of target mRNA. Therefore, in this current study, the potential of RNAi was harnessed to construct siRNA molecules that target the consensus regions of specific structural proteins associated genes of SARS-CoV-2, such as the envelope protein gene (E), membrane protein gene (M), nucleocapsid phosphoprotein gene (N), and surface glycoprotein gene (S) which are important for the viral pathogenesis. Conserved sequences of 811 SARS-CoV-2 strains from around India were collected to design 21 nucleotides long siRNA duplex based on various computational algorithms and parameters targeting E, M, N and S genes. The proposed siRNA molecules possessed sufficient nucleotide-based and other features for effective gene silencing and BLAST results revealed that siRNAs targets have no significant matches across the whole human genome and hence, siRNAs were found to have no off-target effects on the genome, ruling out the possibility of off-target silencing. Finally, out of 157 computationally identified siRNAs, only 4 effective siRNA molecules were selected for each target gene which is proposed to exert the best action based on GC content, free energy of folding, free energy of binding, melting temperature, heat capacity and molecular docking analysis with Human AGO2 protein. Our engineered siRNA candidates could be used as a genome-level therapeutic treatment against various sequenced SARS-CoV-2 strains in India. However, future applications will necessitate additional validations in vitro and in vivo animal models.


title: Computational study and design of effective siRNAs to silence structural proteins associated genes of Indian SARS-CoV-2 strains


title: Plasticity in structure and assembly of SARS-CoV-2 nucleocapsid protein

abstract: Worldwide SARS-CoV-2 sequencing efforts track emerging mutations in its spike protein, as well as characteristic mutations in other viral proteins. Besides their epidemiological importance, the observed SARS-CoV-2 sequences present an ensemble of viable protein variants, and thereby a source of information on viral protein structure and function. Charting the mutational landscape of the nucleocapsid (N) protein that facilitates viral assembly, we observe variability exceeding that of the spike protein, with more than 86% of residues that can be substituted, on average by 3-4 different amino acids. However, mutations exhibit an uneven distribution that tracks known structural features but also reveals highly protected stretches of unknown function. One of these conserved regions is in the central disordered linker proximal to the N-G215C mutation that has become dominant in the Delta variant, outcompeting G215 variants without further spike or N-protein substitutions. Structural models suggest that the G215C mutation stabilizes conserved transient helices in the disordered linker serving as protein-protein interaction interfaces. Comparing Delta variant N-protein to its ancestral version in biophysical experiments, we find a significantly more compact and less disordered structure. N-G215C exhibits substantially stronger self-association, shifting the unliganded protein from a dimeric to a tetrameric oligomeric state, which leads to enhanced co-assembly with nucleic acids. This suggests that the sequence variability of N-protein is mirrored by high plasticity of N-protein biophysical properties, which we hypothesize can be exploited by SARS-CoV-2 to achieve greater efficiency of viral assembly, and thereby enhanced infectivity.


title: Olverembatinib inhibits SARS-CoV-2-Omicron variant-mediated cytokine release

abstract:
The Omicron variant has become dominant in the U.S. and around the world. This variant is found to be 2-fold more infectious than the Delta variant, posing a significant threat of severe cases and death. We and others have recently reported that the N-terminus domain (NTD) of the SARS-CoV-2 of various variants is responsible for inducing cytokine release in human PBMCs. Here, we demonstrate that the NTD of the Omicron variant remains highly effective at inducing cytokine release in human PBMCs. Furthermore, we show that Ponatinib and a novel compound, Olverembatinib, are potent Omicron NTD-mediated cytokine release inhibitors. Target profiling revealed that Olverembatinib blocks most of the previously identified kinases responsible for cytokine release. Together, we propose that Ponatinib and Olverembatinib may represent an attractive therapeutic option for treating moderate to severe COVID-19 cases.

HIGHLIGHTSO_LIThe N-terminus domain (NTD) of the SARS-CoV-2 Omicron variant strongly induces multiple inflammatory molecules in PBMCs, unaffected by the mutations observed in the NTD.
C_LIO_LIThe cytokine release mediated by the Omicron variant is comparable to the Delta variant.
C_LIO_LIOlverembatinib, a clinical-stage multi-kinase inhibitor, potently inhibits Omicron NTD-mediated cytokine release.
C_LIO_LIOlverembatinib could relieve severe symptoms associated with COVID-19 Omicron and Delta variants.
C_LI


title: The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters

abstract: A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs.


title: Aerosol Formation During Processing of Potentially Infectious Samples on Roche Immunochemistry Analyzers (cobas e analyzers) and in an End-to-End Laboratory Workflow to Model SARS-CoV-2 Infection Risk for Laboratory Operators

abstract:
BackgroundThis study assessed formation of potentially infectious aerosols during processing of infectious samples in a real-world laboratory setting, which could then be applied in the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

MethodsThis two-part study assessed aerosol formation when using cobas e analyzers only and in an end-to-end laboratory workflow. To estimate aerosol formation, recombinant hepatitis B surface antigen (HBsAg) was used as a surrogate marker for infectious virus particles to evaluate the potential risk of SARS-CoV-2 infection to laboratory operators. Using the HBsAg model, air sampling was performed at different positions around the cobas e analyzers and in four scenarios reflecting critical handling and/or transport locations in an end-to-end laboratory workflow. Aerosol formation of HBsAg was quantified using the Elecsys(R) HBsAg II quant II assay. The model was then applied to a SARS-CoV-2 context using SARS-CoV-2 infection-specific parameters to calculate viral RNA copies.

ResultsFollowing application to SARS-CoV-2, the mean HBsAg uptake per hour when recalculated into viral RNA copies was 1.9 viral RNA copies across the cobas e analyzers and 0.87 viral RNA copies across all tested scenarios in an end-to-end laboratory workflow. This corresponds to a maximum aspiration rate of <16 viral RNA copies during an 8-hour shift when using cobas e analyzers and/or in an end-to-end laboratory workflow.

ConclusionsThe low production of marker-containing aerosol when using cobas e analyzers and in an end-to-end laboratory workflow is consistent with a remote risk of laboratory-acquired SARS-CoV-2 infection for laboratory operators.

SummaryThis study investigated the formation of potentially infectious aerosols during processing of infectious samples in a model using hepatitis B surface antigen (HBsAg) as a marker for infectious virus particles. The risk to laboratory operators of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was then inferred. Air sampling was performed around cobas e analyzers and in an end-to-end laboratory workflow, after which HBsAg was quantified and applied to SARS-CoV-2 using SARS-CoV-2 infection-specific parameters. The maximum aspiration rate of <16 viral RNA copies/8-hour shift, when applied to a SARS-CoV-2 context, poses a remote risk of SARS-CoV-2 infection to laboratory operators.


title: Spontaneous and ART-induced large offspring syndrome: similarities and differences in DNA methylome

abstract: Large/abnormal offspring syndrome (LOS/AOS) is a congenital overgrowth syndrome reported in ruminants produced by assisted reproduction (ART-LOS) which exhibit global disruption of the epigenome and transcriptome. LOS/AOS shares phenotypes and epigenotypes with the human congenital overgrowth condition Beckwith-Wiedemann syndrome. We have reported that LOS occurs spontaneously (SLOS), however, to date, no study has been conducted to determine if SLOS has the same methylome epimutations as ART-LOS. In this study, we performed whole genome bisulfite sequencing to examine global DNA methylation in SLOS and ART-LOS tissues. We observed unique patterns of global distribution of differentially methylated regions (DMRs) over different genomic contexts, such as promoters, CpG islands and surrounding regions, and repetitive sequences from different treatment groups. In addition, we identified hundreds of LOS-vulnerable DMRs across different cattle breeds when analyzing four idependent LOS experiments together. Specifically, there are 25 highly vulnerable DMRs that could potentially serve as molecular markers for the diagnosis of LOS, including at the promoters of DMRT2 and TBX18, at the imprinted gene bodies of IGF2R, PRDM8, and BLCAP/NNAT, and at multiple CpG islands. We also observed tissue-specific DNA methylation patterns between muscle and blood, and conservation of ART-induced DNA methylation changes between muscle and blood. We conclude that as in ART-LOS, alterations of the epigenome are involved in the etiology of SLOS. In addition, SLOS and ART-LOS share some similarities in methylome epimutations.

category: developmental biology


title: Functional substitutability of native herbivores by livestock for long-term soil carbon sequestration depends on microbial decomposers.

abstract:
The influence of native mammalian herbivores on ecosystems is declining as livestock have displaced them to become the dominant grazers across the world. Alongside rewilding, restoration, and conservation, it is now important to address whether livestock can assume the functional roles of displaced native herbivores in grasslands, savannas, and steppes. If yes, this has implications for the future of biodiversity, ecosystem functions and services, food-security, and other social and environmental dimensions. We test competing alternative hypotheses with a replicated, long-term, landscape-level experiment to ask whether livestock in the Trans-Himalayan ecosystem can match decadal-scale (2006-17) soil-C sequestration under native herbivores. Despite comparable settings, livestock were imperfect substitutes and attained three-fourths of soil-C stocks under native herbivores. We evaluate multiple lines of evidence surrounding this difference with a grazing-exclusion experiment (nutrient-cycling, microbial metabolism, and e-DNA), and asses their inter-relationships. Livestock and native herbivores differed in their effects on several soil microbial variables. Structural equation models showed that livestock alter soil microbial communities towards lower fungal abundance, which is detrimental for microbial carbon use efficiency (CUE), and ultimately also for soil-C. Herbivore-identity influenced ecosystem functions even when total amount of herbivory was comparable. Lags in soil-C storage can be addressed through microbial restoration and rewilding of soil. Therefore, the idea of functional substitutability must be leveraged alongside microbial rewilding to restore and conserve ecosystem functions. Together they can reconcile conflicting demands from food-security and ecosystem services in ways that have eluded conventional approaches to become an integral part of nature-based climate solutions.

Significance StatementGrazing by large mammalian herbivores affords an important natural climate solution via soil-C storage. But native grazers are progressively being replaced by domestic livestock to accommodate changing human diets. Even though livestock are now the dominant grazers around the world, it remains unclear whether they can be functional substitutes for native herbivores in sequestering soil-C. And if so, then to what extent? We find that livestock could only attain three-fourths of soil-C stocks under native herbivores, because they shift the microbial decomposer community in soil towards lower fungal abundance. This reduces microbial carbon use efficiency, and ultimately is detrimental for soil-C storage. So, microbial rewilding may help livestock production systems to emulate the ecosystem services provided by native herbivores.


title: Narrow transmission bottlenecks and limited within-host viral diversity during a SARS-CoV-2 outbreak on a fishing boat

abstract: The long-term evolution of viruses is ultimately due to viral mutants that arise within infected individuals and transmit to other individuals. Here we use deep sequencing to investigate the transmission of viral genetic variation among individuals during a SARS-CoV-2 outbreak that infected the vast majority of crew members on a fishing boat. We deep-sequenced nasal swabs to characterize the within-host viral population of infected crew members, using experimental duplicates and strict computational filters to ensure accurate variant calling. We find that within-host viral diversity is low in infected crew members. The mutations that did fix in some crew members during the outbreak are not observed at detectable frequencies in any of the sampled crew members in which they are not fixed, suggesting viral evolution involves occasional fixation of low-frequency mutations during transmission rather than persistent maintenance of within-host viral diversity. Overall, our results show that strong transmission bottlenecks dominate viral evolution even during a superspreading event with a very high attack rate.


title: Flavobacterium johnsoniae Tyrosine Ammonia Lyase (FjTAL) in-silico Structure Prediction and Molecular Docking to L-tyrosine, p-coumaric acid (pHCA) and Caffeic Acid

abstract: Responsible for the conversion of L-tyrosine to p-coumaric acid in Flavobacterium johnsoniae, FjTAL has drawn the attention of many biochemical engineers who wish to carry out a sustainable biosynthetic scheme for the production of aromatic compounds. In this study, with the aid of various computational tools, the secondary and tertiary structures of FjTAL have been predicted. The results suggest that FjTAL forms a homo-tetramer when active as a cytosolic enzyme and it is mostly consisted of alpha helices. With the aid of molecular docking, one can hypothesize that FjTAL is likely to bind to L-tyrosine, p-coumaric acid, and caffeic acid with a similar molecular mechanism and thus, p-coumaric acid and caffeic acid may exhibit a negative feedback response toward the enzyme and inhibit its activity competitively. Two distinct binding pockets have been discovered, one of which contains highly conserved residues among several species. The residues which form the prosthetic group 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) also emerge in the evolutionary conserved binding pocket. The other discovered cavity, could either be a second binding site for the ligands or simply an artifact of the molecular docking task.


title: Flavobacterium johnsoniae Tyrosine Ammonia Lyase (FjTAL) in-silico Structure Prediction and Molecular Docking to L-Tyrosine, p-Coumaric Acid (pHCA) and Caffeic Acid


title: The structure, binding, and function of a Notch transcription complex involving RBPJ and the epigenetic reader protein L3MBTL3

abstract: The highly conserved Notch pathway transmits signals between neighboring cells to elicit distinct downstream transcriptional programs. In given contexts, Notch is a major regulator of cell fate specification, proliferation, and apoptosis, such that aberrant Notch signaling leads to a pleiotropy of human diseases, including developmental disorders and cancers. The canonical pathway signals through the transcription factor CSL (RBPJ in mammals), which forms a transcriptional activation complex with the intracellular domain of the Notch receptor and the coactivator Mastermind. CSL can also function as a transcriptional repressor by forming complexes with one of several different corepressor proteins, such as FHL1 or SHARP in mammals and Hairless in Drosophila. Recently, we identified the malignant brain tumor (MBT) family member L3MBTL3 as a bona fide RBPJ binding corepressor that recruits the repressive lysine demethylase LSD1/KDM1A to Notch target genes. Here we define the RBPJ-interacting domain (RBP-ID) of L3MBTL3 and report the 2.06 [A] crystal structure of the complex formed between RBPJ, the RBP-ID of L3MBTL3 and DNA. The structure reveals the molecular interactions underlying L3MBTL3 complexation with RBPJ, which we comprehensively analyze with a series of L3MBTL3 and RBPJ mutations that span the binding interface. Compared to other RBPJ-binding proteins, we find that L3MBTL3 interacts with RBPJ via an unusual binding motif, which is sensitive to mutations throughout its RBPJ-interacting region. We also show that these disruptive mutations affect RBPJ and L3MBTL3 function in cells, providing further insights into Notch mediated transcriptional regulation.


title: Auto-Immunoproteomics Analysis of COVID-19 ICU Patients Revealed Increased Levels of Autoantibodies Related to Male Reproductive System

abstract:
The role of autoantibodies in coronavirus disease (COVID-19) complications is not yet fully understood. The current investigation screened two independent cohorts of 97 COVID-19 patients (Discovery (Disc) cohort from Qatar (n = 49) and Replication (Rep) cohort from New York (n = 48)) utilizing high-throughput KoRectly Expressed (KREX) immunome protein-array technology. Autoantibody responses to 57 proteins were significantly altered in the COVID-19 Disc cohort compared to healthy controls (P [≤] 0.05). The Rep cohort had altered autoantibody responses against 26 proteins compared to non-COVID-19 ICU patients that served as controls. Both cohorts showed substantial similarities (r2 = 0.73) and exhibited higher autoantibodies responses to numerous transcription factors, immunomodulatory proteins, and human disease markers. Analysis of the combined cohorts revealed elevated autoantibody responses against SPANXN4, STK25, ATF4, PRKD2, and CHMP3 proteins in COVID-19 patients. KREX analysis of the specific IgG autoantibody responses indicates that the targeted host proteins are supposedly increased in COVID-19 patients. The autoantigen-autoantibody response was cross-validated for SPANXN4 and STK25 proteins using Uniprot BLASTP and sequence alignment tools. SPANXN4 is essential for spermiogenesis and male fertility, which may predict a potential role for this protein in COVID-19 associated male reproductive tract complications and warrants further research.

Significance StatementCoronavirus disease (COVID-19), caused by the SARS-CoV-2 virus, has emerged as a global pandemic with a high morbidity rate and multiorgan complications. It is observed that the host immune system contributes to the varied responses to COVID-19 pathogenesis. Autoantibodies, immune system proteins that mistakenly target the bodys own tissue, may underlie some of this variation. We screened total IgG autoantibody responses against 1,318 human proteins in two COVID-19 patient cohorts. We observed several novel markers in COVID-19 patients that are associated with male fertility, such as sperm protein SPANXN4, STK25, and the apoptotic factor ATF4. Particularly, elevated levels of autoantibodies against the testicular tissue-specific protein SPANXN4 offer significant evidence of anticipating the protein role in COVID-19 associated male reproductive complications.


title: The efficacy of commercial decontamination agents differs between standardised test settings and real-world usage for a variety of bacterial species

abstract: Decontamination of surfaces and items plays an important role in reducing the spread of infectious microorganisms in many settings including hospitals and research institutes. Regardless of the location, appropriate decontamination procedures are required for maintaining biosafety and biosecurity. For example, effective decontamination of microbial cultures is essential to ensure proper biocontainment and safety within microbiological laboratories. To this end, many commercial decontamination agents are available which have been tested to a prescribed standard to substantiate their efficacy. However, these standardised tests are unlikely to accurately reflect all conditions encountered in medical and biomedical research. Despite this, laboratory workers and other users of decontamination agents may assume that all decontamination agents will work in all situations. We tested commonly used commercial decontamination agents against a range of bacterial species to determine their efficacy under real-world laboratory conditions. As each decontamination agent has a different recommended dilution for use, to compare their efficacy we calculated their  effective ratio which reflects the difference between the manufacturer-recommended dilution and the dilution needed to achieve decontamination under real-world laboratory conditions. Effective ratios above 1 indicate that the agent was active at a dilution more dilute than recommended whereas effective ratios lower than 1 indicate that the agent required a higher concentration than recommended. Our results show that the quaternary ammonium agents TriGene Advance and Chemgene HLD4L were the most active out of the agents tested, with biocidal activity measured at up to 64 times the recommended dilution. In contrast, hypochlorite (bleach) and Prevail (stabilised hydrogen peroxide) had the lowest effective ratios amongst the tested agents. In conclusion, our data suggest that not all decontamination agents will work at the recommended dilutions under real-world laboratory conditions. We recommend that the protocols for the use of decontamination agents are verified under the specific conditions required to ensure they are fit for purpose.


title: Termination factor Rho mediates transcriptional reprogramming of Bacillus subtilis stationary phase.

abstract: Reprogramming of gene expression during transition from exponential growth to stationary phase is crucial for bacterial survival. In the model Gram-positive bacterium Bacillus subtilis, this process is mainly governed by the activity of the global transcription regulators AbrB, CodY and Spo0A. We recently showed that the transcription termination factor Rho, known for its ubiquitous role in the inhibition of antisense transcription, is involved in Spo0A-mediated regulation of differentiation programs specific to the stationary phase in B. subtilis. To identify other aspects of the regulatory role of Rho during adaptation to starvation, we have constructed a B. subtilis strain that expresses rho at a relatively stable high level in order to circumvent its decrease occurring in the wild-type cells entering the stationary phase. We show that B. subtilis cells stably expressing Rho fail to sporulate and to develop genetic competence, which is largely, but not exclusively, due to abnormally low expression of the master regulator Spo0A. Moreover, in addition to a global decrease of antisense transcription, these cells exhibit genome-wide alterations of sense transcription. A significant part of these alterations affects genes from global regulatory networks of cellular adaptation to the stationary phase and reflects the attenuated de-repression of the AbrB and CodY regulons and the weakened stringent response. Accordingly, stabilization of Rho level reprograms stationary phase-specific physiology of B. subtilis cells, negatively affects cellular adaptation to nutrient limitations and alters cell-fate decision-making to such an extent that it blocks development of genetic competence and sporulation. Taken together, these results indicate that the activity of termination factor Rho constitutes a previously unknown layer of control over the stationary phase and post-exponential adaptive strategies in B. subtilis, from the adjustment of cellular metabolism to the activation of survival programs.