title: BirdFlow: Learning Seasonal Bird Movements from Citizen Science Data

abstract: Large-scale monitoring of seasonal animal movement is integral to science, conservation, and outreach. However, gathering representative movement data across entire species ranges is frequently intractable. Citizen science databases collect millions of animal observations throughout the year, but it is challenging to infer individual movement behavior solely from observational data. We present BO_SCPLOWIRDC_SCPLOWFO_SCPLOWLOWC_SCPLOW, a probabilistic modeling framework that draws on citizen science data from the eBird database to model the population flows of migratory birds. We apply the model to 11 species of North American birds, using GPS and satellite tracking data to tune and evaluate model performance. We show that BO_SCPLOWIRDC_SCPLOWFO_SCPLOWLOWC_SCPLOW models can accurately infer individual seasonal movement behavior directly from eBird relative abundance estimates. Supplementing the model with a sample of tracking data from wild birds improves performance. Researchers can extract a number of behavioral inferences from model results, including migration routes, timing, connectivty, and forecasts. The BO_SCPLOWIRDC_SCPLOWFO_SCPLOWLOWC_SCPLOW framework has the potential to advance migration ecology research, boost insights gained from direct tracking studies, and serve a number of applied functions in conservation, disease surveillance, aviation, and public outreach.


title: Coupling phenotype stability to growth rate overcomes limitations of bet-hedging strategies

abstract: Microbes in the wild face highly variable and unpredictable environments, and are naturally selected for their average growth rate across environments. Apart from using sensory-regulatory systems to adapt in a targeted manner to changing environments, microbes employ bet-hedging strategies where cells in an isogenic population switch stochastically between alternative phenotypes. Yet, bet-hedging suffers from a fundamental trade-off: increasing the phenotype switching rate increases the rate at which maladapted cells explore alternative phenotypes, but also increases the rate at which cells switch out of a well-adapted state. Consequently, it is currently believed that bet-hedging strategies are only effective when the number of possible phenotypes is limited and when environments last for sufficiently many generations. However, recent experimental results show that gene expression noise generally decreases with growth rate, suggesting that phenotype switching rates may systematically decrease with growth rate. We here show that such growth rate dependent stability (GRDS) can almost completely overcome the trade-off that limits bet-hedging, allowing for effective adaptation even when environments are diverse and change rapidly. GRDS allows cells to be more explorative when maladapted, and more phenotypically stable when well-adapted. We further show that even a small decrease in switching rates of faster growing phenotypes can substantially increase long-term fitness of bet-hedging strategies. Together, our results suggest that stochastic strategies may play an even bigger role for microbial adaptation than hitherto appreciated.

category: evolutionary biology


title: Climatic niche conservatism and ecological diversification in the Holarctic cold-dwelling butterfly genus Erebia

abstract: The evolution and distribution of alpine species have been modulated by their climatic niches. However, the relative roles of climatic niche conservatism promoting geographical speciation and of climatic niche diversification are poorly understood in diverse temperate groups. Here, we describe the evolution of climatic niche in a species rich butterfly genus, Erebia, which occurs across cold regions of the Holarctic and its diversity centre lies in European mountains. We inferred a nearly complete molecular phylogeny and modelled the climatic niche evolution using 25 thousand geo-referenced occurrence records. First, we reconstructed the evolution of the climatic niche. Second, we tested how the species climatic niche width changes across the occupied climate gradient and compared the two main Erebia clades, the European and the Asian clade. Third, we explored climatic niche overlaps among species. We revealed that the evolution of Erebia has been shaped by climatic niche conservatism, supported by a strong phylogenetic signal and niche overlap in sister species, promoting allopatric speciation and by ecological speciation driven by specialization of climatic niches. However, the two main clades evolved their climatic niches toward different local optima. In addition, niche width decreases in extreme conditions of low and high temperatures and species in the diverse European clade have narrower niches compared to the Asian clade. This may be related to the gradient of climate seasonality, with lower European seasonality favouring the evolution of narrower niches. Our findings, coupled with declining diversification rates through time, support a diversity-dependent radiation scenario.


title: When blue turns to grey - Paleogenomic insights into the evolutionary history and extinction of the blue antelope (Hippotragus leucophaeus)

abstract: The blue antelope (Hippotragus leucophaeus) is the only large African mammal to have become extinct in historical times, yet no nuclear genomic information is available for this species. A recent study showed that many alleged blue antelope museum specimens are either roan (H. equinus) or sable (H. niger) antelopes, further reducing the possibilities for obtaining genomic information for this extinct species. While the blue antelope has a rich fossil record from South Africa, climatic conditions in the region are unfavourable to the preservation of ancient DNA. Nevertheless, we recovered two blue antelope draft genomes, one at 3.4x coverage from a historical specimen (~19th century) and one at 2.1x coverage from a fossil specimen dating to 9,800-9,300 cal BP, making it currently the oldest paleogenome from Africa. Phylogenomics show that blue and sable antelope are sister species, confirming previous mitogenomic results, and demonstrate ancient gene flow from roan into blue antelope. We show that blue antelope genomic diversity was much lower than in roan and sable antelopes, indicative of a low population size since at least the early Holocene. This supports observations from the fossil record documenting major decreases in the abundance of blue antelope after the Pleistocene-Holocene transition. Finally, the persistence of this species throughout the Holocene despite low population size suggests that colonial-era human impact was likely a major factor in the blue antelopes extinction.


title: 3D-equivariant graph neural networks for protein model quality assessment

abstract:
MotivationQuality assessment of predicted protein tertiary structure models plays an important role in ranking and using them. With the recent development of deep learning end-to-end protein structure prediction techniques of generating highly confident tertiary structures for most proteins, it is important to explore corresponding quality assessment strategies to evaluate and select the structural models predicted by them since these models have better quality and different properties than the models predicted by traditional tertiary structure prediction methods.

ResultsWe develop EnQA, a novel graph-based 3D-equivariant neural network method that is equivariant to rotation and translation of 3D objects to estimate the accuracy of protein structural models by leveraging the structural features acquired from the state-of-the-art tertiary structure prediction method - AlphaFold2. We train and test the method on both traditional model datasets (e.g., the datasets of the Critical Assessment of Techniques for Protein Structure Prediction (CASP)) and a new dataset of high-quality structural models predicted only by AlphaFold2 for the proteins whose experimental structures were released recently. Our approach achieves state-of-the-art performance on protein structural models predicted by both traditional protein structure prediction methods and the latest end-to-end deep learning method - AlphaFold2. It performs even better than the model quality assessment scores provided by AlphaFold2 itself. The results illustrate the 3D-equivariant graph neural network is a promising approach to the evaluation of protein structural models. AlphaFold2 features are important for improving protein model quality assessment and are complimentary with the geometric property features extracted from structural models.

AvailabilityThe source code is available at https://github.com/BioinfoMachineLearning/EnQA.

Contactchengji@missouri.edu

Supplementary informationSupplementary data are available.

category: bioinformatics


title: Inflammation induced Vascular Smooth Muscle Cell expressing Angiopoietin-like 2 to promote the development of abdominal aortic aneurysm

abstract:
ObjectiveThe objective of this study is to investigate the expression of angiopoietin-like protein 2 (Angptl2) in abdominal aortic aneurysm (AAA) and the expression of Angptl2 in vascular smooth muscle cells (VSMC) under inflammation stimuli.

MethodsThe mouse AAA model was induced by applying calcium chloride method based the literature and the expression of Angptl2 in AAA was investigated by immunohistochemical staining. The primary VSMCs were cultured and identified following with inflammatory stimuli by Escherichia coli lipopolysaccharide (LPS) for 30 minutes and 24 hours. The expression of Angptl2 were assessed by RT-qPCR and ELISA.

ResultsThe mouse AAA model was successfully established after being induced by calcium chloride. The inflammatory cell infiltration in the arterial tissue of the mice in the experimental group was obvious, and the elastic fiber layer was thinned and broken. Immunohistochemical staining showed that the expression of Angptl2 in AAA was up-regulated; After LPS treatment, both the mRNA and protein expressions of Angptl2 were significantly up-regulated in VSMCs in the experimental group compared with control group.

ConclusionAngptl2 expressed in VSMCs promotes the development of calcium chloride induced AAA.

category: pathology


title: Identification of two bZIP transcription factors that regulate development of pavement and trichome cells in Arabidopsis thaliana by single-cell RNA-sequencing

abstract:
Epidermal cells are the main avenue for signal and material exchange between plants and the environment. Leaf epidermal cells primarily include pavement cells (PCs), guard cells, and trichomes cells (TCs), which differentiate from protodermal cells or meristemoids. The development and distribution of different epidermal cells are tightly regulated by a complex transcriptional regulatory network mediated by phytohormones, including jasmonic acid (JA), and transcription factors. Understanding how the fate of leaf epidermal cells is determined, however, is still largely unknown due to the diversity of cell types and the complexity of its regulation. Here, we characterized the transcriptional profiles of epidermal cells in 3-day-old true leaves of Arabidopsis thaliana using single-cell RNA-sequencing. We identified two genes encoding BASIC LEUCINE-ZIPPER (bZIP) transcription factors, namely the bZIP25 and bZIP53, which are highly expressed in PCs and early-stage meristemoid cells. Densities of PCs and TCs were found to increase and decrease, respectively, in bzip25 and bzip53 mutants, compared with wild-type plants. This trend was more pronounced in the presence of JA, suggesting that these transcription factors regulate the development of TCs and PCs in response to JA.

IN A NUTSHELLO_ST_ABSBackgroundC_ST_ABSLeaf epidermal cells, comprised of trichome cells (TCs), guard cells (GCs), and pavement cells (PCs), are responsible for exchanging materials and information between plants and the surrounding aerial environment. Many genes have been identified in Arabidopsis thaliana and confirmed to be involved in the initiation and differentiation of TCs and PCs. The fate determination of TCs and PCs is tightly regulated by positive and negative regulators at the cellular level. The precise underlying molecular mechanisms responsible for the fate determination of TCs and PCs, however, are still unclear at this time.

QuestionWhat are the transcriptomic profiles of different leaf epidermal cell types? Can we dissect the genes that are specifically expressed in certain epidermal cell types? What kinds of transcription factors are involved in regulating the fate determination of TCs and PCs?

FindingsWe performed single cell RNA-seq to investigate the transcriptomic profiles of different leaf epidermal cell types and identified differentially expressed genes in each cell type. We found that genes that are involved in jasmonic acid signaling are highly expressed in early-stage meristemoid (EM) cells which can act as the precursor of PCs and perhaps of TCs. To investigate the regulatory mechanisms underlying EM development, we identified the transcription factors (TFs) in EM cells and found that two bZIP TF genes, bZIP25 and bZIP53, are highly expressed in EMs. Further analyses of these two genes using both loss-of-function and gain-of-function approaches indicated that bZIP25 and bZIP53 are functionally involved in promoting trichome formation but inhibit pavement cell development in response to jasmonic acid.

Next stepsBesides of bZIP25 and bZIP53, we also identified other key genes, for example FES1B, in leaf epidermal cells. Our next step will be to explore the regulation of other key genes involved in the fate determination of different cell types in leaf epidermis.

category: plant biology


title: Mutation of Arabidopsis SME1 and Sm core assembly improves oxidative stress resilience

abstract: Alternative splicing is a key posttranscriptional gene regulatory process, acting in diverse adaptive and basal plant processes. Splicing of precursor-messenger RNA (pre-mRNA) is catalyzed by a dynamic ribonucleoprotein complex, designated the spliceosome. In a suppressor screen, we identified a nonsense mutation in the Sm protein SME1 to alleviate photorespiratory H2O2-dependent cell death in catalase deficient plants. Similar attenuation of cell death was observed upon chemical inhibition of the spliceosome, suggesting pre-mRNA splicing inhibition to be responsible for the observed cell death alleviation. Furthermore, the sme1-2 mutants showed increased tolerance to the reactive oxygen species inducing herbicide methyl viologen. Both an mRNA-seq and shotgun proteomic analysis in sme1-2 mutants displayed a constitutive molecular stress response, together with extensive alterations in pre-mRNA splicing of transcripts encoding metabolic enzymes and RNA binding proteins, even under unstressed conditions. Using SME1 as a bait to identify protein interactors, we provide experimental evidence for almost 50 homologs of mammalian spliceosome-associated protein to reside in the Arabidopsis thaliana spliceosome complexes and propose roles in pre-mRNA splicing for four uncharacterized plant proteins. Furthermore, like in sme1-2, a mutant in the Sm core assembly protein ICLN resulted in a decreased sensitivity to methyl viologen. Taken together, these data show that both a perturbed Sm core composition and assembly results in the activation of a defense response and enhanced resilience to oxidative stress.


title: Optimal enzyme utilization suggests concentrations and thermodynamics favor condition-specific saturations and binding mechanisms

abstract: Understanding the dynamic responses of living cells upon genetic and environmental perturbations is crucial to decipher the metabolic functions of organisms. The rates of enzymatic reactions and their evolution are key to this understanding, as metabolic fluxes are limited by enzymatic activity. In this work, we investigate the optimal modes of operations for enzymes with regard that the evolutionary pressure drives enzyme kinetics toward increased catalytic efficiency. We use an efficient mixed-integer formulation to decipher the principles of optimal catalytic properties at various operating points. Our framework allows assessing the distribution of the thermodynamic forces and enzyme states, providing detailed insight into the mode of operation. Our results confirm earlier theoretical studies on the optimal kinetic design using a reversible Michaelis-Menten mechanism. The results further explored the optimal modes of operation for random-ordered multi-substrate mechanisms. We show that optimal enzyme utilization is achieved by unique or alternative modes of operations depending on the reactants concentrations. Our novel formulation allows investigating the optimal catalytic properties of all enzyme mechanisms with known elementary reactions. We propose that our novel framework provides the means to guide and evaluate directed evolution studies and estimate the limits of the direct evolution of enzymes.

category: systems biology


title: Estimating Brain Functional Connectivity from Common Subspace Mapping Between Structural and Functional Connectomes

abstract: Understanding the connection between the brains structural connectivity and its functional connectivity is of immense interest in computational neuroscience. Although some studies have suggested that whole brain functional connectivity is shaped by the underlying structure, the rule by which anatomy constrains brain dynamics remains an open question. In this work, we introduce a computational framework that identifies a joint subspace of eigenmodes for both functional and structural connectomes. We found that a small number of those eigenmodes are sufficient to reconstruct functional connectivity from the structural connectome, thus serving as low-dimensional basis function set. We then develop an algorithm that can estimate the functional eigenspectrum in this joint space from the structural eigenspectrum. By concurrently estimating the joint eigenmodes and the functional eigenspectrum, we can reconstruct a given subjects functional connectivity from their structural connectome. We perform elaborate experiments and demonstrate that the proposed algorithm for estimating function connectivity from the structural connectome using joint space eigenmodes is superior to all other existing benchmark methods.

category: neuroscience


title: Spatial reasoning via recurrent neural dynamics in mouse retrosplenial cortex

abstract: From visual perception to language, sensory stimuli change their meaning depending on prior experience. Recurrent neural dynamics can interpret stimuli based on externally cued context, but it is unknown whether similar dynamics can compute and employ internal hypotheses to resolve ambiguities. Here, we show that mouse retrosplenial cortex (RSC) can form hypotheses over time and perform spatial reasoning through recurrent dynamics. In our task, mice navigated using ambiguous landmarks that are identified through their mutual spatial relationship, requiring sequential refinement of hypotheses. Neurons in RSC and in artificial neural networks encoded mixtures of hypotheses, location, and sensory information, and were constrained by robust low dimensional dynamics. RSC encoded hypotheses as locations in activity space with divergent trajectories for identical sensory inputs, enabling their correct interpretation. Our results indicate that interactions between internal hypotheses and external sensory data in recurrent circuits can provide a substrate for complex sequential cognitive reasoning.


title: Axonal plasticity in response to active forces generated through magnetic nano-pulling

abstract: Mechanical force is crucial in guiding axon outgrowth, before and after synapse formation. This process is referred to as "stretch-growth". However, how neurons transduce mechanical inputs into signaling pathways remains poorly understood. Another open question is how stretch-growth is coupled in time with the intercalated addition of new mass along the entire axon. Here, we demonstrate that active mechanical force generated by magnetic nano-pulling induces a remodeling of the axonal cytoskeleton. Specifically, the increase in the axonal density of microtubules leads to an accumulation of organelles and signaling vesicles which, in turn, promotes local translation by increasing the probability of assembly of the "translation factories". The modulation of axonal transport and local translation sustains enhanced axon outgrowth and synapse maturation.


title: Carbon dioxide regulates Mycobacterium tuberculosis PhoPR signaling and virulence

abstract: The Mycobacterium tuberculosis (Mtb) two-component regulatory system, PhoPR, is implicated in pH-sensing within the macrophage because it is strongly induced by acidic pH both in vitro and the macrophage phagosome. The carbonic anhydrase (CA) inhibitor ethoxzolamide (ETZ) inhibits PhoPR signaling supporting the hypothesis that CO2 may also play a role in regulating PhoPR. Here, we show that increasing CO2 concentration induces PhoPR signaling, and its induction is independent of medium pH. We also show that at acidic pH 5.7, a normally strong inducer of PhoPR signaling, that increasing CO2 from 0.5% to 5% further induces the pathway. Based on these findings, we propose that PhoPR functions as a CO2 sensor. Mtb has three CA (CanA, CanB, and CanC) and using CRISPR interference knockdowns and gene deletion mutants, we assessed which CAs regulate PhoPR signaling and virulence. We first examined if CA played a role in Mtb pathogenesis and observed that CanB was required for virulence in macrophages, where the knockdown strain had ~1 log reduction in virulence. To further define the interplay of CO2 and Mtb signaling, we conducted transcriptional profiling experiments at varying pH and CO2 concentrations. As hypothesized, we observed the induction of PhoPR at acidic pH is dependent on CO2 concentration, with a subset of core PhoPR regulon genes dependent on both 5% CO2 and acidic pH for their induction, including expression of the ESX-1 secretion system. Transcriptional profiling also revealed core CO2 responsive genes that were differentially expressed independently of the PhoPR regulon or the acidic pH-inducible regulon. Notably, genes regulated by a second two component regulatory system, TrcRS, are associated with adaptation to changes in CO2.

category: microbiology


title: Avian H7N9 influenza viruses are evolutionarily constrained by stochastic processes during replication and transmission in mammals

abstract: H7N9 avian influenza viruses (AIV) have caused over 1,500 documented human infections since emerging in 2013. Although wild type H7N9 AIV can transmit by respiratory droplets in ferrets, they have not yet caused widespread outbreaks in humans. Previous studies have revealed molecular determinants of H7N9 AIV virus host-switching, but little is known about potential evolutionary constraints on this process. Here we compare patterns of sequence evolution for H7N9 AIV and mammalian H1N1 viruses during replication and transmission in ferrets. We show that three main factors - purifying selection, stochasticity, and very narrow transmission bottlenecks - combine to severely constrain the ability of H7N9 AIV to effectively adapt to mammalian hosts in isolated, acute spillover events. We find rare evidence of natural selection favoring new or mammalian-adapting mutations within ferrets, but no evidence of natural selection acting during transmission. We conclude that human-adapted H7N9 viruses are unlikely to emerge during typical spillover infections. Our findings are instead consistent with a model in which the emergence of a human-transmissible virus would be a rare and unpredictable, though highly consequential, "jackpot" event. Strategies to limit the total number of spillover infections will limit opportunities for the virus to win this evolutionary lottery.


title: From individual behaviors to collective outcomes: fruiting body formation in Dictyostelium as a group-level phenotype

abstract: Collective phenotypes, which arise from the interactions among individuals, can be important for the evolution of higher levels of biological organization. However, how a groups composition determines its collective phenotype remains poorly understood. When starved, cells of the social amoeba Dictyostelium discoideum cooperate to build a multicellular fruiting body, and the morphology of the fruiting body is likely advantageous to the surviving spores. We assessed how the number of strains, as well as their genetic and geographic relationships to one another, impact the groups morphology and productivity. We find that some strains consistently enhance or detract from the productivity of their groups, regardless of the identity of the other group members. We also detect extensive pairwise and higher-order genotype interactions, which collectively have a large influence on the group phenotype. Whereas previous work in Dictyostelium has focused almost exclusively on whether spore production is equitable when strains cooperate to form multicellular fruiting bodies, our results suggest a previously unrecognized impact of chimeric co-development on the group phenotype. Our results demonstrate how interactions among members of a group influence collective phenotypes and how group phenotypes might in turn impact selection on the individual.


title: An Efficient CRISPR-Cas9 DNA Editing Methodology Applicable for iPSC Disease Modelling

abstract: The capability to generate induced pluripotent cell (iPSC) lines, in combination with the CRISPR-Cas9 DNA editing technology, offers great promise to understand the underlying genetic mechanisms of human disease. However, technical impediments including, but not limited to, low transfection efficiency, single-cell survival, and high clonal heterogeneity, limit the potential of these techniques. Here we provide an efficient methodology addressing these challenges, resulting in high transfection efficiency exceeding 97% with an increased single cell clone survival rate (up to 70%). These enhancements were accompanied by a high editing efficiency in the range of 48.6 to 57.5%, comparable to existing methods.

category: genetics


title: Adding gene transcripts into genomic prediction improves accuracy and reveals sampling time dependence

abstract: Recent developments allowed generating multiple high quality  omics data that could increase predictive performance of genomic prediction for phenotypes and genetic merit in animals and plants. Here we have assessed the performance of parametric and non-parametric models that leverage transcriptomics in genomic prediction for 13 complex traits recorded in 478 animals from an outbred mouse population. Parametric models were implemented using best linear unbiased prediction (BLUP), while non-parametric models were implemented using the gradient boosting machine algorithm (GBM). We also propose a new model named GTCBLUP that aims to remove between-omics-layer covariance from predictors, whereas its counterpart GTBLUP does not do that. While GBM models captured more phenotypic variation, their predictive performance did not exceed the BLUP models for most traits. Models leveraging gene transcripts captured higher proportions of the phenotypic variance for almost all traits when these were measured closer to the moment of measuring gene transcripts in the liver. In most cases, the combination of layers was not able to outperform the best single-omics models to predict phenotypes. Using only gene transcripts, the GBM model was able to outperform BLUP for most traits except body weight, but the same pattern was not observed when using both SNP genotypes and gene transcripts. Although the GTCBLUP model was not able to produce the most accurate phenotypic predictions, it showed highest accuracies for breeding values for 9 out of 13 traits. We recommend using the GTBLUP model for prediction of phenotypes and using the GTCBLUP for prediction of breeding values.


title: Convolutional networks for supervised mining of molecular patterns within cellular context

abstract: Cryo-electron tomograms capture a wealth of structural information on the molecular constituents of cells and tissues. We present DeePiCt (Deep Picker in Context), an open-source deep-learning framework for supervised structure segmentation and macromolecular complex localization in cellular cryo-electron tomography. To train and benchmark DeePiCt on experimental data, we comprehensively annotated 20 tomograms of Schizosaccharomyces pombe for ribosomes, fatty acid synthases, membranes, nuclear pore complexes, organelles and cytosol. By comparing our method to state-of-the-art approaches on this dataset, we show its unique ability to identify low-abundance and low-density complexes. We use DeePiCt to study compositionally-distinct subpopulations of cellular ribosomes, with emphasis on their contextual association with mitochondria and the endoplasmic reticulum. Finally, by applying pre-trained networks to a HeLa cell dataset, we demonstrate that DeePiCt achieves high-quality predictions in unseen datasets from different biological species in a matter of minutes. The comprehensively annotated experimental data and pre-trained networks are provided for immediate exploitation by the community.


title: Validation of genetic variants from NGS data using Deep Convolutional Neural Networks

abstract: Accurate somatic variant calling from next-generation sequencing data is one most important tasks in personalised cancer therapy. The sophistication of the available technologies is ever-increasing, yet, manual candidate refinement is still a necessary step in state-of-the-art processing pipelines. This limits reproducibility and introduces a bottleneck with respect to scalability. We demonstrate that the validation of genetic variants can be improved using a machine learning approach resting on a Convolutional Neural Network, trained using existing human annotation. In contrast to existing approaches, we introduce a way in which contextual data from sequencing tracks can be included into the automated assessment. A rigorous evaluation shows that the resulting model is robust and performs on par with trained researchers following published standard operating procedure.


title: Origin of Protein Quake: Energy Waves Conducted by a Precise Mechanical Machine

abstract: A long-standing challenge in protein biophysics is to understand protein quake in myoglobin--the structural dynamics responsible for redistributing the excess heme energy after photolysis. Despite extensive efforts, the molecular mechanism of this process remains elusive. Using the energy flow theory, we uncovered a fundamental new phenomenon: the heme energy is redistributed by energy waves with a ubiquitous fundamental frequency and two overtones. Energy waves emanate from the heme into the myoglobin backbone via a conduit of five consecutive dihedrals of the proximal histidine, then travel quickly along the backbone to reach sidechains across the protein. This mechanism is far more effective than the diffusion-based mechanism from previous studies because waves are systematic while diffusion is random. To propagate energy waves, coordinates must cooperate, resulting in collective modes that are singular vectors of the generalized work functional. These modes show task partitioning: a handful of high-energy modes generate large-scale breathing motion, which loosens up the protein matrix to enable hundreds of low-energy vibrational modes for energy transduction.

category: biophysics


title: Acute myeloid leukemia expresses a specific group of olfactory receptors

abstract: Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Although new drugs for specific molecular subsets of AML have recently emerged, the 5-year overall survival rate is still approximately 25%. The treatment options for AML have remained stagnant for decades, and novel molecular targets for this disease are needed. Olfactory receptors (ORs) are seven transmembrane G-protein coupled receptors preferentially expressed in sensory neurons, in which they play a critical role in recognizing odorant molecules. Recent studies have revealed ectopic expression and putative function of ORs in nonolfactory tissues and pathologies, including AML. Here, we comprehensively investigated OR expression in 151 AML samples, 51 healthy tissues (approximately 11,200 samples), and 15 other cancer types (6,400 samples). Our analyses identified a group of 19 ORs with a distinct and major expression pattern in AML. The expression of these ORs was experimentally validated in an independent set of AML samples and cell lines. We also identified an OR signature with prognostic value for AML patients. Finally, we identified cancer-related genes that were coexpressed with the ORs in the AML samples. In summary, we conducted a high-throughput computational study to identify ORs that can be used as novel biomarkers for the diagnosis of AML and as potential drug targets. The same approach may be used to investigate OR expression in other types of cancer.

category: cancer biology


title: The effect of biohanin A isolated from the callus culture of meadow clover (Trifolium pratense L.) on the life expectancy and survival of Caenorhabditis elegans

abstract:
Oxidative stress, in which healthy aging is limited, is a problem, since in the process there is an uncontrolled production of reactive radicals that negatively affect cells. Heat stress is closely related to oxidative stress, as it stimulates the production of radicals. Meadow clover (Trifolium pratense L.) is a promising source of isoflavonoids that have various positive effects on the body. The aim of the work is to study the effect of biohanin A, isolated from Trifolium pratense L. callus cultures, on the life expectancy of the C. elegans model organism, as well as its survival under oxidative and thermal stress.

It was found that biohanin A increased the lifespan of C. elegans worms. The best concentration was determined - 100 microns, at which the survival rate of nematodes after 61 days of incubation was 16.0%. After 48 hours, the survival rate of worms was the highest (87.1%) at a concentration of biohanin A of 200 microns. Other concentrations 10, 50, 100 (81,2, 83,0, 82,4 %) they also showed a higher survival rate compared to the control (74.0%). In addition, the survival rate under heat stress of C. elegans was higher by 12.0; 8.4; 4.0% compared with the control. Thus, the results show the antioxidant potential of biohanin A from plant material under oxidative and thermal stress. The substance also had a positive effect on the lifespan of C. elegans.


title: The effect of trans-cinnamic acid isolated from the root cultures of the Baikal skullcap (Scutellaria baicalensis) on the life expectancy and survival of Caenorhabditis elegans

abstract: Oxidative stress is an increase in reactive oxygen species, which are signaling molecules of various pathologies in the body of living beings. Baikal skullcap (Scutellaria baicalensis) is a valuable source of biologically active substances with various pharmacological activity. The aim of the study is to study the effect of trans-cinnamic acid isolated from root cultures of Scutellaria baicalensis on the lifespan of a model organism of C. elegans, as well as its survival under conditions of oxidative and thermal stress. It was shown that trans-cinnamic acid increased the lifespan of nematodes, while the best concentration of phytomaterials was 50 microns. In addition, all tested concentrations (10-200) had a positive effect on nematodes under oxidative stress caused by paraquat. No positive effect of trans-cinnamic acid was detected during thermal exposure. In general, the results show the antioxidant potential of trans-cinnamic acid from plant material under oxidative stress, as well as the positive effect of the substance on the lifespan of C. elegans.


title: Evolution of cell size control is canalized towards adders or sizers by cell cycle structure and selective pressures

abstract: Cell size is controlled to be within a specific range to support physiological function. To control their size, cells use diverse mechanisms ranging from  sizers, in which differences in cell size are compensated for in a single cell division cycle, to  adders, in which a constant amount of cell growth occurs in each cell cycle. This diversity raises the question why a particular cell would implement one rather than another mechanism? To address this question, we performed a series of simulations evolving cell size control networks. The size control mechanism that evolved was influenced by both cell cycle structure and specific selection pressures. Moreover, evolved networks recapitulated known size control properties of naturally occurring networks. If the mechanism is based on a G1 size control and an S/G2/M timer, as found for budding yeast and some human cells, adders likely evolve. But, if the G1 phase is significantly longer than the S/G2/M phase, as is often the case in mammalian cells in vivo, sizers become more likely. Sizers also evolve when the cell cycle structure is inverted so that G1 is a timer, while S/G2/M performs size control, as is the case for the fission yeast S. pombe. For some size control networks, cell size consistently decreases in each cycle until a burst of cell cycle inhibitor drives an extended G1 phase much like the cell division cycle of the green algae Chlamydomonas. That these size control networks evolved such self-organized criticality shows how the evolution of complex systems can drive the emergence of critical processes.


title: Radioactive Contamination in Feral Dogs in the Chernobyl Exclusion Zone: Population Body-Burden Survey and Implications for Human Radiation Exposure

abstract: This report describes a two-year effort to survey the internal 137Cs and external {beta}-emitter contamination present in the feral dog population near the Chernobyl nuclear power plant (ChNPP) site, and to quantify the potential for human radiation exposure from this contamination. This work was performed as an integral part of the radiation safety and control procedures of an animal welfare oriented trap-neuter-release (TNR) program. The measurement program employed handheld {beta}-sensitive probes, and a simple whole-body counter to measure internal 137Cs burden during post-surgical observation and recovery. External {beta} contamination surveys performed during intake showed that 21/288 animals had significant, removable external contamination. Measurements with the whole-body counter indicated internal 137Cs body burdens ranging from undetectable (minimum detection level [~]100 Bq/kg in 2017, [~] 30 Bq/kg in 2018) to approximately 30,000 Bq/kg. A total of 33 animals had 137Cs body-burdens above 1 kBq/kg. We observe that internal contamination levels are positively correlated with capture locations within ChNPP boundaries. The large variation in the 137Cs concentration in these animals is not well-understood, could be due to prey selection, access to human food scraps, or extended residence in highly contaminated areas. These internally-contaminated animals are unlikely to pose an exposure hazard despite their large body-burdens due to their limited exposure to humans. However, the small minority of animals with external contamination may pose a contamination hazard to workers, tourists, and others interacting with the dogs, as evidenced by total quantity of removable activity and incidents of transfer to materials used in animal capture.

category: zoology


title: Defining tertiary sulci in lateral prefrontal cortex in chimpanzees using human predictions

abstract: Similarities and differences in brain structure and function across species is of major interest in systems neuroscience, comparative biology, and brain mapping. Recently, increased emphasis has been placed on tertiary sulci, which are shallow indentations of the cerebral cortex that appear last in gestation, continue to develop after birth, and are largely either human- or hominoid-specific. While tertiary sulcal morphology in lateral prefrontal cortex (LPFC) has been linked to functional representations and cognition in humans, it is presently unknown if LPFC tertiary sulci also exist in non-human hominoids. To fill this gap in knowledge, we leveraged two freely available multimodal datasets to address the following main question: Can LPFC tertiary sulci be defined in chimpanzee cortical surfaces from human predictions? We found that 1-3 components of the posterior middle frontal sulcus (pmfs) in the posterior middle frontal gyrus are identifiable in nearly all chimpanzee hemispheres. In stark contrast to the consistency of the pmfs components, we could only identify components of the paraintermediate frontal sulcus (pimfs) in two chimpanzee hemispheres. LPFC tertiary sulci were relatively smaller and shallower in chimpanzees compared to humans. In both species, two of the pmfs components were deeper in the right compared to the left hemisphere. As these results have direct implications for future studies interested in the functional and cognitive role of LPFC tertiary sulci across species, we share probabilistic predictions of the three pmfs components to guide the definitions of these sulci in future studies.


title: Tuned inhibitory firing rate and connection weights as emergent network properties

abstract: Excitatory cortical neurons show clear tuning to stimulus features, but the tuning properties of inhibitory neurons are ambiguous and have been the subject of a long debate. While inhibitory neurons have been considered to be largely untuned [1-4], recent studies show that some parvalbumin expressing (PV) neurons do show feature selectivity and participate in co-tuned subnetworks with pyramidal cells in which PV cells show high response similarity to the excitatory (E) neurons [5, 6]. Given shared input from layer 4 that drives feature tuning in excitatory subnetworks, we demonstrate that homeostatic regulation of postsynaptic firing rate governing the synaptic dynamics of the connections from PV to E cells, in combination with heterogeneity in the excitatory postsynaptic potentials (EPSP) that impinge on PV cells, results in the self-organization of PV subnetworks. We reconcile different experimental findings by showing that feature tuning of PV cells is an emerging network property that may be driven by synaptic heterogeneity, and can be inferred using population-level measures, while pairwise individual-level measures may fail to reveal inhibitory tuning. We show that such co-tuning can enhance network stability at the cost of response salience.


title: Conserved cell-type specific signature of resilience to Alzheimer's disease nominates role for excitatory cortical neurons

abstract: Alzheimers disease (AD), the leading cause of dementia, affects millions of people worldwide. With no disease-modifying medication currently available, the human toll and economic costs are rising rapidly. Under current standards, a patient is diagnosed with AD when both cognitive decline and pathology (amyloid plaques and neurofibrillary tangles) are present. Remarkably, some individuals who have AD pathology remain cognitively normal. Uncovering factors that lead to "cognitive resilience" to AD is a promising path to create new targets for therapies. However, technical challenges discovering novel human resilience factors limit testing, validation, and nomination of novel drugs for AD. In this study, we use single-nuclear transcriptional profiles of postmortem cortex from human individuals with high AD pathology who were either cognitively normal (resilient) or cognitively impaired (susceptible) at time of death, as well as mouse strains that parallel these differences in cognition with high amyloid. Our cross-species discovery approach highlights a novel role for excitatory layer 4/5 cortical neurons in promoting cognitive resilience to AD, and nominates several resilience genes that include ATP1A1, GABRB1, PTK2, and ROCK2. Nominated resilience genes were tested for replication in orthogonal data sets and confirmed to be correlated with cognitive resilience. Additionally, we identified several potential mechanisms of resilience, including regulation of membrane potential, axonal and dendritic growth, and general increase of protein cycle, potentially of membrane proteins. Because our discovery of resilience-associated genes in layer 4/5 cortical neurons originates from an integrated human and mouse transcriptomic space from susceptible and resilient individuals, we are positioned to test causality and perform mechanistic, validation, and pre-clinical studies in our human-relevant AD-BXD mouse panel.


title: Reduced ventral attention network connectivity is linked to the accelerated maturation of adult-like cortical organization in childhood

abstract: Across the transition from childhood to adolescence the human brain experiences profound functional changes, shifting from an organizational framework anchored within somatosensory/motor and visual regions into one that is balanced through interactions with later-maturing aspects of association cortex. Here, we provide evidence linking this profile of large-scale functional reorganization to developmental shifts in attention network connectivity. We demonstrate that changes in functional connectivity between childhood and adolescence are preferential to the salience/ventral attention network. Excluding these network-linked vertices from analysis effectively removes developmental changes in cortical organization, while connectivity within the ventral attention network predicts intellectual ability. Children with low ventral attention network connectivity exhibit adolescent-like topographical profiles, suggesting attentional systems may be critically important for understanding how brain functions are refined across development. These data highlight a core role for the ventral attention network in supporting age-dependent shifts in the macroscale organization of cortex across development.


title: Polar growth protein Wag31 undergoes changes in homo-oligomeric network topology, and has distinct functions at both cell poles and the septum

abstract:
Mycobacterial cell elongation occurs at the cell poles; however, it is not clear how cell wall insertion is restricted to the pole and organized. Wag31 is a pole-localized cytoplasmic protein that is essential for polar growth, but its molecular function has not been described. Wag31 homo-oligomerizes in a network at the poles, but it is not known how the structure of this network affects Wag31 function. In this study we used a protein fragment complementation assay to identify Wag31 residues involved in homo-oligomeric interactions, and found that amino acids all along the length of the protein mediate these interactions. We then used both N-terminal and C-terminal splitGFP fusions to probe Wag31 network topology at different sites in the cell, and found that Wag31 C-terminal-C-terminal interactions predominate at the septa, while C-terminal-C-terminal and C-terminal-N-terminal interactions are found equally at the poles. This suggests the Wag31 network is formed through an ordered series of associations. We then dissected Wag31s functional roles by phenotyping a series of wag31 alanine mutants; these data show that Wag31 has separate functions in not only new and old pole elongation, but also inhibition of both septation and new pole elongation. This work establishes new functions for Wag31, and indicates that changes in Wag31 homo-oligomeric network topology may contribute to cell wall regulation in mycobacteria.

ImportanceMany bacteria restrict cell wall elongation to their cell poles, but it is not known how polar growth is affected on the molecular level. Wag31 is a protein that is required for this polar elongation. In this work, we show that Wag31 actually has at least four distinct functions in regulating the cell wall: it promotes elongation at both poles in different ways, and it can also inhibit cell wall metabolism at the new pole and the septum. In addition, we propose a new model for how Wag31 self-associates into a protein network. This work is important because it shows that a DivIVA homolog can have distinct functions depending on cell context. And, this work clarifies that Wag31 is doing several different things in the cell, and gives us genetic tools to disentangle its functions.


title: Autism-associated mutation in Hevin/Sparcl1 induces endoplasmic reticulum stress through structural instability

abstract: Hevin is a secreted extracellular matrix protein that is encoded by SPARCL1 gene. Recent studies show that Hevin plays an important role in regulating synaptogenesis and synaptic plasticity. Mutations in SPARCL1 gene increase the risk of autism spectrum disorder (ASD). However, the molecular basis of how mutations in SPARCL1 increase the risk of ASD has not been fully understood. In this study, we show that one of SPARCL1 mutations associated with ASD impairs normal Hevin secretion. We identified Hevin mutants lacking the EF-hand motif through analyzing ASD-related mice with vulnerable spliceosome functions. Hevin deletion mutants accumulate in the ER, leading to the activation of unfolded protein responses. We also found that a single amino acid substitution of Trp647 with Arg in the EF-hand motif associated with a familial case of ASD causes a similar phenotype with the EF-hand deletion mutant. Importantly, molecular dynamics (MD) simulation revealed that this single amino acid substitution triggers exposure of hydrophobic amino acid to the surface, increasing the binding of Hevin with a molecular chaperon, BIP. Taken together, these data suggest that the integrity of EF-hand motif in Hevin is crucial for proper folding and ASD-related mutation impairs an export of Hevin from the endoplasmic reticulum (ER). Our data provide a novel mechanism linking a point mutation in SPARCL1 gene to the molecular and cellular characteristics involved in ASD.

category: molecular biology


title: Resolving subcellular pH with a quantitative fluorescent lifetime biosensor

abstract: Changes in sub-cellular pH play a key role in metabolism, cell growth, membrane transport, and can also be exploited to control cargo release from therapeutic delivery systems. Most methods to measure pH rely on intensity changes of pH sensitive fluorophores, however these measurements are hampered by high uncertainty in the inferred pH and the need for multiple fluorophores. To address this, we have developed a method to accurately quantify sub-cellular pH in individual vesicles using fluorescent lifetime imaging microscopy (pHLIM). pHLIM exploits the linear pH dependant lifetime of the fluorescent protein mApple and uses deep learning models to automatically identify and measure the pH of subcellular compartments. We have engineered mApple fusion proteins to measure the pH of the cytosol, endosomes, lysosomes and demonstrated the utility of pHLIM by measuring pH changes induced by drugs (bafilomycin A1) and polyethylenimine (a common transfection reagent). pHLIM is a simple and quantitative method to measure sub-cellular pH that has the potential to help with the design of the next generation of controlled drug release systems and to understand drug action and disease progression.

category: cell biology


title: Regulation of Store-Operated Ca2+ Entry by IP3 Receptors Independent of Their Ability to Release Ca2+

abstract: Loss of ER Ca2+ activates store-operated Ca2+ entry (SOCE) by causing STIM1 to unfurl domains that activate Orai1 channels in the plasma membrane at membrane contact sites (MCS). In human neurons, SOCE evoked by thapsigargin to deplete ER Ca2+ is attenuated by loss of IP3Rs, and restored by expression of IP3Rs even when they cannot release Ca2+, but only if the IP3Rs can bind IP3. In cells expressing poredead IP3Rs, IP3 enhances SOCE evoked by partial store depletion without enhancing Ca2+ release. Proximity ligation assays establish that IP3Rs enhance association of STIM1 with Orai1 in cells with empty stores; this requires an IP3-binding site, but not a pore. Over-expressing STIM1, or extended synaptotagmin-1 to exaggerate MCS, circumvents the need for IP3Rs. Thus, IP3 binding to IP3Rs stimulates SOCE by both mediating ER Ca2+ release and promoting STIM1-Orai1 interactions. Convergent regulation by IP3Rs may tune SOCE to respond selectively to IP3.


title: The use of alternative spawning habitats by the American horseshoe crab, Limulus polyphemus

abstract: For animals that develop externally, habitats where environmental conditions are optimal for embryonic development are sometimes assumed to represent the highest recruitment potential and thus support the majority of reproductive output for a species. However, organisms may spawn in areas considered sub-optimal for embryonic development. Thus, understanding spawning habitat selection decisions and their potential impacts on recruitment and ecological interactions is necessary for predicting population status and identifying critical habitats to inform sustainable conservation decisions and effective management approaches. The American horseshoe crab, Limulus polyphemus, is ecologically, economically, and biomedically important. Females come ashore to spawn in the sediment where eggs develop for 2 - 4 weeks. Horseshoe crabs have been thought to primarily use sandy beach habitat for spawning in part because this habitat has been shown to be optimal for embryonic development. Horseshoe crab eggs on sandy beaches are an essential part of the diet of many organisms, including shorebirds such as the rufa red knot which requires the eggs to fuel their migration to arctic spawning grounds. While horseshoe crabs have been observed spawning in alternative habitats such as salt marshes and peat beds, this behavior has been assumed to be rare and non-adaptive. In this study, we compare the use of beach and alternative habitats by horseshoe crabs for spawning. To do so, we conducted adult horseshoe crab spawning surveys and horseshoe crab egg surveys in beach and Spartina-dominated salt marsh alternative habitats in South Carolina, Connecticut, and New Hampshire, U.S.A. While spawning horseshoe crabs were more likely to be observed on beach habitats than in alternative habitats, potentially due to logistical constraints surveying alternative habitats, we found similar densities of spawning horseshoe crabs in both habitat types. We also tended to find more eggs in alternative habitats than on beaches. Taken together, these results suggest that alternative habitats likely represent a significant source of horseshoe crab spawning activity and recruitment that had not previously been quantified. We recommend this information be incorporated into horseshoe crab population assessments, habitat protections, and more directed research at understanding variability in habitat-specific horseshoe crab spawning and its relationship to migratory shorebirds.

category: ecology


title: Legacy effect of microplastics on plant-soil feedbacks

abstract:
Microplastics are a complex contaminant suite that are now understood to affect plants and soil biota and the processes they drive. However, the role of microplastic in plant-soil feedbacks, a key feature in plant-soil interactions, is still unknown. We address this here, using soil from a previous experiment, which has been conditioned with 12 different microplastic types including fibers, films, foams, and fragments. To evaluate the feedback effect, we grew a native and a range-expanding plant species with inocula extracted from each one of these soils. At harvest, plant biomass and root morphological traits were measured.

Films gave rise to a positive feedback on shoot mass (higher mass with soil inocula conditioned with microplastics than without), likely via negative effects on harmful soil biota. Foams and fragments also caused positive feedback on shoot mass likely via effects on enzymatic activities and mutualistic soil biota. Fibers led to negative feedback on root mass as they may promote the abundance of soil pathogens.

Microplastics also have a legacy effect on root traits: Daucus had thicker roots probably for promoting mycorrhizal associations while Calamagrostis had reduced root diameter probably for diminishing pathogenic infection. Microplastic legacy on plants is species-specific and may affect plant biomass primarily via root traits. Microplastics, as a function of their shape and polymer type, have a feedback effect on plant performance.


title: Interpreting the pervasive observation of U-shaped Site Frequency Spectra

abstract: The standard neutral model of molecular evolution has traditionally been used as the null model for population genomics. We gathered a collection of 45 genome-wide site frequency spectra from a diverse set of species, most of which display an excess of low and high frequency variants compared to the expectation of the standard neutral model, resulting in U-shaped spectra. We show that multiple merger coalescent models often provide a better fit to these observations than the standard Kingman coalescent. Hence, in many circumstances these under-utilized models may serve as the more appropriate reference for genomic analyses. We further discuss the underlying evolutionary processes that may result in the widespread U-shape of frequency spectra.


title: Estimation of Left Ventricular Mechanical Activation Times from Motion-Corrected Cardiac 4DCT Images

abstract:
BackgroundMechanical activation times of the left ventricle (LV) are strongly correlated with patient response to cardiac resynchronization therapy (CRT); however, there remains an unmet clinical need for a simple, robust imaging modality that yields accurate and reproducible estimates of LV mechanical activation times. Modern four-dimensional computed tomography (4DCT) imaging systems with effective motion correction could fulfill this need.

PurposeTo demonstrate the clinical utility of a newly developed cardiac motion correction algorithm called ResyncCT and to investigate its effect on estimating mechanical activation times of the LV.

MethodsTwenty-four subjects with full cardiac volume, full cardiac cycle 4DCT images were retrospectively analyzed in this study. The reconstructed 4DCT images exhibited typical motion artifact characteristics that were dependent on the direction of LV wall motion with respect to the gantry position at each cardiac phase; these artifacts rotated synchronously with the gantry. The motion corrupted images, referred to as the uncorrected images, were processed with a novel cardiac motion correction algorithm called ResyncCT to yield motion corrected images (referred to as the ResyncCT images). Regional shortening (RSCT) of the LV was calculated over a 72-segment model for both the uncorrected and the ResyncCT images; each segment contained RSCT vs time data. From these RSCT vs time curves, LV mechanical activation was estimated as the time point at which the RSCT vs time curve shortened by 10% of its full dynamic range during systolic contraction; we refer to this time point as the time to onset of shortening (TOS). A shift in TOS > |35| ms has a significant effect on reclassifying patients based on their probabilities of responding to CRT; we investigated the effect of ResyncCT on the measured values of TOS over the LV.

ResultsResyncCT had a pronounced effect on the TOS estimates; ResyncCT could potentially reclassify 23/24 (96%) subjects as either responders or non-responders to CRT. In 16/24 (67%) subjects, the differences in TOS that were sufficient to bring about a reclassification of CRT response were at least as large in surface area of the LV as one entire American Heart Association segment.

ConclusionsWe demonstrated the clinical utility of ResyncCT in estimating LV mechanical activation times as  times to onset of shortening in 24 human subjects. ResyncCT has a pronounced effect on the estimation of LV mechanical activation times; the differences in activation times between the ResyncCT and uncorrected images are heterogenous and subject specific. The effect of ResyncCT could potentially reclassify 96% of the subjects used in this study based on their probabilities of responding to CRT. The results reported in this study highlight the potential utility of ResyncCT in estimating timing of mechanical events of interest of the LV for CRT planning.

category: bioengineering


title: Knowledge-graph-based cell-cell communication inference for spatially resolved transcriptomic data with SpaTalk

abstract: Spatially resolved transcriptomics (ST) provides genetic information in space toward elucidation of the spatial architecture in intact organs and the spatially resolved cell-cell communications mediating tissue homeostasis, development, and disease. To facilitate inference of spatially resolved cell-cell communications from ST data, we here present SpaTalk, which relies on a graph network and knowledge graph to model and score the ligand-receptor-target signaling network between spatially proximal cells, decomposed from ST data through a non-negative linear model and spatial mapping between single-cell RNA-sequencing and ST data. The performance of SpaTalk benchmarked on public single-cell ST datasets was superior to that of existing cell-cell communication inference methods. SpaTalk was then applied to STARmap, Slide-seq, and 10X Visium data, revealing the in-depth communicative mechanisms underlying normal and disease tissues with spatial structure. SpaTalk can uncover spatially resolved cell-cell communications for single-cell and spot-based ST data universally, providing new insights into spatial inter-cellular dynamics.


title: Modeling vital rates and age-sex structure of Pacific arctic phocids: influence on aerial survey correction factors

abstract: To estimate abundance, surveys of marine mammals often rely on samples of satellite-tagged individuals to correct counts for the proportion of animals that are unavailable to be detected. However, naive application of this correction relies on the key assumption that availability of the tagged sample resembles that of the population. Here, we show how matrix population models can be used to estimate the stable stage structure of bearded seals (Erignathus barbatus), ribbon seals (Histriophoca fasciata), ringed seals (Pusa hispida), and spotted seals (Phoca largha) in the Bering and Chukchi Seas, and how these proportions can be used to adjust aerial survey correction factors so that they represent population-level availability. We find that correction factors ignoring age-sex composition can positively bias spotted seal abundance by an average of 13% and negatively bias ribbon seal abundance by an average of 5%. Note that we did not examine potential bias for bearded or ringed seals due to low sample sizes; as such, we urge caution in interpretation of abundance estimates for these species.


title: Nav1.2 and BK channels interaction shapes the action potential in the axon initial segment

abstract: In neocortical layer-5 pyramidal neurons, the action potential (AP) is generated in the axon initial segment (AIS) when the membrane potential (Vm) reaches the threshold for activation of two diverse voltage-gated Na+ channels, that differ in spatial distribution and biophysical properties. Yet, the understanding of specific functional differences between these two channels remains elusive. Here, using ultrafast Na+, Vm and Ca2+ imaging in combination with partial block of Nav1.2 by a recent peptide, we demonstrate an exclusive role of Nav1.2 in shaping the generating AP. Precisely, Ca2+ influx via Nav1.2 activates BK Ca2+-activated K+ channels determining the amplitude and the early phase of repolarisation of the AP in the AIS. We mimicked this result with a NEURON model where the role of the different ion channels tested reproduced the experimental evidence. The exclusive role of Nav1.2 reported here is important for understanding the physiology and pathology of neuronal excitability.


title: Reelin deficiency exacerbates cocaine-induced hyperlocomotion by enhancing neuronal activity in the dorsomedial striatum.

abstract:
The Reln gene encodes for the extracellular glycoprotein Reelin, which regulates several brain functions from development to adulthood, including neuronal migration, dendritic growth and branching, and synapse formation and plasticity. Human studies have implicated Reelin signaling in several neurodevelopmental and psychiatric disorders. Mouse studies using the heterozygous Reeler (HR) mice have shown that reduced levels of Reln expression are associated with deficits in learning and memory and increased disinhibition. Although these traits are relevant to substance use disorders, the role of Reelin in cellular and behavioral responses to addictive drugs remains largely unknown. Here, we compared HR mice to wild-type (WT) littermate controls to investigate the contribution of Reelin signaling to the hyper-locomotor and rewarding effects of cocaine. After a single cocaine injection, HR mice showed enhanced cocaine-induced locomotor activity compared to WT controls. After repeated injections of cocaine, Reelin deficiency also led to increased cocaine-induced locomotor sensitization, which persisted after withdrawal. In contrast, Reelin deficiency did not affect the rewarding effects of cocaine measured in the conditioned place preference assay. The elevated cocaine-induced hyper-locomotion in HR mice resulted in increased Fos expression in the dorsal medial striatum (DMS) compared to WT. Lastly, we found that Reln was highly co-expressed with the Drd1 gene, which encodes for the dopamine receptor D1, in the DMS.

These findings demonstrated that Reelin signaling contributes to the locomotory effects of cocaine and improved our understanding of the neurobiological mechanisms underlying the cellular and behavioral effects of cocaine.


title: Representational integration and differentiation in the human hippocampus following goal-directed navigation

abstract: As we learn, dynamic memory processes build structured knowledge across our experiences. Such knowledge enables the formation of internal models of the world that we use to plan, make decisions, and act. Recent theorizing posits that mnemonic mechanisms of differentiation and integration - which at one level may seem to be at odds - both contribute to the emergence of structured knowledge. We tested this possibility using fMRI as human participants learned to navigate within local and global virtual environments over the course of three days. Pattern similarity analyses on entorhinal cortex, hippocampus, and ventromedial prefrontal cortex patterns revealed evidence that differentiation and integration work concurrently to build local and global environmental representations, and that variability in integration relates to differences in navigation efficiency. These results offer new insights into the neural machinery and the underlying mechanisms that translate experiences into structured knowledge that allows us to navigate to achieve goals.


title: Improving the fidelity of uridine analog incorporation during in vitro transcription.

abstract: In vitro transcribed synthetic messenger RNAs (mRNAs) represent a novel therapeutic modality and are currently being evaluated for a wide range of clinical indications. To overcome the inherent immunogenicity of the synthetic mRNAs, as well as to increase the therapeutic efficacy of the molecules, RNA sequence optimization is routinely performed and modified uridine analogs--such as pseudouridine ({Psi}) and N1-methyl-pseudouridine (m1{Psi}), are incorporated in the synthetic mRNA. To decipher the fidelity with which these modifications are incorporated during the in vitro transcription (IVT) process, here, we compared, the incorporation fidelity of uridine, {Psi}, or m1{Psi} in multiple RNA sequences with different single-subunit DNA-dependent RNA polymerases (ssRNAPs). By comparing the incorporation of each modified base to that of the unmodified equivalent, we demonstrate that m1{Psi} is incorporated with higher fidelity than {Psi}. Furthermore, the various ssRNAPs exhibit different error rates; however, the spectrum of mutations observed between the RNAPs is similar. We also show that the array of nucleotide misincorporation is not dependent on the template DNA sequence context and that the distribution of these misincorporated nucleotides is not localized to any specific region along the length of the RNA. Based on our findings, we introduce a novel protocol to improve uridine analog incorporation--without affecting total RNA yield--during IVT. Our proof-of-concept experiments and protocol for higher-fidelity incorporation of uridine analogs during IVT provide guidelines when choosing ssRNAPs for the generation of modified uridine--containing mRNAs in vitro.


title: Nuclear transport under stress phenocopies transport defects in models of C9Orf72 ALS

abstract: The nucleus is the hallmark of eukaryotic life and transport to and from the nucleus occurs through the nuclear pore complex (NPC). There is a multitude of data connecting the nuclear transport machinery - i.e. the NPCs and associated nuclear transport factors - to neurodegenerative diseases, but the mechanisms are not well understood. Using Saccharomyces cerevisiae, we systematically studied how the expression of polyPR and polyGA related to C9Orf72 amyotrophic lateral sclerosis impacts the nuclear transport machinery. We measured the abundance and localization of NPC components and transport factors, and assessed the kinetics of import and export by four transport receptors. PolyPR and polyGA cause distinct, and transport receptor dependent effects. We compared the specific changes in transport to those obtained when cells were exposed to different stress situations or mutations. This comparison showed similar patterns of transport defects in cells lacking specific NTRs and cells expressing polyPR. In contrast, polyGA expressing cells bear resemblance to stress conditions where energy maintenance is decreased. The similarity of the patterns of transport deficiencies suggests that polyPR has a direct effect on nuclear transport via NTRs, while polyGA impacts the energy state of the cell and subsequently changes transport.


title: A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag

abstract: Monitoring autophagic flux is necessary for most autophagy studies. The autophagic flux assays currently available for mammalian cells are generally complicated and do not yield highly quantitative results. Yeast autophagic flux is routinely monitored with the GFP-based processing assay, whereby the amount of GFP proteolytically released from GFP-containing reporters (e.g., GFP-Atg8), detected by immunoblotting, reflects autophagic flux. However, this simple and effective assay is typically inapplicable to mammalian cells because GFP is efficiently degraded in lysosomes while the more proteolytically resistant RFP accumulates in lysosomes under basal conditions. Here, we report a HaloTag (Halo)-based reporter processing assay to monitor mammalian autophagic flux. We found that Halo is sensitive to lysosomal proteolysis but becomes resistant upon ligand binding. When delivered into lysosomes by autophagy, pulse-labeled Halo-based reporters (e.g., Halo-LC3 and Halo-GFP) are proteolytically processed to generate Haloligand when delivered into lysosomes by autophagy. Hence, the amount of free Haloligand detected by immunoblotting or in-gel fluorescence imaging reflects autophagic flux. We demonstrate the applications of this assay by monitoring the autophagy pathways, macroautophagy, selective autophagy, and even bulk nonselective autophagy. With the Halo-based processing assay, mammalian autophagic flux and lysosome-mediated degradation can be monitored easily and precisely.


title: A quantitative and spatial analysis of cell cycle regulators during the fission yeast cell cycle

abstract:
We have carried out a systems-level analysis of the spatial and temporal dynamics of cell cycle regulators in the fission yeast Schizosaccharomyces pombe. In a comprehensive single cell analysis we have precisely quantified the levels of 38 proteins previously identified as regulators of the G2 to mitosis transition, and of 7 proteins acting at the G1 to S-phase transition. Only two of the 38 mitotic regulators exhibit changes in concentration at the whole cell level, the mitotic B-type cyclin Cdc13 which accumulates continually throughout the cell cycle, and the regulatory phosphatase Cdc25 which exhibits a complex cell cycle pattern. Both proteins show similar patterns of change within the nucleus as in the whole cell but at higher concentrations. In addition, the concentrations of the major fission yeast cyclin dependent kinase (CDK) Cdc2, the CDK regulator Suc1 and the inhibitory kinase Wee1 also increase in the nucleus peaking at mitotic onset but are constant in the whole cell. The significant increase in concentration with size for Cdc13 supports the model that mitotic B-type cyclin accumulation acts as a cell size sensor. We propose a two-step process for the control of mitosis. First, Cdc13 accumulates in a size-dependent manner which drives increasing CDK activity. Second, from mid G2 the increasing nuclear accumulation of Cdc25 and the counteracting Wee1 introduces a bistability switch that results in a rapid rise of CDK activity at the end of G2 and thus brings about an orderly progression into mitosis.

Significance StatementAcross eukaryotes the increasing level of cyclin dependent kinase (CDK) activity drives progression through the cell cycle. As most cells divide at specific sizes, information responding to the size of the cell must feed into the regulation of CDK activity. In this study, we use fission yeast to precisely measure how proteins that have been previously identified in genome wide screens as cell cycle regulators change in their levels with cell cycle progression. We identify the mitotic B-type cyclin Cdc13 and mitotic inhibitory phosphatase Cdc25 as the only two proteins that change in both whole cell and nuclear concentration through the cell cycle, making them candidates for universal cell size sensors at the onset of mitosis and cell division.


title: Microvesicle-Mediated Tissue Regeneration Mitigates the Effects of Cellular Ageing

abstract: An ageing global population brings with it a significant burden of age-related morbidities. Recently, a novel intervention strategy to mitigate this burden has emerged, involving the use of Extracellular Vesicles (EV), comprising use of Microvesicles (MV) and Exosomes (Exo). These membranous vesicles are secreted by cells and mediate repair of cellular and tissue damage via paracrine mechanisms, involving interaction of their bioactive cargoes with stem cells. The actions of EV under normative and morbid conditions in the context of ageing remains largely unexplored. We now show that MV, but not Exo, from Pathfinder cells (PC), a putative stem cell regulatory cell type, enhance the repair of Human Dermal Fibroblast (HDF) and Mesenchymal Stem Cell (MSC) co-cultures following both mechanical and genotoxic stress. Critically, this effect was found to be both cellular age and stress-specific. Notably, MV treatment was unable to repair mechanical injury in older co-cultures, but still remained therapeutic following genotoxic stress. These observations were further confirmed in HDF and Vascular Smooth Muscle Cell (VSMC) co-cultures of increasing cellular age. In a model of comorbidity, comprising co-cultures of HDF and highly senescent Abdominal Aortic Aneurysm (AAA) VSMC, MV administration appeared to be senolytic following both mechanical and genotoxic stress, prior to enabling regeneration. To our knowledge, this is the first description of EV-based senolysis. It provides novel insight into understanding the biology of EV and the specific roles they play during tissue repair and ageing. These data will potentiate development of novel cell-free therapeutic interventions capable of attenuating age-associated morbidities and avoiding undesired effects. Ultimately, this might act as a possible intervention strategy to extend human healthspan.


title: The meiotic LINC complex component KASH5 is an activating adaptor for cytoplasmic dynein

abstract: Cytoplasmic dynein-driven movement of chromosomes during prophase I of meiosis is essential for synapsis and genetic exchange. Dynein connects to chromosome telomeres via KASH5 and SUN1/2, which span the outer and inner nuclear envelopes, respectively. Here, we show that KASH5 promotes dynein motility in vitro, and cytosolic KASH5 inhibits dyneins interphase functions. KASH5 interacts with either dynein light intermediate chain (DYNC1LI1 or DYNC1LI2) via a conserved linker-helix in the LIC C-terminal, and this region is also needed for dyneins recruitment to other cellular membranes. KASH5s N-terminal EF-hands are essential, as the interaction with dynein is disrupted by mutation of key calcium-binding residues, although it is not regulated by cellular calcium levels. Dynein can be recruited to KASH5 at the nuclear envelope independently of dynactin, while LIS1 is essential for dynactin incorporation into the KASH5-dynein complex. Altogether, we show that the trans-membrane protein KASH5 is an activating adaptor for dynein, and shed light on the hierarchy of assembly of KASH5-dynein-dynactin complexes.


title: Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation

abstract: Stimulated emission depletion (STED) microscopy achieves super-resolution by exciting a diffraction-limited volume and then suppressing fluorescence in its outer parts by depletion. Multiple depletion lasers may introduce misalignment and bleaching. Hence, a single depletion wavelength is preferable for multi-color analyses. However, this limits the number of usable spectral channels. Using cultured cells, common staining protocols, and commercially available fluorochromes and microscopes we exploit that the number of fluorochromes in STED or confocal microscopy can be increased by phasor based fluorescence lifetime separation of two dyes with similar emission spectra but different fluorescent lifetimes. In our multi-color FLIM-STED approach two fluorochromes in the near red (exc. 594 nm, em. 600-630) and two in the far red channel (633/641-680), supplemented by a single further redshifted fluorochrome (670/701-750) were depleted with 775 nm. To the best of our knowledge, these are the first published five color STED images. Generally, this approach doubles the number of fully distinguishable colors in laser scanning microscopy. We provide evidence that eight color FLIM-STED with a single depletion laser would be possible if suitable fluorochromes were identified and we confirm that a fluorochrome may have different lifetimes depending on the molecules to which it is coupled.


title: Sun4 is a type II transmembrane protein of the spermatid inner nuclear membrane that forms heteromeric assemblies with Sun3 and interacts with Lamin B3

abstract:
SUN domain proteins are conserved proteins of the nuclear envelope and key components of the LINC complexes (linkers of the nucleoskeleton and the cytoskeleton). Previous studies have demonstrated that the testis-specific SUN domain protein Sun4 is a vital player in spermatogenesis, critically involved in the directed shaping of the spermatid nucleus. Its molecular properties relating to this crucial function, however, have remained largely unknown. Previous studies presented quite controversial data for the general organization and orientation of Sun4 within the spermatid nuclear envelope. In the present study, we have re-evaluated this issue in detail and present new robust data on the Sun4 topology and its interactions at the nucleo-cytoplasmic junction. We identified Sun4 as an integral protein of the inner nuclear membrane, sharing a classical SUN domain protein topology. Similar to other SUN domain proteins, the C-terminal SUN domain of Sun4 locates to the perinuclear space and the N-terminus is directed to the nucleoplasm, where it interacts with the spermiogenesis specific Lamin B3. We found that Sun4 in its natural environment forms heteromeric assemblies with Sun3 and, beyond this, it is crucially involved in the regulation of Sun3 expression. Together, our results contribute to a better understanding of the specific function of Sun4 at the spermatid nucleo-cytoplasmic junction and the entire process of sperm-head formation.

Summary statementIn our current study, we have analyzed in detail the biochemical and dynamic properties of the testis-specific SUN domain protein Sun4 and we provide novel insights into its interaction behavior at the spermatid nucleo-cytoplasmic junction.


title: Multiple waves of viral invasions in Symbiodiniaceae algal genomes

abstract: Dinoflagellates from the family Symbiodiniaceae are phototrophic marine protists that engage in symbiosis with diverse hosts. Their large and distinct genomes show pervasive gene duplication and large-scale retroposition events. However, little is known about the role and scale of horizontal gene transfer (HGT) in the genomic evolution of this algal family. In other dinoflagellates, higher levels of HGTs have been observed, linked to major genomic transitions, such as the appearance of a viral acquired nucleoprotein that originated via HGT from a large DNA algal virus. Previous work showed Symbiodiniaceae from different hosts being actively infected by several viral groups, such as giant DNA viruses and ssRNA viruses, that may play an important role in coral health. This includes a hypothetical latent viral infection, whereby viruses could persist in the cytoplasm or integrate into the host genome as a provirus. This hypothesis received some experimental support however, the cellular localization of putative latent viruses and their taxonomic affiliation are still unknown. In addition, despite the finding of viral sequences in some genomes of Symbiodiniaceae, viral origin, taxonomic breadth, and metabolic potential have not been explored. To address these questions, we searched for evidence of protein sequences of putative viral origin in 13 Symbiodiniaceae genomes. We found 59 candidate viral-derived HGTs that give rise to 12 phylogenies across 10 genomes. We also describe the taxonomic affiliation of these virus-related sequences, their structure, and genomic context. These results lead us to propose a model to explain the origin and fate of Symbiodiniaceae viral acquisitions.


title: Benchmarking community-wide estimates of growth potential from metagenomes using codon usage statistics

abstract: Trait inference from mixed-species assemblages is a central problem in microbial ecology. Frequently, sequencing information from an environment is available, but phenotypic measurements from individual community members are not. With the increasing availability of molecular data for microbial communities, bioinformatic approaches that map metagenome to (meta)phenotype are needed. Recently, we developed a tool, gRodon, that enables the prediction of the maximum growth rate of an organism from genomic data on the basis of codon usage patterns. Our work and that of other groups suggests that such predictors can be applied to mixed-species communities in order to derive estimates of the average community-wide maximum growth rate. Here, we present an improved maximum growth rate predictor designed for metagenomes that corrects a persistent GC bias in the original gRodon model for metagenomic prediction. We benchmark this predictor with simulated metagenomic datasets to show that it has superior performance on mixed-species communities relative to earlier models. We go on to provide guidance on data pre-processing, and show that calling genes from assembled contigs rather than directly from reads dramatically improves performance. Finally, we apply our predictor to large-scale metagenomic datasets from marine and human microbiomes to illustrate how community-wide growth prediction can be a powerful approach for hypothesis-generation. Altogether, we provide an updated tool with clear guidelines for users about the uses and pitfalls of metagenomic prediction of the average community-wide maximal growth rate.


title: Systematic conformation-to-phenotype mapping via limited deep-sequencing of proteins

abstract: Non-native conformations drive protein misfolding diseases, complicate bioengineering efforts, and fuel molecular evolution. No current experimental technique is well-suited for elucidating them and their phenotypic effects. Especially intractable are the transient conformations populated by intrinsically disordered proteins. We describe an approach to systematically discover, stabilize, and purify native and non-native conformations, generated in vitro or in vivo, and directly link conformations to molecular, organismal, or evolutionary phenotypes. This approach involves high-throughput disulfide scanning (HTDS) of the entire protein. To reveal which disulfides trap which chromatographically resolvable conformers, we devised a limited deep-sequencing method for proteins that identifies the exact sequence positions of both cysteines simultaneously within each polypeptide molecule. HTDS of the abundant E. coli periplasmic chaperone HdeA revealed distinct classes of disordered hydrophobic conformers with variable cytotoxicity depending on where the backbone was cross-linked. HTDS can bridge conformational and phenotypic landscapes for many proteins that function in disulfide-permissive environments.


title: DNA damage response signaling is crucial for effective Chikungunya virus replication

abstract:
Viruses utilize a plethora of strategies to manipulate the host pathways and hijack its machineries for efficient replication. Several DNA as well as handful of RNA viruses are reported to interact with proteins involved in DNA damage responses (DDR). As the DDR pathways have never been explored in Alphaviruses, this investigation intended to determine the importance of the DDR pathways in CHIKV infection through in vitro, in vivo and ex vivo models. The study reveals that CHIKV infection activates the Chk2 and Chk1 proteins associated with DDR signaling pathways and increases DNA damage by 95%. Inhibition of both ATM-ATR kinases by ATM/ATR kinase inhibitor (AAKi) shows drastic reduction in viral particle formation in vitro. Next, the treatment of mice with this drug has been shown to reduce the disease score substantially in CHIKV-infected C57BL/6 mice with 71% decrement in the viral copy and the same has been established in hPBMC-derived monocyte-macrophage populations. Additionally, gene silencing of Chk2 and Chk1 reduces viral progeny formation around 73.7% and 78% respectively. Moreover, it has been demonstrated that CHIKV-nsP2 interacts with Chk2 and Chk1 during CHIKV infection and docking analysis depicts the specific amino acids responsible for these interactions. Further, the data suggests that CHIKV infection induces cell cycle arrest in G1 and G2 phases.

In conclusion, this work demonstrated for the first time the mechanistic insight of the induction of DDR pathways by CHIKV that might contribute to the designing of effective therapeutics for the control of this virus infection in future.

IMPORTANCEViruses being intra-cellular parasite, need several host cell machineries so as to achieve effective replication of their own genome, along with virus-encoded enzymes. One of the strategies is to hijack the DDR pathways. Several DNA as well as handful of RNA viruses interact with the cellular proteins involved in DDR pathways, however, reports with respect to the association of Chk2 and Chk1 in alphavirus infection are scanty. Hence, this study is amongst the first to report that modulation of DDR pathways is crucial for effective CHIKV infection. This work also shows that there is interaction of CHIKV-nsP2 with two crucial host factors, Chk2 and Chk1 for efficient viral infection. Interestingly, CHIKV infection was found to cause DNA damage and arrest cell cycle in G1 and G2 phases to facilitate viral infection. This information might facilitate to develop effective therapeutics for the control of the CHIKV infection in future.


title: Broadly neutralizing and protective nanobodies against diverse sarbecoviruses

abstract: As SARS-CoV-2 Omicron and other variants of concern continue spreading around the world, development of antibodies and vaccines to confer broad and protective activity is a global priority. Here, we report on the identification of a special group of nanobodies from immunized alpaca with exceptional breadth and potency against diverse sarbecoviruses including SARS-CoV-1, Omicron BA.1, and BA.2. Crystal structure analysis of one representative nanobody, 3-2A2-4, revealed a highly conserved epitope between the cryptic and the outer face of the receptor binding domain (RBD). The epitope is readily accessible regardless of RBD in "up" or "down" conformation and distinctive from the receptor ACE2 binding site. Passive delivery of 3-2A2-4 protected K18-hACE2 mice from infection of authentic SARS-CoV-2 Delta and Omicron. This group of nanobodies and the epitope identified should provide invaluable reference for the development of next generation antibody therapies and vaccines against wide varieties of SARS-CoV-2 infection and beyond.

category: immunology


title: SARS-CoV-2 Delta breakthrough infections in vaccinated patients

abstract: The continuous emergence of SARS-CoV-2 variants with increased transmission and immune evasion has caused breakthrough infections in vaccinated population. It is important to determine the threshold of neutralizing antibody titers that permit breakthrough infections. Here we tested the neutralization titers of vaccinated patients who contracted Delta variant. All 75 patients with Delta breakthrough infections exhibited neutralization titers (NT50) of less than 70. Among the breakthrough patients, 76%, 18.7%, and 5.3% of them had the NT50 ranges of <20, 20-50, and 50-69, respectively. These clinical laboratory results have implications in vaccine strategy and public health policy.


title: Structural and biochemical mechanism for increased infectivity and immune evasion of Omicron BA.1 and BA.2 variants and their mouse origins

abstract: The Omicron BA.2 variant has become a dominant infective strain worldwide. Receptor binding studies reveal that the Omicron BA.2 spike trimer have 11-fold and 2-fold higher potency to human ACE2 than the spike trimer from the wildtype (WT) and Omicron BA.1 strains. The structure of the BA.2 spike trimer complexed with human ACE2 reveals that all three receptor-binding domains (RBDs) in the spike trimer are in open conformation, ready for ACE2 binding, thus providing a basis for the increased infectivity of the BA.2 strain. JMB2002, a therapeutic antibody that was shown to have efficient inhibition of Omicron BA.1, also shows potent neutralization activities against Omicron BA.2. In addition, both BA.1 and BA.2 spike trimers are able to bind to mouse ACE2 with high potency. In contrast, the WT spike trimer binds well to cat ACE2 but not to mouse ACE2. The structures of both BA.1 and BA.2 spike trimer bound to mouse ACE2 reveal the basis for their high affinity interactions. Together, these results suggest a possible evolution pathway for Omicron BA.1 and BA.2 variants from human-cat-mouse-human circle, which could have important implications in establishing an effective strategy in combating viral infection.


title: Markers of Fungal Translocation Are Elevated During Post-Acute Sequelae of SARS-CoV-2 Infection and Induce NF-κB Triggered Inflammation.

abstract: Long COVID, a type of Post-Acute Sequelae of SARS CoV-2 infection (PASC), has been associated with sustained elevated levels of immune activation and inflammation. However, the pathophysiological mechanisms that drive this inflammation remain unknown. Inflammation during acute Coronavirus Disease 2019 (COVID-19) could be exacerbated by microbial translocation (from the gut and/or lung) to the blood. Whether microbial translocation contributes to inflammation during PASC is unknown. We found higher levels of fungal translocation - measured as {beta}-glucan, a fungal cell wall polysaccharide - in the plasma of individuals experiencing PASC compared to those without PASC or SARS-CoV-2 negative controls. The higher {beta}-glucan correlated with higher levels of markers of inflammation and elevated levels of host metabolites involved in activating N-Methyl-D-aspartate receptors (such as metabolites within the tryptophan catabolism pathway) with established neuro-toxic properties. Mechanistically, {beta}-glucan can directly induce inflammation by binding to myeloid cells (via the Dectin-1 receptor) and activating Syk/NF-{kappa}B signaling. Using an in vitro Dectin-1/NF-{kappa}B reporter model, we found that plasma from individuals experiencing PASC induced higher NF-{kappa}B signaling compared to plasma from SARS-CoV-2 negative controls. This higher NF-{kappa}B signaling was abrogated by the Syk inhibitor Piceatannol. These data suggest a potential targetable mechanism linking fungal translocation and inflammation during PASC.


title: The Omicron (B.1.1.529) SARS-CoV-2 variant of concern also affects companion animals

abstract: The recent emergence of the Omicron variant (B.1.1.529) has brought with it a large increase in the incidence of SARS-CoV-2 disease worldwide. However, there is hardly any data on the incidence of this new variant in companion animals. In this study, we have detected the presence of this new variant in domestic animals such as dogs and cats living with owners with COVID19 in Spain that have been sampled at the most optimal time for the detection of the disease. None of the RT-qPCR positive animals (10.13%) presented any clinical signs and the viral loads detected were very low. In addition, the shedding of viral RNA lasted a short period of time in the positive animals. Infection with the Omicron variant of concern (VOC) was confirmed by a specific RT-qPCR for the detection of this variant and by sequencing. These outcomes suggest a lower virulence of this variant in infected cats and dogs. This study demonstrates the transmission of this new variant from infected humans to domestic animals and highlights the importance of doing active surveillance as well as genomic research to detect the presence of VOCs or mutations associated with animal hosts.


title: Bacillus thuringiensis Cry1A toxins divert progenitor cell fate toward enteroendocrine lineage by diminishing cell adhesion with intestinal stem cells

abstract: Bacillus thuringiensis subsp. kurstaki (Btk) is a strong pathogen toward lepidopteran larvae thanks to specific Cry toxins causing leaky gut phenotypes. Hence, Btk and its toxins are used worldwide as microbial insecticide and in genetically modified crops, respectively, to fight crop pests. However, Btk belongs to the B. cereus group, some strains of which are well known human opportunistic pathogens. Therefore, ingestion of Btk along with food may threaten organisms not susceptible to Btk infection. Here we show that Cry1A toxins induce enterocyte death and intestinal stem cell (ISC) proliferation in the midgut of Drosophila melanogaster, an organism non-susceptible to Btk. Surprisingly, a high proportion of the ISC daughter cells differentiate into enteroendocrine cells instead of their initial enterocyte destiny. We show that Cry1A toxins weaken the Cadherin-dependent adherens junction between the ISC and its immediate daughter progenitor, leading the latter to adopt an enteroendocrine fate. Hence, though not lethal to non-susceptible organisms, Cry toxins can interfere with conserved cell adhesion mechanisms, thereby disrupting intestinal homeostasis and enteroendocrine functions.


title: A novel PNGase Rc for improved protein N-deglycosylation in bioanalytics and HDX-MS epitope mapping under challenging conditions

abstract:
N-linked glycosylation is a ubiquitous posttranslational modification of proteins. While it plays an important role in the biological function of proteins, it often poses a major challenge for their analytical characterization. Currently available peptide N-glycanases (PNGases) are often inefficient at deglycosylating proteins due to sterically inaccessible N-glycosylation sites. This usually leads to poor sequence coverage in bottom-up analysis using liquid chromatography with tandem mass spectrometry (LC-MS) and makes it impossible to obtain an intact mass signal in top-down MS analysis. In addition, most PNGases operate optimally only in the neutral to slightly acidic pH range and are severely compromised in the presence of reducing and denaturing substances, which limits their use for advanced bioanalysis based on hydrogen-deuterium exchange in combination with mass spectrometry (HDX-MS).

Here, we present a novel peptide N-glycanase from Rudaea cellulosilytica (PNGase Rc) for which we demonstrate broad substrate specificity for N-glycan hydrolysis from multiply occupied and natively folded proteins. Our results show that PNGase Rc is functional even under challenging, HDX quench conditions (pH 2.5, 0 {degrees}C) and in the presence of 0.4 M Tris(2-carboxyethyl)phosphine (TCEP), 4 M urea and 1 M guanidinium chloride (GdmCl). Most importantly, we successfully applied the PNGase Rc in an HDX-MS workflow to determine the epitope of a nanobody targeting the extracellular domain of human signal-regulating protein alpha (SIRP).

category: biochemistry