title: MAPLE: A Hybrid Framework for Multi-Sample Spatial Transcriptomics Data

abstract:
High throughput spatial transcriptomics (HST) technologies have allowed for identification of distinct cell sub-populations in tissue samples, i.e., tissue architecture identification. However, existing methods do not allow for simultaneous analysis of multiple HST samples. Moreover, standard tissue architecture identification approaches do not provide uncertainty measures. Finally, no existing frameworks have integrated deep learning with Bayesian statistical models for HST data analyses. To address these gaps, we developed MAPLE: a hybrid deep learning and Bayesian modeling framework for detection of spatially informed cell spot sub-populations, uncertainty quantification, and inference of group effects in multi-sample HST experiments. MAPLE includes an embedded regression model to explain cell sub-population abundance in terms of available covariates such as treatment group, disease status, or tissue region. We demonstrate the capability of MAPLE to achieve accurate, comprehensive, and interpretable tissue architecture inference through four case studies that spanned a variety of organs in both humans and animal models.

AvailabilityAn R package maple is available at https://github.com/carter-allen/maple.

Contactchung.911@osu.edu

Supplementary informationSupplementary data are available online.


title: P4HA2: A link between tumor-intrinsic hypoxia, partial EMT and collective migration

abstract: Epithelial-to-mesenchymal transition (EMT), a well-established phenomenon studied across pan-cancer types, has long been known to be a major player in driving tumor invasion and metastasis. Recent studies have highlighted the importance of partial EMT phenotypes in metastasis [1]. Initially thought as a transitional state between epithelial and mesenchymal phenotypic states, partial EMT state is now widely recognized as a key driver of intra-tumoral heterogeneity and phenotypic plasticity, further accelerating tumor metastasis and therapeutic resistance. However, how tumor microenvironments regulate partial EMT phenotypes remains unclear. We have developed unique size-controlled three-dimensional microtumor models that recapitulate tumor-intrinsic hypoxia and the emergence of collectively migrating cells. In this study, we further interrogate these microtumor models to understand how tumor-intrinsic hypoxia regulates partial EMT and collective migration in hypoxic large microtumors fabricated from T47D breast cancer cells. We also compared global gene expression profiles of hypoxic, migratory microtumors to that of non-hypoxic, non-migratory microtumors at early and late time-points. Using our microtumor models, we identified unique gene signatures for tumor-intrinsic hypoxia (early versus late), partial EMT and migration (pre-migratory versus migratory phenotype). Through differential gene expression analysis between the microtumor models with an overlap of hypoxia, partial EMT and migration signatures, we identified prolyl 4-hydroxylase subunit 2 (P4HA2), a hypoxia responsive gene, as a central regulator common to hypoxia, partial EMT and collective migration. Further, the inhibition of P4HA2 significantly blocked collective migration in hypoxic microtumors. Thus, using the integrated computational-experimental analysis, we identify the key role of P4HA2 in tumor-intrinsic hypoxia-driven partial EMT and collective migration.

category: cancer biology


title: Cytologic Scoring of Equine Exercise-Induced Pulmonary Hemorrhage (EIPH): Performance of Human Experts and a Deep Learning-Based Algorithm

abstract: Exercise-induced pulmonary hemorrhage (EIPH) is a relevant respiratory disease in sport horses which can be diagnosed by examination of bronchoalveolar lavage fluid (BALF) cells using the total hemosiderin score (THS). The aim of this study was to evaluate the diagnostic accuracy and reproducibility of trained annotators and to validate a deep learning-based algorithm for the THS. Digitized, iron-stained cytological specimens were prepared from 52 equine BALF samples. Ten annotators produced a THS for each slide according to published methods. The reference methods for comparing annotators and algorithmic performance included a ground truth dataset, the mean annotators THSs, and chemical iron measurements. Results of the study showed that annotators had marked inter-observer variability of the THS, which was mostly due to a systematic error between annotators in grading the intracytoplasmatic hemosiderin content of individual macrophages. Regarding overall measurement error between the annotators, 87.7% of the variance of the could be reduced by using standardized grades based on the ground truth. The algorithm was highly consistent with the ground truth in assigning hemosiderin grades. Compared to the ground truth THS, annotators had an accuracy of diagnosing EIPH (THS of < or [≥] 75) of 75.7% whereas the algorithm had an accuracy of 92.3% with no marked difference in correlation to chemical iron measurements. The results show that deep learning-based algorithms are useful for improving reproducibility and routine applicability of the THS. For THS by experts, a diagnostic uncertainty interval of 40 to 110 is proposed. THSs within this interval have insufficient reproducibility regarding the EIPH diagnosis.

category: pathology


title: A Single Multipurpose FSH-Blocking Therapeutic for Osteoporosis and Obesity

abstract: Pharmacological and genetic studies over the past decade have established FSH as an actionable target for diseases affecting millions, notably osteoporosis, obesity and Alzheimers disease (AD). Blocking FSH action prevents bone loss, fat gain and AD-like features in mice. We recently developed a first-in-class, humanized, epitope-specific FSH blocking antibody, MS-Hu6, with a KD of 7.52 nM. Using a GLP-compliant platform, we now report the efficacy of MS-Hu6 in preventing obesity and osteoporosis in mice, and parameters of acute safety in monkeys. Biodistribution studies using 89Zr-labelled, biotinylated or unconjugated MS-Hu6 in mice and monkeys showed localization to bone, bone marrow and fat depots. MS-Hu6 displayed a {beta} phase t[1/2] of 13 days (316 hours) in humanized Tg32 mice, and bound endogenous FSH. We tested 215 variations of excipients using the protein thermal shift assay to generate a final formulation that rendered MS-Hu6 stable in solution upon freeze-thaw and at different temperatures, with minimal aggregation, and without self-, cross-, or hydrophobic interactions or appreciable binding to relevant human antigens. MS-Hu6 showed the same level of "humanness" as human IgG1 in silico, and was non-immunogenic in ELISPOT assays for IL-2 and IFN{gamma} in human peripheral blood mononuclear cell cultures. We conclude that MS-Hu6 is efficacious, durable and manufacturable, and is therefore poised for future human testing as a multipurpose therapeutic.

category: pharmacology and toxicology


title: Knowledge of theory of evolution and beliefs as determinants of dualism in health science students

abstract: It has been proposed that dualistic conceptions on the mind and the body can affect the practice of health professionals. Religious beliefs have already been described as affecting mind-body dualistic thinking. Another factor that may play a role is knowledge of evolutionary theory, but this relationship has not been explored. In this work, data on knowledge of evolutionary theory and supernatural and dualistic beliefs of 287 Psychology and Medicine university students from Argentina were collected through a cross-sectional online survey. Information was analyzed to determine whether an association exists between knowledge of evolutionary theory, supernatural beliefs and dualism. We found significant statistical associations between supernatural beliefs and dualism (positive) and between both and knowledge of evolutionary theory (negative). Also, some heterogeneity was found within dualistic questions between theoretical ones and a hypothetical case. Our results are consistent with the hypothesis that knowledge of evolutionary theory could undermine mind-body dualistic conceptions.

category: scientific communication and education


title: Psilocybin induces spatially constrained alterations in thalamic functional organization and connectivity

abstract:
BackgroundSerotonin 2A receptor (5-HT2AR) agonist psychedelics including psilocybin and LSD ("classic" psychedelics) evoke acute alterations in perception and cognition. Altered thalamocortical connectivity has been proposed to underlie these effects, which is supported by some functional MRI (fMRI) studies. Likely due to sample size limitations, these studies have treated the thalamus as a unitary structure, despite known differential 5-HT2AR expression and functional specificity of different intrathalamic nuclei. Independent Component Analysis (ICA) has been employed to generate functional subdivisions of the thalamus from resting state fMRI (rsfMRI) data. This report utilizes a novel data-sparing ICA approach in order to examine psilocybin-induced changes in intrathalamic functional organization and thalamocortical connectivity.

MethodsBaseline rsfMRI data (n=38) was utilized to generate a template, which was then applied in a novel ICA-based analysis of the acute effects of psilocybin on intra- and extra-thalamic functional organization and connectivity in a smaller sample (n=18). Correlations with subjective reports of drug effect and comparisons with a previously reported analytic approach (treating the thalamus as a single functional unit) were conducted.

ResultsSeveral intrathalamic components showed significant psilocybin-induced alterations in intrathalamic spatial organization, largely localized to the mediodorsal and pulvinar nuclei, and correlated with reported subjective effects. These same components demonstrated alterations in thalamocortical connectivity, largely with visual and default mode networks. Analysis in which the thalamus is treated as a singular unitary structure showed an overall numerical increase in thalamocortical connectivity, consistent with previous literature using this approach, but this increase did not reach statistical significance.

ConclusionsUtilization of a novel analytic approach demonstrated changes in intra- and extra-thalamic functional organization and connectivity of intrathalamic nuclei and cortical networks known to express the 5-HT2AR. Given that these changes were not observed using whole-thalamus analyses, it seems that psilocybin may cause widespread but modest increases in thalamocortical connectivity that are offset by strong focal decreases in functionally relevant intrathalamic nuclei.

category: neuroscience


title: A tale of two connectivities: intra- and inter-subject functional connectivity jointly enable better prediction of social abilities

abstract: Naturalistic functional magnetic resonance imaging (fMRI) paradigms, such as movie viewing, are attracting increased attention, given their ability to mimic the real-world cognitive demands on attention and multimodal sensory integration. Moreover, naturalistic paradigms allow for characterizing brain network responses associated with dynamic social cognition in a model-free manner using inter-subject functional connectivity (ISFC). While intra-subject functional connectivity (FC) characterizes the individuals brain functional architecture, ISFC characterizes the neural coupling driven by time-locked extrinsic dynamic stimuli across individuals. Here, we hypothesized that ISFC and FC provide distinct and complementary information about individual differences in social cognition. To test this hypothesis, we examined a public movie-viewing fMRI dataset with 32 healthy adults and 90 typically developing children. Building three partial least squares regression (PLS) models to predict social abilities using FC and/or ISFC, we compared predictive performance to determine whether combining two connectivity measures could improve the prediction accuracy of individuals social-cognitive abilities measured by a Theory of Mind (ToM) assessment. Our results indicated that the joint model (ISFC+FC) yielded the highest predictive accuracy and significantly predicted individuals social cognitive abilities (rho = 0.34, p < 0.001). We also confirmed that the improved accuracy was not due to the increased feature dimensionality. In conclusion, we demonstrated that intra-/inter-subject connectivity encodes unique information about social abilities, and a joint investigation could help us gain a more complete understanding of the complex processes supporting social cognition.


title: Keratinocyte-tethering modification for biologics enables location-precise treatment in mouse vitiligo

abstract: Despite the central role of IFN{gamma} in vitiligo pathogenesis, systemic IFN{gamma} neutralization is an impractical treatment option due to strong immunosuppression. However, most vitiligo patients present with less than 20% affected body surface area, which provides an opportunity for localized treatments that avoid systemic side effects. After identifying keratinocytes as key cells that amplify IFN{gamma} signaling during vitiligo, we hypothesized that tethering an IFN{gamma} neutralizing antibody to keratinocytes would limit anti-IFN{gamma} effects to the treated skin for the localized treatment. To that end, we developed a bispecific antibody (BsAb) capable of blocking IFN{gamma} signaling while binding to desmoglein expressed by keratinocytes. We characterized the effect of the BsAb in vitro, ex vivo, and in a mouse model of vitiligo. SPECT/CT biodistribution and serum assays after local footpad injection revealed that the BsAb had improved skin retention, faster elimination from the blood, and less systemic IFN{gamma} inhibition than the non-tethered version. Furthermore, the BsAb conferred localized protection almost exclusively to the treated footpad during vitiligo that was not possible by local injection of the non-tethered anti-IFN{gamma} antibody. Thus, keratinocyte-tethering proved effective while significantly diminishing off-tissue effects of IFN{gamma} blockade, offering a new treatment strategy for localized skin diseases, including vitiligo.

category: immunology


title: Beyond resource selection: emergent spatio-temporal distributions from animal movements and stigmergent interactions

abstract: A principal concern of ecological research is to unveil the causes behind observed spatio-temporal distributions of species. A key tactic is to correlate observed locations with environmental features, in the form of resource selection functions or other correlative species distribution models. In reality, however, the distribution of any population both affects and is affected by those surrounding it, creating a complex network of feedbacks causing emergent spatio-temporal features that may not correlate with any particular aspect of the underlying environment. Here, we study the way in which the movements of populations in response to one another can affect the spatio-temporal distributions of ecosystems. We construct a stochastic individual-based modelling (IBM) framework, based on stigmergent interactions (i.e. organisms leave marks which cause others to alter their movements) between and within populations. We show how to gain insight into this IBM via mathematical analysis of a partial differential equation (PDE) system given by a continuum limit. We show how the combination of stochastic simulations of the IBM and mathematical analysis of PDEs can be used to categorise emergent patterns into homogeneous vs. heterogeneous, stationary vs. perpetually-fluctuating, and aggregation vs. segregation. In doing so, we develop techniques for understanding spatial bifurcations in stochastic IBMs, grounded in mathematical analysis. Finally, we demonstrate through a simple example how the interplay between environmental features and between-population stigmergent interactions can give rise to predicted spatial distributions that are quite different to those predicted purely by accounting for environmental covariates.

category: ecology


title: Arctic evergreening - encroaching allelopathy in a management blind spot

abstract: Management blind spots, i.e., influential processes not included in management models, may undermine management objectives and reduce system resilience. Here we address one such blind spot in the management of Rangifer in Norway. The current model is based on regulation of pastures and population vital rates through reindeer density, but lacks validation of the underlying assumption of forage stability, which is a critical blind spot. We report pasture changes over 18 growing seasons, under variable reindeer densities and a changing climate. We show a strong increase of the native evergreen, allelopathic crowberry, contrasted by smaller increases of deciduous dwarf-shrubs and stagnation in herbaceous species. We found no evidence of reindeer density influencing these vegetation changes. Our results unveil a trajectory of Arctic evergreening and increasing allelopathy, and a shift towards lower biodiversity and ecosystem functioning. It exemplifies an ongoing change in a management blind spot that greatly impacts social-ecological systems.


title: A functional trait approach reveals the effects of landscape context on ecosystem services provided by urban birds

abstract: Land use intensification in urban areas can have profound effects on biological communities that provide valuable ecosystem services to urban residents. We used a functional response and effect trait approach to determine how bird species responses to local and landscape-scale habitat of urban green spaces affects the supply of cultural and regulating ecosystem services. We sampled bird communities and habitat variables in urban green spaces that varied in local and landscape-scale habitat composition and compiled a dataset of species response and effect traits related to nesting, foraging, diet, and visual and acoustic aesthetic appeal. Overall, the landscape-scale context of a green space had a stronger influence on species abundances than local-scale habitat. Landscape-scale impervious surface surrounding our study sites interacted with response traits related to nesting in human-built structures, clutch size, and consumption of invertebrates to drive bird species abundances. Because correlations between response and effect traits can influence the effect traits available to provide ecosystem services at a site, we explored the correlation of these three response traits to a suite of effect traits and found the response traits were correlated with several effect traits related to diet and regulating services but correlated with few of the plumage and acoustic traits that produce cultural services. Finally, we found that effect traits associated with cultural and regulating ecosystem services varied strongly along the landscape-scale gradient of urbanization. Sites with high impervious surface cover are expected to have low levels of invertebrate pest control and visual appeal but high levels of acoustic appeal, diet evenness (generalism), and granivory. Overall, our study highlights the key role of landscape-scale habitat in driving bird-mediated ecosystem services and underscores the importance of regional urban planning to create healthy and livable cities.


title: Dynamic molecular evolution of a supergene with suppressed recombination in white-throated sparrows

abstract: In white throated sparrows, two alternative morphs differing in plumage and behavior segregate with a large chromosomal rearrangement. As with sex chromosomes such as the mammalian Y, the rearranged version of chromosome two (ZAL2m) is in a near-constant state of heterozygosity, offering opportunities to investigate both degenerative and selective processes during the early evolutionary stages of  supergenes. Here, we generated, synthesized, and analyzed extensive genome-scale data to better understand the forces shaping the evolution of the ZAL2m chromosome in this species. We found that features of ZAL2m are consistent with substantially reduced recombination and low levels of degeneration. We also found evidence that selective sweeps took place both on ZAL2m and its standard counterpart, ZAL2, after the rearrangement event. Signatures of positive selection were associated with allelic bias in gene expression, suggesting that antagonistic selection has operated on gene regulation. Finally, we discovered a region exhibiting two long-range haplotypes inside the rearrangement on ZAL2m. These two haplotypes appear to have been maintained by balancing selection, retaining genetic diversity within the supergene. Together, our analyses illuminate mechanisms contributing to the evolution of a young chromosomal polymorphism, revealing complex selective processes acting concurrently with genetic degeneration to drive the evolution of supergenes.

category: evolutionary biology


title: Discordant population structure inferred from male- and female-type mtDNAs from Limecola balthica, a bivalve species characterized by doubly uniparental inheritance of mitochondria

abstract: Doubly Uniparental Inheritance (DUI) of mitochondria is a remarkable exception to the Strictly Maternal Inheritance (SMI) in metazoans. In species characterized by DUI --almost exclusively gonochoric bivalve mollusks--, females (F) transmit mitochondria to offspring of both sexes, while males (M) pass on their mitochondria exclusively to their sons. Under DUI, males are heteroplasmic, somatic tissues containing F-transmitted mtDNA and gametic cells containing M-transmitted mtDNAs. The aforementioned transmission routes make M- and F-transmitted mtDNA interesting as sex-specific markers which can differ in their effective population sizes, mutation rates, and selective constraints. For these reasons, looking at both markers can provide significant insight into the genetic structure of populations and investigate its determinants. In this study, we document differences in genetic diversity, divergence, inter-populational genetic differentiation and biogeographic structure between M- and F-type cox1 mt genes in the Baltic tellin (Limecola balthica) to test whether cox1m and cox1f genes bear the marks of similar phylogeographic histories. Both markers were sequenced for 313 male individuals sampled from the Baltic Sea to the Gironde Estuary (Southern France). Haplotype diversity and net divergence were over twice higher in cox1m compared to cox1f. A strong southward decrease in nucleotide diversity was observed only at cox1m. Genetic differentiation between northern and southern populations was nearly 3 times higher at cox1m compared to cox1f (global {Phi}ST = 0.447 and 0.126 respectively) and the geographic localization of the strongest genetic break significantly differed between the markers (Finistere Peninsula at cox1f; Cotentin Peninsula at cox1m). A higher mutation rate, relaxed negative selective pressure and differences in effective population sizes (depending on locations) at cox1m could explain differences in population genetic structure. As both F- and M-type mtDNAs interact with nuclear genes for oxidative phosphorylation and ATP production, geographical discordances in genetic clines could be linked to mito-nuclear genetic incompatibilities in this system.


title: Diversity of sex chromosomes in Sulawesian medaka fishes

abstract: Recent genetic and genomic studies have revealed tremendous diversity in sex chromosomes across diverse taxa. Although theoretical studies predict that sex chromosome evolution can drive the evolution of sexual dimorphism, empirical studies are still limited. A taxonomic group that shows diversity in both sex chromosomes and sexual dimorphism provides excellent opportunity to investigate the link between the evolution of sex chromosomes and sexual dimorphism. The medaka fishes (family Adrianichthyidae) exhibit both rapid sex chromosome turnovers and diversification of sexually dimorphic traits. In the present study, we investigated the sex chromosomes of 13 Oryzias species from Sulawesi, Indonesia, that have diversified in sexual dichromatism. Using pooled sequencing we found sex chromosomes in 9 species that all had XY systems, with a few species being possibly modified by multiple loci. Seven species (O. woworae, O. asinua, O. wolasi, O. matanensis, O. celebensis, O. hadiatyae, and O. dopingdopingensis) share linkage group (LG) 24 as sex chromosomes; however, they differed in the length and magnitude of sequence divergence between the X and Y chromosomes. The sex chromosome of O. eversi was LG4, which has not been reported as a sex chromosome in any other medaka species. In O. sarasinorum, LG16 and LG22 are associated with sex. Although LG16 was found to be sex-linked in another medaka species previously examined, the sex-determining regions did not overlap. Frequent turnovers and the great diversity of the sex chromosomes make Sulawesian medaka species a model system for investigating the roles of sex chromosome evolution in the diversification of sexual dimorphism.


title: New Insights on the Evolutionary Relationships Between the Major Lineages of Amoebozoa

abstract: The supergroup Amoebozoa unites a wide diversity of amoeboid organisms and encompasses enigmatic lineages recalcitrant to modern phylogenetics. Deep divergences, taxonomic placement of some key taxa and character evolution in the group largely remain poorly elucidated or controversial. We surveyed available Amoebozoa genomes and transcriptomes to mine conserved putative single copy genes, which were used to enrich gene sampling and generate the largest supermatrix (824 genes) in the group to date. We recovered a well-resolved and supported tree of Amoebozoa, revealing novel deep level relationships and resolving placement of enigmatic lineages congruent with morphological data. In our analysis the deepest branching group is Tubulinea. A recent proposed major clade Tevosa, uniting Evosea and Tubulinea, is not supported. Based on the new phylogenetic tree, paleoecological and paleontological data as well as data on the biology of presently living amoebozoans, we hypothesize that the evolution of Amoebozoa probably was driven with the need to disrupt and graze on microbial mats - a dominant ecosystem of the mid-Proterozoic period of the Earth history.


title: EpiBERTope: a sequence-based pre-trained BERT model improves linear and structural epitope prediction by learning long-distance protein interactions effectively

abstract:
MotivationEpitopes are the immunogenic regions of antigen that are recognized by antibodies in a highly specific manner to trigger an immune response. Predicting such regions is extremely difficult yet contains profound implications for complex mechanisms of humoral immunogenicity.

ResultsHere, we present a BERT-based epitope prediction model called EpiBERTope, a pre-trained model on the Swiss-Prot protein database, which can predict both linear and structural epitopes using protein sequences only. The model achieves an AUC of 0.922 and 0.667 for linear and structural epitope datasets respectively, outperforming all benchmark classification models including random forest, gradient boosting, naive Bayesian, and support vector machine models. In conclusion, EpiBERTope is a sequence-based model that captures content-based global interactions of antigen sequences, which will be transformative in epitope discovery with high specificity.

Contactminjun.park@standigm.com

category: bioinformatics


title: Network analysis of Differentially Expressed Genes (DEGs) identified in zebrafish after infection with Spring viremia of carp virus (SVCV) - an in silico approach

abstract: Spring viremia of carp virus (SVCV) is a virus that belongs to family of spring viremia of carp (SVC) and frequently causes hemorrhagic symptoms in several types of cyprinids and causes severe economic and environmental losses. Therefore, the mechanism of the infection is not clearly understood. In this study, zebrafish was employed as the infection model to explore the pathogenesis of SVCV. 4 groups of zebrafish tissues were set and RNA sequencing (RNA-Seq) technology was employed to analyze the differentially expressed genes (DEGs) after SVCV-infection. A total of 360,971,498 clean reads were obtained from samples, 382 DEGs in the brain and 926 DEGs in the spleen were identified. These DEGs were annotated into three ontologies after gene ontology (GO) enrichment analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these DEGs were primarily related to Influenza. A pathway and Herpes simplex infection pathway in brain and Tuberculosis and Toxoplasmosis pathways in spleen, and all of these pathways may be involved in response to pathogen invasion. The transcriptome analysis results demonstrated changes and tissue-specific influences caused by SVCV in vivo, which provided us with more information to understand the complex relationships between SVCV and its host.


title: Data-driven large-scale genomic analysis reveals an intricate phylogenetic and functional landscape in J-domain proteins

abstract: The 70 kDalton Heat shock protein (Hsp70) chaperone system is emerging as a central node of the proteostasis network that supervises protein homeostasis in all organisms. Hsp70 recruitment to perform a vast array of different cellular functions is regulated by a family of co-chaperones known as J-domain proteins (JDP) that bear a small namesake J-domain, which is required to interact and drive the ATPase cycle of Hsp70s. Both prokaryotic and in eukaryotic JDPs display a staggering diversity in domain architecture (besides the ubiquitous J-domain), function and cellular localization. This is mirrored by a comparatively small number of Hsp70 paralogs, suggesting a degree of specificity, but also of promiscuity, in their mutual partnering. Very little is known about this family of cochaperones, despite their essential role in cellular proteostasis, development and the link to a broad range of human diseases. The numbers of JDP sequences identified as a consequence of efficient sequencing techniques has exponentially increased where it is now beyond the ability of careful manual curation. In this work, we first provide a broad view of the JDP repertoire accessible from public databases, and then we then use an automated classification scheme, based on Artificial Neural Networks (ANNs), to demonstrate that the sequences of the J-domains carry sufficient information to recover with high reliability the phylogeny, localization and domain composition of the corresponding full length JDP. Exploiting the interpretability of the ANNs employed, we find that several of the sequence positions that are most useful for the classification match to residues that form the interaction interface between the J-domain and Hsp70, revealing that a set of residues within the J-domains have co-evolved with their obligatory Hsp70 partners to build chaperone circuits for specific functions.


title: Deep Learning in Spatial Transcriptomics: A Survey of Deep Learning Methods for Spatially-Resolved Transcriptomics

abstract: Spatial transcriptomics (ST) technologies are rapidly becoming the extension of single-cell RNA sequencing (scRNAseq), holding the potential of profiling gene expression at a single-cell resolution while maintaining cellular compositions within a tissue. Having both expression profiles and tissue organization enables researchers to better understand cellular interactions and heterogeneity, providing insight into complex biological processes that would not be possible with traditional sequencing technologies. The data generated by ST technologies are inherently noisy, high-dimensional, sparse, and multi-modal (including histological images, count matrices, etc.), thus requiring specialized computational tools for accurate and robust analysis. However, many ST studies currently utilize traditional scRNAseq tools, which are inadequate for analyzing complex ST datasets. On the other hand, many of the existing ST-specific methods are built upon traditional statistical or machine learning frameworks, which have shown to be sub-optimal in many applications due to the scale, multi-modality, and limitations of spatially-resolved data (such as spatial resolution, sensitivity and gene coverage). Given these intricacies, researchers have developed deep learning (DL)-based models to alleviate ST-specific challenges. These methods include new state-of-the-art models in alignment, spatial reconstruction, and spatial clustering among others. However, deep-learning models for ST analysis are nascent and remain largely underexplored. In this review, we provide an overview of existing state-of-the-art tools for analyzing spatially-resolved transcriptomics, while delving deeper into the DL-based approaches. We discuss the new frontiers and the open questions in this field and highlight the domains in which we anticipate transformational DL applications.


title: Liquid-liquid phase separation in amino acid mixtures is governed by composition

abstract: Liquid-liquid phase separation (LLPS) has recently come to immense prominence as it is central to the formation of membraneless organelles, leading to a new paradigm of cellular organization. This type of phase transition is mediated by molecular interactions between biomolecules, including nucleic acids and both ordered and disordered proteins. In the latter case, the separation between protein-dense and dilute phases is often interpreted using models adapted from polymer theory. Specifically, the "stickers and spacers" model proposes that LLPS originates from the interplay between two classes of residues and that the main determinants for phase separation are multivalency and sequence patterning. The duality of roles of stickers (aromatics like Phe and Tyr) and spacers (Gly and polar residues) may apply more broadly in protein-like mixtures, and the presence of these two types of components alone may suffice for LLPS to take place. In order to explore this hypothesis, we use atomistic molecular dynamics simulations of capped amino-acid residues as a minimal model system. We study the behaviour of pure amino acids in water for three types of residues that are thought to play critical roles in LLPS, corresponding to the spacer and sticker categories, and their multicomponent mixtures. In agreement with previous observations, we find that the spacer-type amino acids fail to phase-separate on their own, while the sticker is prone to aggregation. However, ternary amino acid mixtures involving both types of amino acids phase-separate into two phases that retain intermediate degrees of compaction and considerable fluidity. Our results suggest that LLPS is an emergent property of amino acid mixtures determined by composition.

category: biophysics


title: Shaping the Genome via Lengthwise Compaction, Phase Separation, and Lamina Adhesion

abstract: The link between genomic structure and biological function is yet to be consolidated, it is, however, clear that physical manipulation of the genome, driven by the activity of a variety of proteins, is a crucial step. To understand the consequences of the physical forces underlying genome organization, we build a coarse-grained polymer model of the genome, featuring three fundamentally distinct classes of interactions: lengthwise compaction, i.e., compaction of chromosomes along its contour, self-adhesion among epigenetically similar genomic segments, and adhesion of chromosome segments to the nuclear envelope or lamina. We postulate that these three types of interactions sufficiently represent the concerted action of the different proteins organizing the genome architecture and show that an interplay among these interactions can recapitulate the architectural variants observed across the tree of life. The model elucidates how an interplay of forces arising from the three classes of genomic interactions can drive drastic, yet predictable, changes in the global genome architecture, and makes testable predictions. We posit that precise control over these interactions in vivo is key to the regulation of genome architecture.


title: Triple Negative Breast Cancer-derived Small Extracellular Vesicles as Modulator of Biomechanics in target cells

abstract: Extracellular vesicle (EV) mediated communication has recently been proposed as one of the pivotal routes in the development of cancer metastasis. EVs are nano-sized vesicles swapped between cells, carrying a biologically active content that can promote tumor-induced immune suppression, metastasis and angiogenesis. Thus, EVs constitute a potential target in cancer therapy. However, their role in triggering the premetastatic niche and in tumor spreading is still unclear. Here, we focused on the EV ability to modulate the biomechanical properties of target cells, known to play a crucial role in metastatic spreading. To this purpose, we isolated and thoroughly characterized triple-negative breast cancer (TNBC)-derived small EVs. We then evaluated variations in the mechanical properties (cell stiffness, cytoskeleton/nuclear/morphology and Yap activity rearrangements) of non-metastatic breast cancer MCF7 cells upon EV treatment. Our results suggest that TNBC-derived small EVs are able to directly modify MCF7 cells by inducing a decrease in cell stiffness, rearrangements in cytoskeleton, focal adhesions and nuclear/cellular morphology, and an increase in Yap downstream gene expression. Testing the biomechanical response of cells after EV addition might represent a new functional assay in metastatic cancer framework that can be exploited for future application both in diagnosis and in therapy.


title: Case Study of Single-cell Protein Activity Based Drug Prediction for Precision Treatment of Cholangiocarcinoma

abstract: Cholangiocarcinoma is a rare, aggressive malignancy with limited treatment options, due to a paucity of actionable mutations and low response to immune checkpoint inhibitors. Furthermore, its extreme heterogeneity prevents identification of actionable dependencies from bulk-tissue profiles. To address these challenges, we introduce a highly generalizable, single-cell framework for the mechanism-based prioritization of drugs to treat rare, highly heterogeneous tumors. Analysis of transformed cells, accounting for only 10% of a cholangiocarcinoma patient biopsy revealed three molecularly-distinct subpopulations, predicted to be sensitive to four drugs by regulatory network-based analysis. Validation in a low-passage, patient-derived xenograft (PDX) from the same patient confirmed tumor growth rate control by two of these drugs (plicamycin and dacinostat) and further validated predicted subpopulation-specific effects, suggesting they may represent promising candidates for follow-up clinical trials, either alone or in combination with current standard-of-care chemotherapies. The proposed approach can be generalized to elucidate complementary dependencies of rare, heterogeneous tumors, at the single cell level.


title: Addition of soluble fiber in low fat purified diets improves gut and metabolic health compared to traditional AIN diets

abstract:
BackgroundPurified diets (PDs) contain refined ingredients with one main nutrient, allowing for greater control relative to grain-based diets (GBDs), which contain unrefined grains and animal byproducts. Traditional PDs like the AIN-76A (76A) and AIN-93G (93G) can negatively impact metabolic and gut health when fed long-term, in part due to lower total fiber, no soluble fiber, and higher sucrose content.

ObjectiveTwo studies were conducted to determine how PDs with reduced sucrose and increased fiber (soluble and insoluble) influence metabolic and gut health in mice compared to traditional AIN PDs or GBDs.

MethodsIn study 1, C57Bl/6N mice consumed a GBD (5002), 76A, 93G, or 2 Open standard PDs (OSDs) with reduced sucrose and higher fiber for 88 days. Body composition and metabolic parameters were assessed. In study 2, C57Bl/6N mice consumed either 2 GBDs (5001 or 5002) or OSDs with different types/levels of fiber for 14 days. Microbiome alterations and predicted functional metagenomic changes were measured.

ResultsOSD marginally influenced body weight and adiposity, but improved glucose tolerance relative to 93G (p = 0.0131) and 76A (p = 0.0014). Cecal and colonic weights were lower in mice fed cellulose-based PDs compared to those fed GBDs and soluble fiber PDs. Soluble fiber diets reduced alpha diversity and showed similar beta diversity, which differed from cellulose fed PDs and GBDs. Certain genera associated with improved gut health such as Bifidobacteria and Akkermansia were significantly elevated by soluble fibers PDs (p[≤]0.01). Some metabolic pathways related to carbohydrate and fatty acid metabolism were affected by PDs.

ConclusionPDs formulated with lower sucrose and increased fiber content, particularly soluble fiber, blunted elevations in metabolic parameters and favorably impacted microbiota and metagenome in C57BL/6N mice.

category: physiology


title: The endoplasmic reticulum in perisynaptic astrocytic processes: shape, distribution and effect on calcium activity

abstract: Astrocytes recently emerged as key regulators of information processing in the brain. Ca2+ signals in perisynaptic astrocytic processes (PAPs) notably allow astrocytes to fine-tune neurotransmission at so-called tripartite synapses. As most PAPs are below the diffraction limit, their content in Ca2+ stores and the contribution of the latter to astrocytic Ca2+ activity is unclear. Here, we reconstruct tripartite synapses in 3D from electron microscopy and find that 75% of PAPs contain some endoplasmic reticulum (ER), a major astrocytic Ca2+ store, displaying strikingly diverse geometrical properties. To investigate the role of such spatial properties, we implemented an algorithm that creates 3D PAP meshes of various ER distributions and constant shape. Reaction-diffusion simulations in those meshes reveal that astrocyte activity is shaped by a complex interplay between the location of Ca2+ channels, Ca2+ buffering, ER shape and distribution. Overall, this study sheds new light into mechanisms regulating signal transmission in the brain.


title: Visual Perception of 3D Space and Shape in Time - Part II: 3D Space Perception with Holographic Depth

abstract:
Visual perception plays a critical role in navigating 3D space and extracting semantic information crucial to survival. Even though visual stimulation on the retina is fundamentally 2D, we seem to perceive the world around us in vivid 3D effortlessly. This reconstructed 3D space is allocentric and faithfully represents the external 3D world. How can we recreate stable 3D visual space so promptly and reliably?

To solve this mystery, we have developed new concepts MePMoS (Memory-Prediction-Motion-Sensing) and NHT (Neural Holography Tomography). These models state that visual signal processing must be primarily top-down, starting from memory and prediction. Our brains predict and construct the expected 3D space holographically using traveling alpha brainwaves. Thus, 3D space is represented by the three time signals in three directions.

To test this hypothesis, we designed reaction time (RT) experiments to observe predicted space-to-time conversion, especially as a function of distance. We placed LED strips on a horizontal plane to cover distances from close up to 2.5 m or 5 m, either using a 1D or a 2D lattice. Participants were instructed to promptly report observed LED patterns at various distances. As expected, stimulation at the fixation cue location always gave the fastest RT. Additional RT delays were proportional to the distance from the cue. Furthermore, both covert attention (without eye movements) and overt attention (with eye movements) created the same RT delays, and both binocular and monocular views resulted in the same RTs. These findings strongly support our predictions, in which the observed RT-depth dependence is indicative of the spatiotemporal conversion required for constructing allocentric 3D space. After all, we perceive and measure 3D space by time as Einstein postulated a century ago.


title: In vivo physiology of foveal retinal ganglion cells in Macaca fascicularis

abstract: The primate fovea is specialized for high-resolution spatial and chromatic vision, possessing a unique topography characterized by a pit in which the inner retina and associated vasculature have been excavated, the highest density of cones anywhere in the retina, and a disproportionately large representation in visual cortex. The crucial visual properties conferred by the fovea are mediated by retinal ganglion cells (RGCs) the somas of which lie at the margin of the foveal pit. Microelectrode recordings of these centermost retinal ganglion cells have been challenging due to the fragility of the fovea in the excised retina. This has been overcome in this study by combining high resolution fluorescence adaptive optics ophthalmoscopy with calcium imaging to optically record functional responses of foveal RGCs to visual stimuli in the living eye. Here we use this approach to study the chromatic responses of RGCs to uniform fields modulated in different directions in color space and achromatic responses with monochromatic drifting gratings. We repeated these recordings in many cells in each of three macaca fasicularis primates over weeks or months. We find that most of the L/M chromatically opponent cells serving the most central cones respond to high spatial frequencies, reflecting strong surround inhibition that sacrifices sensitivity at low frequencies in favor of the transmission of fine detail in the retinal image. In addition, we find the L/M cells vary in spatial frequency bandwidths, suggesting that L/M chromatic opponency, often considered largely due to input from single cones, has more diverse receptive field structure than previously thought. By characterizing the functional properties of retinal ganglion cells in vivo through adaptive optics, we provide a lower bound on the response characteristics of these cells in situ and their role in vision processing.


title: Chronic methamphetamine administration produces cognitive deficits through augmentation of GABAergic synaptic transmission in the prefrontal cortex

abstract:
BACKGROUNDChronic methamphetamine (METH) abuse is associated with the emergence of cognitive deficits and hypofrontality, a pathophysiological marker of many neuropsychiatric disorders that is produced by altered balance of local excitatory and inhibitory synaptic transmission. However, there is a dearth of information regarding the cellular and synaptic mechanisms underlying METH-induced cognitive deficits and associated hypofrontal states.

METHODSRats went through a METH sensitization regime or saline (SAL) consisting of 14 days of METH treatment (day 1 and 14, 1 mg/kg; days 2-13, 5 mg /kg) followed by 7-10 days of home cage abstinence. Temporal Order Memory and Working Memory tests, chemogenetic experiments as well as whole-cell patch recordings on prelimbic PFC ex vivo slices were performed during abstinence.

RESULTSWe find here that repeated METH administration in rats produces deficits in working memory and increases in inhibitory synaptic transmission onto pyramidal neurons in the prefrontal cortex (PFC). The increased PFC inhibition is detected by an increase in spontaneous and evoked inhibitory postsynaptic synaptic currents (IPSCs), an increase in GABAergic presynaptic function, and a shift in the excitatory-inhibitory balance onto PFC deep-layer pyramidal neurons. We find that pharmacological blockade of D1 dopamine receptor function reduces the METH-induced augmentation of IPSCs, suggesting a critical role for D1 dopamine signaling in METH-induced hypofrontality. In addition, chronic METH administration increases the intrinsic excitability of parvalbumin-positive interneurons, a key local interneuron population in PFC that controls inhibitory tone. Using a cell type-specific chemogenetic approach, we show that increasing PV+FSI activity in the PFC is necessary and sufficient to cause deficits in temporal order memory similar to those induced by METH.

CONCLUSIONTogether, our findings reveal that chronic METH exposure increases PFC inhibitory tone through a D1 dopamine signaling-dependent potentiation of inhibitory synaptic transmission, and that reduction of PV+FSI activity can rescue METH-induced cognitive deficits, suggesting a potential therapeutic approach to treating cognitive symptoms in patients suffering from methamphetamine use disorder.


title: Transcranial random noise stimulation of the primary visual cortex but not retina modulates visual contrast sensitivity.

abstract:
Transcranial random noise stimulation (tRNS) has been shown to significantly improve visual perception. Previous studies demonstrated that tRNS delivered over cortical areas acutely enhances visual contrast detection of stimuli when tRNS intensity is optimized for the individual. However, it is currently unknown whether tRNS-induced signal enhancement could be achieved within different neural substrates along the retino-cortical pathway and whether the beneficial effect of optimal tRNS intensities can be reproduced across sessions.

In 3 experimental sessions, we tested whether tRNS applied to the primary visual cortex (V1) and/or to the retina improves visual contrast detection. We first measured visual contrast detection threshold (VCT; N=24, 16 females) during tRNS delivery separately over V1 (no tRNS, 0.75, 1, 1.5mA) and over the retina (no tRNS, 0.1, 0.2, 0.3mA), determined the optimal tRNS intensities for each individual (ind-tRNS), and retested the effects of ind-tRNS within the sessions. We further investigated whether we could reproduce the ind-tRNS-induced modulation on a different session (N=19, 14 females). Finally, we tested whether the simultaneous application of ind-tRNS to the retina and V1 causes additive effects.

We found that at the group level tRNS of 0.75mA decreases VCT compared to baseline when delivered to the V1. Beneficial effects of ind-tRNS could be replicated when retested within the same experimental session but not when retested in a separate session. Applying tRNS to the retina did not cause a systematic reduction of VCT, irrespective of whether the individually optimized intensity was considered or not. We also did not observe consistent additive effects of V1 and retina stimulation.

Our findings demonstrate that V1 seems to be more sensitive than the retina to tRNS-induced modulation of visual contrast processing.

Significance statementOur findings confirm previous evidence showing acute online benefits of tRNS of V1 on visual contrast detection in accordance with the stochastic resonance phenomenon. We further extend it, demonstrating that the optimal tRNS intensity varies among participants, but when individually tailored it can improve visual processing when re-tested within the experimental session. The tRNS-induced enhancement in visual sensitivity seems to be specific for cortical contrast processing as stimulation of the retina did not lead to systematic effects.


title: Turning the head while biking makes older people lose cycling direction and balance

abstract:
BackgroundBike safety in older subjects is a major concern in multiple countries given the strong increase in the number of cyclists that are admitted to the hospital due to single-sided bicycle accidents in older populations. The increase in single-sided accident rate in older subjects suggests that older adults have more difficulty controlling balance while bicycling in traffic, which has been related to various age-related changes in the sensorimotor system. Yet, the impact of these age-related sensorimotor deficits on the ability of older people to remain stable on their bike during traffic situations such as turning their upper body to check on potential road users, remains unknown.

MethodsWe instructed a group of 40 young (22.86 {+/-} 1.53 years) and 41 older participants (62.73 {+/-} 1.57 years) to bike in a straight line while performing a shoulder check movement in order to identify the color of an object presented behind them. We recorded the task-errors (lost balance, errors in identifying the color, cycling outside the lane) and computed the steering angle and rotation of the frame, pelvis and torso from IMU recordings.

ResultsWe observed that older adults made task-errors (one third of the older participants failed at the task by making errors such as cone-identification errors, loss of balance and cycling outside the lane). In the successful trials, we observed an increase in steering angle and rotation of the pelvis with respect to the frame in the older subjects compared to the young subjects.

SignificanceOlder cyclists lose balance while turning their head, which might contribute to the increased number of single sided bike accidents in older subjects. The use of simple devices, such as using a mirror, should be encouraged in older subjects to replace the shoulder check maneuver.


title: Synergistic effect of mutations in the Parkinson's disease-linked proteins synaptojanin 1 and auxilin

abstract: Parkinsons disease (PD) is a neurodegenerative disorder characterized by defective dopaminergic (DAergic) input to the striatum. Mutations in two genes encoding synaptically-enriched clathrin-uncoating factors, synaptojanin 1 (SJ1) and auxilin, have been implicated in PD. SJ1 knock-in (SJ1-KIRQ) mice carrying PD mutation display neurological manifestation reminiscent of PD. Here we report that auxilin knockout (Aux-KO) mice display dystrophic changes of a subset of nigrostriatal DAergic terminals similar to those of SJ1-KIRQ mice. Furthermore, Aux-KO/SJ1-KIRQ double mutant mice have shorter lifespan and more severe synaptic defects than single mutant mice. These include increase in dystrophic striatal nerve terminals positive for DAergic markers and for the PD risk protein SV2C, as well as adaptive changes in striatal interneurons. The synergistic effect of the two mutations demonstrates a special lability of DAergic neurons to defects in clathrin uncoating, with implications for PD pathogenesis in at least some forms of this condition.


title: Synergistic effect of mutations in the Parkinson's disease-linked endocytic proteins synaptojanin 1 (PARK20) and auxilin (PARK19)


title: Genetic Variation and Disease Severity of Respiratory Syncytial Viruses

abstract:
Respiratory Syncytial Virus (RSV) disease in newborns ranges from mild symptoms to severe disease requiring hospitalization. RSV is classified into two subtypes (RSVA and RSVB) based on antigenic and genetic differences. The role these genomic variations play in disease severity remains unknown. Genome sequences were obtained using next-generation RNA sequencing on archived frozen nasal swabs of young children (< 8 months-old) infected with RSV in Rochester, NY between 1977-1998. Samples were chosen from both children hospitalized with severe RSV disease (inpatient) and those presenting with mild symptoms (outpatient) during their first cold-season. Both A and B subtypes demonstrated significant differences in the phylogeny and primary-protein structure during this time period. We found a significant association between RSV phylogeny over this time period and disease severity. For both subtypes, the G-protein demonstrated the greatest amino acid substitutions, although the number of amino acid substitutions was higher in the RSVA subtype. We found a significant association between G-protein variation and disease severity for RSVA, but not RSVB. For both subtypes, variation in the M2-2 protein was significantly associated with disease severity. These results suggest that the genetic variability of RSV proteins may contribute to disease severity in humans.

ImportanceEach cold-season Respiratory Syncytial Virus (RSV) infects thousands of children in the US. Some will display mild cold symptoms while others develop severe disease, sometimes resulting in lifelong lung problems or fatality. RSV initiates infection and replicates in the nasopharynx. Substitutions in the RSV genome can be found in clinically isolated nasal-swab samples of RSV infected children. Whether these genome variations contribute to severe disease is unknown. Here we found a statistically significant association between RSV phylogeny and disease severity. Furthermore, we found specific RSV proteins (G and M2-2) whose amino acid variation was statistically associated with severe disease, although which protein was associated depended on subtype. Taken together, our results suggest that RSV genotype contributed to disease severity over this time period.

category: microbiology


title: LIN-35 is necessary in both the soma and germline for preserving fertility in Caenorhabditis elegans under moderate temperature stress

abstract: Maintenance of germline function under stress conditions is crucial for species survival. The germline across many species is especially sensitive to elevated temperature stress. We have investigated the role that the pocket protein LIN-35 plays in preserving fertility in C. elegans under moderate high temperature stress. Here we show that lin-35 mutants have a number of temperature sensitive germline defects. The decreased brood size seen at elevated temperatures is more severe in lin-35 mutants than wild type. This loss of fertility under temperature stress is primarily due to loss of zygotic but not maternal LIN-35. Additionally, we have found that expression of LIN-35 is necessary in both the germline and soma for the preserving fertility at elevated temperature. Specifically, germline expressed LIN-35 is primarily what is important for maintaining fertility in hermaphrodites, but broad somatic expression of LIN-35 is also necessary for oocyte formation and/or function at elevated temperatures. Additionally, we found that overexpression of LIN-35 in the germline only can stabilize the formation of P granules in primordial germ cells. Our data adds to the emerging critical role that LIN-35 plays in buffering tissues against stress.

category: genetics


title: Discovery of compounds that inhibit SARS-CoV-2 Mac1-ADP-ribose binding by high-throughput screening

abstract: The emergence of several zoonotic viruses in the last twenty years, especially the pandemic outbreak of SARS-CoV-2, has exposed a dearth of antiviral drug therapies for viruses with pandemic potential. Developing a diverse drug portfolio will be critical for our ability to rapidly respond to novel coronaviruses (CoVs) and other viruses with pandemic potential. Here we focus on the SARS-CoV-2 conserved macrodomain (Mac1), a small domain of non-structural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that cleaves mono-ADP-ribose (MAR) from target proteins, protects the virus from the anti-viral effects of host ADP-ribosyltransferases, and is critical for the replication and pathogenesis of CoVs. In this study, a luminescent-based high-throughput assay was used to screen [~]38,000 small molecules for those that could inhibit Mac1-ADP-ribose binding. We identified 5 compounds amongst 3 chemotypes that inhibit SARS-CoV-2 Mac1-ADP-ribose binding in multiple assays with IC50 values less than 100{micro}M, inhibit ADP-ribosylhydrolase activity, and have evidence of direct Mac1 binding. These chemotypes are strong candidates for further derivatization into highly effective Mac1 inhibitors.

category: biochemistry


title: Flipped Over U: Structural Basis for dsRNA Cleavage by the SARS-CoV-2 Endoribonuclease

abstract: Coronaviruses generate double-stranded (ds) RNA intermediates during viral replication that can activate host immune sensors. To evade activation of the host pattern recognition receptor MDA5, coronaviruses employ Nsp15, which is uridine-specific endoribonuclease. Nsp15 is proposed to associate with the coronavirus replication-transcription complex within double-membrane vesicles to cleave these dsRNA intermediates. How Nsp15 recognizes and processes dsRNA is poorly understood because previous structural studies of Nsp15 have been limited to small single-stranded (ss) RNA substrates. Here we present cryo-EM structures of SARS-CoV-2 Nsp15 bound to a 52nt dsRNA. We observed that the Nsp15 hexamer forms a platform for engaging dsRNA across multiple protomers. The structures, along with site-directed mutagenesis and RNA cleavage assays revealed critical insight into dsRNA recognition and processing. To process dsRNA Nsp15 utilizes a base-flipping mechanism to properly orient the uridine within the active site for cleavage. Our findings show that Nsp15 is a distinctive endoribonuclease that can cleave both ss- and dsRNA effectively.


title: GraphBio: a shiny web app to easily perform popular visualization analysis for omics data

abstract:
SummaryHere, we present GraphBio, a shiny web app to easily perform visualization analysis for omics data. GraphBio provides 15 popular visualization analysis methods, including heatmap, volcano plots, MA plots, network plots, dot plots, chord plots, pie plots, four quadrant diagrams, venn diagrams, cumulative distribution curves, PCA, survival analysis, ROC analysis, correlation analysis and text cluster analysis. It enables experimental biologists without programming skills to easily perform popular visualization analysis and get publication-ready figures.

Availability and implementationGraphBio, as an online web application, is freely available at http://www.graphbio1.com/en/ (English version) and http://www.graphbio1.com/ (Chinese version).

Contactdatabio@163.com


title: Fly-casting with ligand-sliding and orientational selection to support the complex formation of a GPCR and a middle-sized flexible molecule

abstract: To elucidate computationally a binding mechanism of a middle-sized flexible molecule, bosentan, to a GPCR protein, human endothelin receptor type B (hETB), a GA-guided multidimensional virtual-system coupled molecular dynamics (GA-mD-VcMD) simulation was performed. This method is one of generalized ensemble methods and produces a free-energy landscape of the ligand-receptor binding by searching large-scale motions accompanied with stably keeping the fragile cell-membrane structure. All molecular components (bosentan, hETB, membrane, and solvent) were represented with an all-atom model, and sampling was carried out from conformations where bosentan was distant from the binding site in the hETBs binding pocket. The deepest basin in the resultant free-energy landscape was assigned to the native-like complex conformation. The obtained binding mechanism is as follows. First, bosentan fluctuating randomly in solution is captured by a tip region of the flexible N-terminal tail of hETB via nonspecific attractive interactions (fly-casting). Bosentan then occasionally slides from the tip to root of the N-terminal tail (ligand-sliding). In this sliding, bosentan passes the gate of the binding pocket from outside to inside of the pocket with accompanying a quick reduction of the molecular orientational variety of bosentan (orientational selection). Last, in the pocket, ligand-receptor attractive native contacts are formed, and eventually the native-like complex is completed. The bosentan-captured conformations by the tip- and root-regions of the N-terminal tail correspond to two basins in the free-energy landscape, and the ligand-sliding corresponds to overcoming a free-energy barrier between the basins.


title: Human hippocampal responses to network stimulation vary with theta phase

abstract: Hippocampal memory function is thought to be supported by distinct connectivity states, with strong extrahippocampal input benefitting encoding and weak extrahippocampal input benefitting retrieval. Previous research in rodents suggests that the hippocampal theta oscillation orchestrates the transition between these states, with opposite phase angles respectively predicting minimal and maximal extrahippocampal input. However, it is unclear whether this phase dependence exists in humans. We used network-targeted stimulation to investigate whether hippocampal receptivity to network input varies with theta phase in humans of both sexes. Intracranial local field potentials were recorded from individuals with epilepsy undergoing medically necessary stereotactic electroencephalographic recording. In each subject, biphasic bipolar direct electrical stimulation was delivered to lateral temporal sites with demonstrated connectivity to hippocampus. Stimulation evoked ipsilateral hippocampal potentials with distinct early and late negative components. Using evoked-component amplitude to measure functional connectivity, we assessed whether different phases of hippocampal theta reflected distinct connectivity states. Stimulation enhanced both early and late component amplitudes when delivered during the observed theta peak relative to trough. Analysis of estimated theta phase delays between stimulation locations and hippocampus suggested that hippocampal response amplitude was maximized when stimulation influenced hippocampal activity at approximately 90{degrees} before the trough of its theta rhythm. The pattern of theta phase dependence observed in hippocampus was not identified for amygdala and orbitofrontal cortex control locations. These findings demonstrate that theta phase reflects connectivity states of human hippocampal networks, confirming a putative mechanism by which oscillations modulate local hippocampal processing.


title: Localizing temporal lobe seizure onset zones using deep learning on SEEG cortico-cortical evoked potentials

abstract: In drug resistant temporal lobe epilepsy, automated tools for seizure onset zone (SOZ) localization using brief interictal recordings would supplement presurgical evaluations and improve care. Thus, we sought to localize SOZs by training a multi-channel convolutional neural network on stereo-EEG (SEEG) cortico-cortical evoked potentials. We performed single pulse electrical stimulation with 10 drug resistant temporal lobe epilepsy patients implanted with SEEG. Using the 500,000 unique post-stimulation SEEG epochs, we trained a multi-channel one-dimensional convolutional neural network to determine whether an SOZ was stimulated. SOZs were classified with a mean leave-one-patient-out testing sensitivity of 78.1% and specificity of 74.6%. To achieve maximum accuracy, the model requires a 0-350 ms post stimulation time period. Post-hoc analysis revealed that the model accurately classified unilateral vs bilateral mesial temporal lobe seizure onset, and neocortical SOZs. This is the first demonstration, to our knowledge, that a deep learning framework can be used to accurately classify SOZs using cortico-cortical evoked potentials. Our findings suggest accurate classification of SOZs relies on a complex temporal evolution of evoked potentials within 350 ms of stimulation. Validation in a larger dataset could provide a practical clinical tool for the presurgical evaluation of drug resistant epilepsy.


title: Neuronal-spiking-based closed-loop stimulationduring cortical ON and OFF states in freely moving mice

abstract: The slow oscillation (SO) is a central neuronal dynamic during sleep and is generated by alternating periods of high and low neuronal activity (ON and OFF states). Mounting evidence causally links the SO to sleeps functions, and it has recently become possible to manipulate the SO non-invasively and phase-specifically. These developments represent promising clinical avenues, but they also highlight the importance of improving our understanding of how ON/OFF states affect incoming stimuli and what role they play in neuronal plasticity. Most studies using closed-loop stimulation rely on the electroencephalogram (EEG) and local field potential (LFP) signals, which reflect neuronal ON and OFF states only indirectly. Here we develop an online detection algorithm based on spiking activity recorded from laminar arrays in mouse motor cortex. We find that online detection of ON and OFF states reflects specific phases of spontaneous LFP SO. Our neuronal-spiking-based closed-loop procedure offers a novel opportunity for testing the functional role of SO in sleep-related restorative processes and neural plasticity.


title: Reconstruction and simulation of thalamoreticular microcircuitry

abstract: Thalamoreticular circuitry is known to play a key role in attention, cognition and the generation of sleep spindles, and is implicated in numerous brain disorders, but the cellular and synaptic mechanisms remain intractable. Therefore, we developed the first detailed computational model of mouse thalamus and thalamic reticular nucleus microcircuitry that captures morphological and biophysical properties of [~]14,000 neurons connected via [~]6M synapses, and recreates biological synaptic and gap junction connectivity. Simulations recapitulate multiple independent network-level experimental findings across different brain states, providing a novel unifying cellular and synaptic account of spontaneous and evoked activity in both wakefulness and sleep. Furthermore, we found that: 1.) inhibitory rebound produces frequency-selective enhancement of thalamic responses during wakefulness, in addition to its role in spindle generation; 2.) thalamic interactions generate the characteristic waxing and waning of spindle oscillations; and 3.) changes in thalamic excitability (e.g. due to neuromodulation) control spindle frequency and occurrence. The model is openly available and provides a new tool to interpret spindle oscillations and test hypotheses of thalamoreticular circuit function and dysfunction across different network states in health and disease.


title: Thalamic control of sensory enhancement and sleep spindle properties in a biophysical model of thalamoreticular microcircuitry


title: Peptidoglycan recognition protein S2 is crucial for activation the Toll pathway against Israeli acute paralysis virus infection in honey bee Apis mellifera

abstract:
Although honey bee responses to pathogens have been systematically described in the past decades, antiviral signalling pathways mechanisms are not thoroughly characterized. To decipher direct antiviral roles of an immune pathway, we firstly used the infectious clone of Israeli acute paralysis virus (IAPV) to screen 42 immune genes involved in mTOR, MAPK, Toll, Endocytosis, Jak-STAT pathway and homeobox protein, heat shock protein, as well as antimicrobial peptides (AMPs), and found that Toll pathway was a potential predominant immune pathway in Apis mellifera. Consistent with this, only dsRNA-PGRP-S2 treated A. mellifera significantly exhibited impaired activation of Toll pathway, promoting susceptibility to the IAPV infection. Finally, immunofluorescence results confirmed that the Toll pathway was initiated by peptidoglycan recognition protein S2 (PGRP-S2) interacting with Toll protein. Co-immunoprecipitation findings also further preliminarily confirmed PGRP-S2 directly interacting with viral capsid protein IAPV-VP3 to induce the activation of the Toll pathway in A. mellifera. These findings highlight that the Toll pathway is demanded efficient inhibitions of IAPV replication as a specific antiviral pathway in A. mellifera, and PGRP-S2, acting as a pattern recognition receptor, could be a new approach for control of the viral disease.

Author summaryHoney bee viruses, particularly IAPV, had been implicated in the colony decline with a global distribution resulting in insufficient pollination services. However, little is known about the antiviral mechanism of honey bee. In this study, we found that the Toll pathway was required for A. mellifera against IAPV infection and initiated by PGRP-S2. We also confirmed that dsRNA-PGRP-S2 treated A. mellifera exhibited impaired Toll pathway activation and promoted susceptibility to the IAPV infection. As a result, we employed co-immunoprecipitation technique to identify the interaction between the PGRP-S2 with Toll. Moreover, it was found the PGRP-S2 directly recognized IAPV-VP3 to activate the immune pathway against IAPV infection. Our work provides novel evidence that honey bees own a specific antiviral immune pathway and suggests that targeting PGRP-S2 could be a new approach for controlling the viral disease.


title: Identification of highly cross-reactive mimotopes for a public T cell response in murine melanoma

abstract: While immune checkpoint blockade results in durable responses for some patients, many others have not experienced such benefits. These treatments rely upon reinvigorating specific T cell-antigen interactions. However, it is often unknown what antigens are being recognized by T cells or how to potently induce antigen-specific responses in a broadly applicable manner. Here, we characterized the CD8+ T cell response to a murine model of melanoma following combination immunotherapy to determine the basis of tumor recognition. Sequencing of tumor-infiltrating T cells revealed a repertoire of highly homologous TCR sequences that were particularly expanded in treated mice and which recognized an antigen from an endogenous retrovirus. While vaccination against this peptide failed to raise a protective T cell response in vivo, engineered antigen mimotopes induced a significant expansion of CD8+ T cells cross-reactive to the original antigen. Vaccination with mimotopes resulted in killing of antigen-loaded cells in vivo yet showed modest survival benefit in a prophylactic vaccine paradigm. Together, this work demonstrates the identification of a dominant tumor-associated antigen and generation of mimotopes which can induce robust functional T cell responses that are cross-reactive to the endogenous antigen across multiple individuals.


title: Rapid hypermutation B cell trajectory recruits previously primed B cells upon third SARS-CoV-2 mRNA vaccination

abstract:
High antibody affinity against the ancestral SARS-CoV-2 strain seems to be necessary (but not always sufficient) for the control of emerging immune-escape variants. Therefore, aiming at strong B cell somatic hypermutation - not only at high antibody titers - is a priority when utilizing vaccines that are not targeted at individual variants. Here, we developed a next-generation sequencing based SARS-CoV-2 B cell tracking protocol to rapidly determine the level of immunoglobulin somatic hypermutation at distinct points during the immunization period. The percentage of somatically hypermutated B cells in the SARS-CoV-2 specific repertoire was low after the primary vaccination series, evolved further over months and increased steeply after boosting. The third vaccination mobilized not only naive, but also antigen-experienced B cell clones into further rapid somatic hypermutation trajectories indicating increased affinity. Together, the strongly mutated post-booster repertoires and antibodies deriving from this may explain why the booster, but not the primary vaccination series, offers some protection against immune-escape variants such as Omicron B.1.1.529.

Brief summaryPriming SARS-CoV-2 vaccinations generate antibodies from low-level matured B cells while the third vaccination strongly boosts somatic hypermutation potentially explaining different protection from immune-escape variants.


title: Functional characterization of the thrombospondin-related paralogous proteins rhoptry discharge factor 1 and 2 unveils phenotypic plasticity in Toxoplasma gondii rhoptry exocytosis

abstract: To gain access to the intracellular cytoplasmic niche essential for their growth and replication, apicomplexan parasites such as Toxoplasma gondii rely on the timely secretion of two types of apical organelles named micronemes and rhoptries. Rhoptry proteins are key to host cell invasion and remodelling, however, the molecular mechanisms underlying the thight control of rhoptry discharge are poorly understood. Here, we report the identification and functional characterization of two novel T. gondii thrombospondin-related proteins implicated in rhoptry exocytosis. The two proteins, already annotated as MIC15 and MIC14, were renamed rhoptry discharge factor 1 (RDF1) and rhoptry discharge factor 2 (RDF2) and found to be exclusive of the Coccidia class of apicomplexan parasites. Furthermore, they were shown to have a paralogous relationship and share a C-terminal transmembrane domain followed by a short cytoplasmic tail. Immunofluorescence analysis of T. gondii tachyzoites revealed that RDF1 presents a diffuse cytoplasmic localization not reminiscent of any know subcellular compartment, whereas RDF2 was not detected. Using a conditional knockout approach, we demonstrated that RDF1 loss caused a marked growth defect. The lack of the protein did not affect parasite gliding motility, host cell attachment, replication and egress, whereas invasion was dramatically reduced. Notably, while RDF1 depletion did not result in altered microneme exocytosis, rhoptry discharge was found to be heavily impaired. Interestingly, rhoptry secretion was partially reversed by spontaneous upregulation of the RDF2 gene in knockdown parasites grown under constant RDF1 repression. Collectively, our results identify RDF1 and RDF2 as additional key players in the pathway controlling rhoptry discharge. Furthermore, this study unveils a new example of compensatory mechanism contributing to phenotypic plasticity in T. gondii.


title: Longitudinal analysis of the gut microbiome in the 5xfAD mouse model of Alzheimer's disease

abstract:
INTRODUCTIONMicrobial exposures impact Alzheimers disease (AD), and a baseline understanding of AD model microbiomes is vital for improving model efficacy. We describe the evaluation of the microbiome and metabolome in longitudinal samples from the 5xfAD transgenic mouse model.

METHODSCecal and fecal samples from 5xfAD and wild-type B6J (WT) animals from 4- 18 months of age were subjected to DNA extraction and shotgun Illumina sequencing. Metabolomics was performed on plasma and feces from a subset of animals.

RESULTSSignificant sex, age, and cage-specific differences were observed in the microbiome. Eight bacteria species were elevated in older 5xfAD mice and nine were depleted, including Turicibacter spp. Contradicting published findings covering persons with AD, plasma measurements revealed elevated serotonin in 18 month 5xfAD animals.

DISCUSSIONTaken together, these findings strengthen the link between Turicibacter abundance and AD and provide a basis for further microbiome studies of murine models for AD.


title: Deletion of proU suppresses proQ phenotypes in Escherichia coli

abstract: The ProQ protein interacts as an RNA chaperone with diverse RNA molecules in Escherichia coli. ProQ is implicated in the bacterial osmotic stress response. When the osmotic pressure is high, cells maintain their hydration by accumulating organic solutes denoted osmolytes. Transporters ProP and ProU (which is ProVWX) mediate osmolyte accumulation by Escherichia coli. Mutations at proQ impair ProP activity by reducing ProP levels (the ProQ transport phenotype) but do not impair ProU activity or reduce the level of ProX. The proQ- bacteria are longer than proQ+ bacteria during growth in either low or high salinity medium and they grow slowly at high salinity (the ProQ growth phenotype). In addition, spherical cells with crescent-shaped, nucleic acid-rich foci appear and cells lyse (the ProQ morphological phenotypes). In this work, the proQ transport phenotype was suppressed by deletions of proU, or by an insertion of IS5 in proU, when proP was expressed from the chromosome or from the heterologous, plasmid-based PBAD promoter. A point mutation disrupting the Walker B motif of ProV inactivated ProU but did not suppress the transport phenotype. ProP activities and ProP levels varied in parallel, so proQ and proU act at the same level to regulate ProP expression. Deletion of the proU operon also suppressed the growth and morphological phenotypes. The proU locus may overlap the gene encoding a regulatory sRNA that acts with ProQ, contributing to cellular morphogenesis and osmotic stress tolerance, or the relationship between ProQ and proU may be indirect.


title: Combining transgenesis with paratransgenesis to fight malaria

abstract:
Malaria is among the deadliest infectious diseases and Plasmodium, the causative agent, needs to complete a complex development cycle in its vector mosquito for transmission to occur. Two promising strategies to curb transmission are transgenesis, consisting of genetically engineering mosquitoes to express anti-malarial effector molecules and paratransgenesis, consisting of introducing into the mosquito, commensal bacteria engineered to express anti-malarial effector molecules. Although both approaches restrict parasite development in the mosquito, it is not known how their effectiveness compares. Here we provide an in-depth assessment of transgenesis and paratransgenesis and evaluate the combination of the two approaches. Using the Q-system to drive gene expression, we engineered mosquitoes to produce and secrete two effectors - scorpine and the MP2 peptide - into the mosquito gut and salivary glands. We also engineered Serratia, a commensal bacterium capable to spread through mosquito populations, to secrete the same two effectors into the mosquito gut. Whereas both mosquito-based and bacteria-based approaches strongly reduced the oocyst and sporozoite intensity, a substantially stronger reduction of P. falciparum development was achieved when transgenesis and paratransgenesis were combined. Most importantly, transmission of P. berghei from infected to naive mice was maximally inhibited by the combination of the two approaches. Combining these two strategies promise to become a powerful approach to combat malaria.

SignificanceMalaria kills hundreds of thousand persons yearly. Clearly, new approaches are needed to fight this disease. Two promising approaches are based on the concept of genetically modifying the mosquito to make it a poor vector for the parasite: 1) transgenesis (engineering the mosquito to deliver anti-malarial compounds) and 2) paratransgenesis (engineering mosquito symbiotic bacteria to deliver anti-malarial compounds). The key questions addressed by this manuscript are: which of the two is the most promising approach? And because transgenesis and paratransgenesis are not mutually exclusive, would the combination of both be the most effective strategy? Our results argue for the combination of the two, showing the additive impact that these two strategies may have in controlling malaria transmission in the field.


title: Remdesivir, Zidovudine (AZT) and Nevirapine inhibit Chandipura virus replication through high energy interactions with the RdRp domain of the polymerase protein L

abstract: Chandipura Virus (CHPV), a rhabdovirus belonging to mononegavirales, is an emerging pathogen in Indian subcontinent. The virus infection causes fever, brain encephalitis among the young children below 14 yrs of age. In recent past, several outbreaks and deaths among children were reported from in India. There are no targeted drugs or vaccines available against CHPV and symptomatic treatments are the only option. In this background, we aimed to investigate the inhibitory effects of some priviously known viral RNA polymerase inhibitor drugs on CHPV replication. First, we examined remdesivir, which is known to inhibit HCV, Ebola and SARS-CoV-2 replication and close structural similarity along with conserved residues in the finger region of RNA dependent RNA polymerase (RdRp) domain is the basis of replication inhibition. Our results showed that remdesivir inhibits CHPV replication in vero E6 cells to a significant level. In this study we have also included non-nucleoside anti-retroviral inhibitor (NNRTI) drug nevirapine, and nucleoside inhibitor (NRTI) drug AZT (Zidovudine) to determine if these are also able to inhibit CHPV replication. Interestingly, we observed inhibition of CHPV replication by both nevirapine and AZT (in the order nevirapine>AZT), albeit to a lesser extent compared to remdesivir. We next performed molecular docking and modeling study to get an insight about the interactions of these drugs with CHPV polymerase protein. Modeling study predicts that remdesivir has most favourable CHPV polymerase binding energy among these three drugs. Both remdesivor and AZT binds near the polymerase active site through interctions with residues in finger and palm regions of RdRp. In contrast, nevirapine binds to the N-terminal domain (NTD) of the RdRp. In summary, we found remdesivir as a potent inhibitor of CHPV. A combination therapy including remdesivir, nevirapine and AZT may be a better drug cocktail to treat CHPV disease. Our findings warrant further studies of these drugs against CHPV in animal models for clinical use in near future.


title: Comparative pathogenicity of SARS-CoV-2 Omicron and Delta variants in Syrian hamsters mirrors the attenuated clinical outlook of Omicron in COVID-19 irrespective of age

abstract: Coronavirus disease 2019 continues to batter the world with the unceasing introduction of new variants of the causative virus, SARS-CoV-2. In order to understand differences in disease caused by variants of concern and to develop variant-specific vaccines, suitable small animal models are required that mimic disease progression in humans at various stages of life. In this study, we compared the dynamics of infection with two SARS-CoV-2 variants of concern (Delta and Omicron) in aged (>1 year 3 months old) and young (<5 weeks old) Syrian hamsters (Mesocricetus auratus). We show that no weight loss occurred in Omicron infected groups regardless of age, while infection with the Delta variant caused weight loss of up to 10% by day 7 post-infection with slower and incomplete recovery in the aged group. Omicron replicated to similar levels as Delta in the lungs, trachea and nasal turbinates, with no significant differences in the tissue viral loads of aged versus young animals for either variant. In contrast to rare necrosis observed in Omicron-infected animals regardless of age, severe necrosis was observed in the olfactory epithelium in Delta-infected animals. Omicron infection also resulted in mild pulmonary disease in both young and aged animals compared to the moderate acute necrotizing bronchointerstitial pneumonia seen in Delta-infected animals. These results suggest that Omicron infection results in an attenuated clinical disease outlook in Syrian hamsters compared to infection with the Delta variant irrespective of age.


title: Comparative infection and pathogenesis of SARS-CoV-2 Omicron and Delta variants in aged and young Syrian hamsters


title: The 3D structure of lipidic fibrils of α-synuclein

abstract: -synuclein (Syn) is abundant in neurons, but its misfolding and abnormal fibrillization are associated with severe neurodegenerative diseases. Although interactions between Syn and phospholipid membranes are relevant during Syn fibril assembly, insights into the interactions of Syn fibrils with phospholipids have remained elusive. Here, we present six novel polymorphic atomic structures of Syn fibrils aggregated in the presence of phospholipids. The structures reveal that phospholipids favor a novel protofilament fold, mediate an unusual arrangement of protofilaments, and fill the central cavities between the protofilaments. These findings provide a structural rationale for fibril-induced lipid extraction, a mechanism likely to be involved in the development of -synucleinopathies.

category: molecular biology


title: Nematode histone H2A variant evolution reveals diverse histories of retention and loss, and evidence for conserved core-like variants

abstract:
Histone variants replace canonical histones in nucleosomes, sometimes changing nucleosome function. Histone variant evolution is poorly characterized, and, as we show here, reconstruction of histone protein evolution can be challenging given large differences in rates across gene lineages and across sites. The positions of introns that interrupt genes can provide complementary phylogenetic information. We combined sequence and intron data to reconstruct the evolution of three histone H2A variants in Caenorhabditis elegans to reveal disparate histories. For the variant HIS-35 (which differs from H2A by only a single glycine-to-alanine C-terminal change), we find no evidence for the hypothesis of distinct protein function: the HIS-35 alanine is ancestral and common across canonical Caenorhabditis H2A sequences, with one species encoding identical HIS-35 and canonical H2A proteins. We propose instead that HIS-35 allows for H2A expression outside of the S-phase. Genes encoding such "backup" functions could be functionally important yet readily replaceable; consistent with this notion, both HTAS-1 and HIS-35 exhibit phylogenetic patterns that combine long-term evolutionary persistence and recurrent loss. Finally, the H2A.Z homolog, HTZ-1, exhibits recurrent intron loss and gain, suggesting that it is intron presence, rather than a specific intron sequence or position, that may be important in histone variant expression.

SIGNIFICANCEHistone variants are proteins that replace the core canonical histones in the nucleosomes and often confer specific structural and functional features. Histone variants represent a unique opportunity to study the functional significance of gene duplicates. The origin of new variants by core histone gene duplication is generally a highly asymmetrical affair marked by variant-specific changes in gene processing signals and accelerated evolution. The functions and evolutionary significance of the diverse variants found in available eukaryotic genomes remain almost entirely obscure, with only a few characterized case studies. By using a novel method that leverages intron position conservation, we reconstructed the evolutionary history of H2A variants within C. elegans and relatives. Our findings provide several insights that run counter to current paradigms of histone variant function and evolution.


title: Effects of Sanhuang plaster on expression of MyD88, TRAF6, MIP-1β and IL-23 in rats infected by MRSA

abstract: Objective: To investigate the effects of Sanhuang plaster on expression of myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor-related factor 6 (TRAF6), macrophage inflammatory protein-l{beta} (MIP-1{beta}) and its mediated cytokine interleukin-23 (IL-23) in soft tissues of rats infected by methicillin-resistant Staphylococcus aureus (MRSA). Methods: Ninety-six healthy rats were randomly divided into normal control group, model control group, Mupirocin group, high, medium and low dose groups of Sanhuang plaster, with 16 rats in each group. MRSA bacterial liquid was used to make skin and soft tissue infection models. The rats in the normal control group and the model control group were not given any treatment measures. The high, medium, and low dose groups of Sanhuang plaster were given to the affected area with Sanhuang plaster, and the Mupirocin group was given to the affected area for treatment for 10 days. The wound pathological changes were observed. The levels of MIP-1{beta} and IL-23 in serum and infected tissues of rats in each group were measured by ELISA. The mRNA expressions of MyD88, TRAF6, MIP-l{beta} and IL-23 were measured by Quantitative Real-time PCR, and Western blot was used to measure MyD88 and TRAF6 protein expression. Results: Compared with the model control group, the general condition of the Sanhuang plaster groups was significantly improved, and the pathological damage was reduced. The MIP-1{beta} and IL-23 levels in the serum and infected tissues of the high dose group of Sanhuang plaster, the mRNA expressions of MyD88, TRAF6, MIP-1{beta} and IL-23and in large and medium dose groups of Sanhuang plaster, and the protein expressions of Myd88 and TRAF6 in high dose of Sanhuang plaster were significantly decreased (p<0.05 or p<0.01). Conclusion: Sanhuang plaster may play a role in promoting healing of infected wounds by down-regulating the expression levels of MyD88, TRAF6, and MIP-1{beta}, and inhibiting the abnormal secretion of cytokine IL-23.


title: Decoding COVID-19 mRNA Vaccine Immunometabolism in Central Nervous System: human brain normal glial and glioma cells by Raman imaging

abstract:
The paper presents the effect of COVID-19 mRNA (Pfizer/BioNT) vaccine on in vitro glial cells of the brain studied by means of Raman spectroscopy and imaging.. The results obtained for human brain normal and tumor glial cells of astrocytes, astrocytoma, glioblastoma incubated with the Covid-19 mRNA vaccine Pfizer/BioNT vaccine show alterations in the reduction-oxidation pathways associated with Cytochrome c.

We found that the Pfizer/BioNT vaccine down regulate the concentration of cytochrome c in mitochondria upon incubation with normal and tumorous glial cells. Concentration of oxidized form of cytochrome c in brain cells has been shown to decrease upon incubation the mRNA vaccine. Lower concentration of oxidized cytochrome c results in lower effectiveness of oxidative phosphorylation (respiration), reduced apoptosis and lessened ATP production. Alteration of Amide I concentration, which may reflect the decrease of mRNA adenine nucleotide translocator. Moreover, mRNA vaccine leads to alterations in biochemical composition of lipids that suggest the increasing role of signaling. mRNA vaccine produce statistically significant changes in cell nucleus due to histone alterations. The results obtained for mitochondria, lipid droplets, cytoplasm may suggest that COVID-19 mRNA (Pfizer/BioNT) vaccine reprograms immune responses. The observed alterations in biochemical profiles upon incubation with COVID-19 mRNA in the specific organelles of the glial cells are similar to those we observe for brain cancer vs grade of aggressiveness.

category: cell biology


title: Alcam-a and Pdgfr-α are essential for the development of sclerotome derived stromal cells that support hematopoiesis in vivo.

abstract: Mesenchymal stromal cells are essential components of hematopoietic stem and progenitor cell (HSPC) niches, regulating HSPC proliferation and fate decisions. Their developmental origins are largely unknown. In zebrafish, we previously found that the stromal cells of the caudal hematopoietic tissue (CHT), a niche functionally homologous to the fetal liver in mammals, arise from the ventral part of caudal somites. We have now discovered that this ventral domain is actually the sclerotome, and that two typical markers of mammalian mesenchymal stem/stromal cells, Alcam and Pdgfr-, are distinctively expressed there and instrumental for the emergence and migration of stromal cell progenitors, which in turn conditions the proper assembly of the vascular component of the CHT niche. Furthermore, we find that the trunk somites are similarly dependent on Alcam and Pdgfr- to produce mesenchymal stromal cells that foster the initial emergence of HSPCs from the dorsal aorta. Thus the sclerotome contributes essential stromal cells for each of the key steps of developmental hematopoiesis, and likely is the embryological origin of most if not all mesenchymal stem/stromal cells found in non-cephalic tissues.

category: developmental biology


title: Double knockout CRISPR screen in cancer resistance to T cell cytotoxicity

abstract: Immunotherapy has transformed cancer treatments; however, a large fraction of patients encounter resistance. Such resistance is mediated by complex factors, often involving interactions between multiple genes. Thus, it is crucially important to identify genetic interactions between genes that are significantly mutated in cancer patients and those involved in immune responses, ideally the ones that currently have chemical compounds for direct targeting. To systematically interrogate such genetic interactions that mediate cancer cells response to T cell killing, we designed an asymmetric CRISPR/Cas9 dual perturbation library targeting the matched combinations between significantly mutated tumor suppressors and immune resistance genes. We performed a combinatorial double knockout screen on 1,159 gene pairs and identified those where joint loss-of-function renders altered cellular response to T cell cytotoxicity. With individual double knockout constructs we validated these genetic interactions including Jak1-Trp53, Jak1-Kmt2d, and Ifngr1-Kmt2d. Interactions between significantly mutated tumor suppressors and potentially druggable immune resistance genes may offer insights on potential new concepts of how to target clinically relevant cancer mutations with currently available agents. This study also provides a technology platform and an asymmetric CRISPR double knockout library for interrogating genetic interactions between cancer mutations and immune resistance pathways under various settings.

category: genomics


title: Genome-Wide DNA Methylation Profiling of the Failing Human Heart with Mechanical Unloading Identifies LINC00881 as an Essential Regulator of Calcium Handling in the Cardiomyocyte

abstract:
BackgroundHuman heart failure is characterized by global alterations in the myocardial DNA methylation profile, yet little is known about epigenetic regulation of non-coding transcripts and potential reversibility of DNA methylation with left ventricular assist device (LVAD) support.

MethodGenome-wide mapping of myocardial DNA methylation was performed in 36 patients with end-stage heart failure at the time of LVAD implant, 8 patients at the time of LVAD explant, and 7 non-failing controls using high-density bead array platform. Transcriptomic and functional studies were performed in human induced pluripotent stem cell derived cardiomyocytes (iPSCs).

ResultsEtiology-specific analysis revealed 2079 differentially methylated positions (DMPs) in ischemic cardiomyopathy (ICM) and 261 DMPs in non-ischemic cardiomyopathy (NICM). 192 DMPs were common to ICM and NICM. Analysis of paired samples before and after LVAD support demonstrated reverse methylation of only 3.2% of HF-specific DMPs. Methylation-expression correlation analysis yielded several protein-coding genes that are hypomethylated and upregulated (HTRA1, FAM65A, FBXO16, EFCAB13, AKAP13, RPTOR) or hypermethylated and downregulated (TBX3) in ICM and NICM patients. A novel cardiac-specific super-enhancer lncRNA (LINC00881) is hypermethylated and downregulated in the failing human heart. LINC00881 is an upstream regulator of sarcomere and calcium channel gene expression including MYH6, CACNA1C, and RYR2. LINC00881 knockdown significantly reduced peak calcium amplitude in the beating human iPSCs.

ConclusionsFailing human heart exhibits etiology-specific changes in DNA methylation including coding and non-coding regions, which are minimally reversible with mechanical unloading. Epigenetic reprogramming may be necessary to achieve transcriptional normalization and sustained clinical recovery from heart failure.