Biology subjects

Chang, Y.

Publications and source records attributed to Chang, Y..

At least 127 records · Page 7Linked to original sources

bioRxiv2021

Direct induction of hemogenic endothelial progenitors from hPSCs by defined factors revealed by single-cell transcriptome analysis

Transcription factors (TFs) play critical roles in stem cell maintenance and differentiation. Using single cell RNA sequencing, we investigated TFs expressed in hemogenic endothelial (HE) progenitors differentiated from human pluripotent stem cells (hPSCs) and identified upregulated expression of SOXF factors SOX7, SOX17, and SOX18 in the HE population. To test whether overexpression of these factors increases HE differentiation efficiency, we established inducible hPSC lines and found only SOX17 improved differentiation. Temporal expression analysis further revealed SOX17 was turned on immediately before VE-Cadherin, indicating SOX17 may be a causative factor for HE differentiation. Upon SOX17 knockdown via CRISPR-Cas13d, HE differentiation was significantly abrogated. Strikingly, we discovered SOX17 overexpression alone is sufficient to generate more than 50% CD34+VE-cadherin+CD73- cells that could be directed to hematopoietic progenitors, which emerged via an endothelial-to-hematopoietic transition and significantly upregulated definitive hematopoietic transcriptional programs. Functional assays showed that these progenitors can differentiate into blood cells from multiple lineages. Our analyses reveal an uncharacterized function of SOX17 in directing hPSCs differentiation towards HE cells. Significance StatementHemogenic endothelial (HE) cells have been generated from human pluripotent stem cells (hPSCs) to study blood development. However, their full transcriptomic characterization and key genes involving in directing HE differentiation is unclear. Utilizing single cell RNA-seq analysis, we find that SOX17 is solely expressed in HE cells and is also required for HE differentiation. Strikingly, we find that overexpression of SOX17 alone is sufficient to program hPSCs into CD34+VE-cadherin+CD73-HE cells, which could further differentiate into blood progenitors. Our research reveals that SOX17 is sufficient to direct hPSCs differentiation to HE cells. ClassificationPhysical Sciences/Engineering; Biological Sciences/Cell Biology.

bioengineering↗

bioRxiv2021

Spatial and temporal plasticity of neoantigen-specific T-cell responses bases on characteristics associated to antigen and TCR

Neoantigens derived from somatic mutations have been demonstrated to correlate with therapeutic responses mediated by treatment with immune checkpoint inhibitors. Neoantigens are therefore highly attractive targets for the development of personalized medicine approaches although their quality and associated immune responses is not yet well understood. In a case study of metastatic malignant melanoma, we performed an in-depth characterization of neoantigens and respective T-cell responses in the context of immunotherapy with Ipilimumab. Three neoantigens identified either by immunopeptidomics or in silico prediction were investigated using binding affinity analyses and structural simulations. We isolated seven T-cell receptors (TCRs) from the patient immune repertoire recognizing these antigens. TCRs were compared in-vitro and in-vivo with multi-parametric analyses. Identified immunogenic peptides showed similar binding affinities to the human leukocyte antigen (HLA) complex and comparable differences to their wildtype counterparts in molecular dynamic simulations. Nevertheless, isolated TCRs differed substantially in functionality and frequency. In fact, TCRs with comparably lower functional avidity and higher potential for cross-reactivity provided at least equal anti-tumor immune responses in vivo. Of note, these TCRs showed a reduced activation pattern upon primary in vitro stimulation. Exploration of the TCR-{beta} repertoire in blood and in different tumor-related tissues over three years, offered insights on the high frequency and particular long-term persistence of low-avidity TCRs. These data indicate that qualitative differences of neoantigen-specific TCRs and their impact on function and longevity need to be considered for neoantigen targeting by adoptive T-cell therapy using TCR-transgenic T cells. Statement of translational relevanceImmunotherapy has demonstrated high efficacy in diverse malignancies. Neoantigens derived from mutations provide promising targets for safe and highly tumor-specific therapeutic approaches. Yet, single determinants of an effective and enduring T-cell mediated tumor rejection are still not well understood. We analyzed in detail seven neoantigen-specific T-cell receptors (TCRs) derived from a melanoma patient targeting three different altered peptide ligands identified by mass spectrometry and prediction analyses. Functional characterization of these TCRs revealed potent anti-tumor reactivity of all TCRs. Of special interest, TCRs with comparably lower affinity demonstrated effective in vivo activity as well as dominant spatial and temporal distribution in blood and tissue. Functional differences of TCR may require further T-cell and/or TCR engineering and should be considered for future clinical trial designs.

immunology↗

bioRxiv2021

DBPOM: a comprehensive database of pharmaco-omics for cancer precision medicine

During the course of cancer treatment, both efficacy and the adverse effects of drugs on patients should be taken into account. Although some public databases and modeling frameworks have been developed through studies on drug response, the negative effects of drugs are always neglected. Furthermore, most of them only considered the ramifications of the drug on the cell line, but the effects on the patient still require a huge amount of work to integrate data from various databases and calculations, especially in relation to precision treatment. In order to address these issues, we developed the DBPOM (http://www.dbpom.net/, a comprehensive database of pharmaco-omics for cancer precision medicine), which explores various drugs efficacy levels by calculating their potency in reverse, or enhancing cancer-associated gene expression changes. When compared with existing databases, the DBPOM could estimate the effectiveness of a drug on individual patients through the mapping of various cell lines to each person according to their genetic mutation similarities. The DBPOM is an easy-to-use and one-stop database for clinicians and drug researchers to search and analyze the overall effect of a drug or a drug combination on cancer patients as well as the biological functions that they target. We anticipate that DBPOM will become an important resource and analysis platform for drug development, drug mechanism studies and the discovery of new therapies.

bioinformatics↗

bioRxiv2020

IRIS-FGM: an integrative single-cell RNA-Seq interpretation system for functional gene module analysis

SummarySingle-cell RNA-Seq (scRNA-Seq) data is useful in discovering cell heterogeneity and signature genes in specific cell populations in cancer and other complex diseases. Specifically, the investigation of functional gene modules (FGM) can help to understand gene interactive networks and complex biological processes. QUBIC2 is recognized as one of the most efficient and effective tools for FGM identification from scRNA-Seq data. However, its limited availability to a C implementation restricted its application to only a few downstream analyses functionalities. We developed an R package named IRIS-FGM (Integrative scRNA-Seq Interpretation System for Functional Gene Module analysis) to support the investigation of FGMs and cell clustering using scRNA-Seq data. Empowered by QUBIC2, IRIS-FGM can effectively identify co-expressed and co-regulated FGMs, predict cell types/clusters, uncover differentially expressed genes, and perform functional enrichment analysis. It is noteworthy that IRIS-FGM can also takes Seurat objects as input, which facilitate easy integration with existing analysis pipeline. Availability and ImplementationIRIS-FGM is implemented in R environment (as of version 3.6) with the source code freely available at https://github.com/OSU-BMBL/IRIS-FGM Contactqin.ma@osumc.edu Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2020

Extensive MET alterations confer clinical response to MET inhibitors in gliomas

Activating alterations of the MET gene are well-characterized oncogenic drivers, and MET inhibitors could successfully treat several tumor types with MET alterations, including gliomas with PTPRZ1-MET fusion. However, the full diversity and prevalence of MET alterations in gliomas are still lacking to accurately identify a subset of patients likely to benefit from MET inhibitor treatment. Here, we interrogated genomic profiles of 1,351 gliomas, and further identify 60 cases harboring MET alterations, including MET fusions and various MET exon skipping events. MET RNA alterations, but not MET amplification, are highly enriched in the secondary glioblastomas (sGBM) with significantly worse prognosis. Further molecular analysis has shown that MET RNA alterations acting an additive effects of MET overexpression are induced in the course of glioma evolution. In vitro and clinical studies indicate cells and patients harboring MET alterations have better response to MET inhibitors. Collectively, these data suggest that a subgroup of gliomas harboring MET alterations likely to have benefit from MET-targeted therapy.

neuroscience↗

bioRxiv2020

A CRISPR interference platform for selective downregulation of gene expression in Borrelia burgdorferi

The spirochete Borrelia burgdorferi causes Lyme disease, an increasingly prevalent infection. While previous studies have provided important insight into B. burgdorferi biology, many aspects, including basic cellular processes, remain underexplored. To help speed up the discovery process, we adapted a CRISPR interference (CRISPRi) platform for use in B. burgdorferi. For efficiency and flexibility of use, we generated various CRISPRi template constructs that produce different basal and induced levels of dcas9 and carry different antibiotic resistance markers. We characterized the effectiveness of our CRISPRi platform by targeting the motility and cell morphogenesis genes flaB, mreB, rodA, and ftsI, whose native expression levels span two orders of magnitude. For all four genes, we obtained gene repression efficiencies of at least 95%. We showed by darkfield microscopy and cryo-electron tomography that flagellin (FlaB) depletion reduced the length and number of periplasmic flagella, which impaired cellular motility and resulted in cell straightening. Depletion of FtsI caused cell filamentation, implicating this protein in cell division in B. burgdorferi. Finally, localized cell bulging in MreB- and RodA-depleted cells matched the locations of new peptidoglycan insertion specific to spirochetes of the Borrelia genus. These results therefore implicate MreB and RodA in the particular mode of cell wall elongation of these bacteria. Collectively, our results demonstrate the efficiency and ease of use of our B. burgdorferi CRISPRi platform, which should facilitate future genetic studies of this important pathogen. IMPORTANCEGene function studies are facilitated by the availability of rapid and easy-to-use genetic tools. Homologous recombination-based methods traditionally used to genetically investigate gene function remain cumbersome to perform in B. burgdorferi, as they often are relatively inefficient. In comparison, our CRISPRi platform offers an easy and fast method to implement as it only requires a single plasmid transformation step and IPTG addition to obtain potent (>95%) downregulation of gene expression. To facilitate studies of various genes in wild-type and genetically modified strains, we provide over 30 CRISPRi plasmids that produce distinct levels of dcas9 expression and carry different antibiotic resistance markers. Our CRISPRi platform represents a useful and efficient complement to traditional genetic and chemical methods to study gene function in B. burgdorferi.

microbiology↗

bioRxiv2020

Genome-scale phylogenetic analyses confirm Olpidium as the closest living zoosporic fungus to the non-flagellated, terrestrial fungi

The zoosporic obligate endoparasites, Olpidium, hold a pivotal position to the reconstruction of the flagellum loss in fungi, one of the key morphological transitions associated with the colonization of land by the early fungi. We generated genome and transcriptome data from non-axenic zoospores of Olpidium bornovanus and used a metagenome approach to extract phylogenetically informative fungal markers. Our phylogenetic reconstruction strongly supported Olpidium as the closest zoosporic relative of the non-flagellated terrestrial fungi. Super-alignment analyses resolved Olpidium as sister to the non-flagellated terrestrial fungi, whereas a super-tree approach recovered different placements of Olpidium, but without strong support. Further investigations detected little conflicting signal among the sampled markers but revealed a potential polytomy in early fungal evolution associated with the branching order among Olpidium, Zoopagomycota and Mucoromycota. The branches defining the evolutionary relationships of these lineages were characterized by short branch lengths and low phylogenetic content and received equivocal support for alternative phylogenetic hypotheses from individual markers. These nodes were marked by important morphological innovations, including the transition to hyphal growth and the loss of flagellum, which enabled early fungi to explore new niches and resulted in rapid and temporally concurrent Precambrian diversifications of the ancestors of several phyla of fungi.

evolutionary biology↗

bioRxiv2020

A genome-scale phylogeny of Fungi; insights into early evolution, radiations, and the relationship between taxonomy and phylogeny

Phylogenomic studies based on genome-scale amounts of data have greatly improved understanding of the tree of life. Despite their diversity, ecological significance, and biomedical and industrial importance, large-scale phylogenomic studies of Fungi are lacking. Furthermore, several evolutionary relationships among major fungal lineages remain controversial, especially those at the base of the fungal phylogeny. To begin filling these gaps and assess progress toward a genome-scale phylogeny of the entire fungal kingdom, we compiled a phylogenomic data matrix of 290 genes from the genomes of 1,644 fungal species that includes representatives from most major fungal lineages; we also compiled 11 additional data matrices by subsampling genes or taxa based on filtering criteria previously shown to improve phylogenomic inference. Analyses of these 12 data matrices using concatenation- and coalescent-based approaches yielded a robust phylogeny of the kingdom in which [~]85% of internal branches were congruent across data matrices and approaches used. We found support for several relationships that have been historically contentious (e.g., for the placement of Wallemiomycotina (Basidiomycota), as sister to Agaricomycotina), as well as evidence for polytomies likely stemming from episodes of ancient diversification (e.g., at the base of Basidiomycota). By examining the relative evolutionary divergence of taxonomic groups of equivalent rank, we found that fungal taxonomy is broadly aligned with genome sequence divergence, but also identified lineages, such as the subphylum Saccharomycotina, where current taxonomic circumscription does not fully account for their high levels of evolutionary divergence. Our results provide a robust phylogenomic framework to explore the tempo and mode of fungal evolution and directions for future fungal phylogenetic and taxonomic studies.

evolutionary biology↗

bioRxiv2020

scGNN: a novel graph neural network framework for single-cell RNA-Seq analyses

Single-cell RNA-sequencing (scRNA-Seq) is widely used to reveal the heterogeneity and dynamics of tissues, organisms, and complex diseases, but its analyses still suffer from multiple grand challenges, including the sequencing sparsity and complex differential patterns in gene expression. We introduce the scGNN (single-cell graph neural network) to provide a hypothesis-free deep learning framework for scRNA-Seq analyses. This framework formulates and aggregates cell-cell relationships with graph neural networks and models heterogeneous gene expression patterns using a left-truncated mixture Gaussian model. scGNN integrates three iterative multi-modal autoencoders and outperforms existing tools for gene imputation and cell clustering on four benchmark scRNA-Seq datasets. In an Alzheimers disease study with 13,214 single nuclei from postmortem brain tissues, scGNN successfully illustrated disease-related neural development and the differential mechanism. scGNN provides an effective representation of gene expression and cell-cell relationships. It is also a novel and powerful framework that can be applied to scRNA-Seq analyses.

bioinformatics↗

bioRxiv2020

Genus-wide characterization of bumblebee genomes reveals variation associated with key ecological and behavioral traits of pollinators

Bumblebees are a diverse group of globally important pollinators in natural ecosystems and for agricultural food production. With both eusocial and solitary lifecycle phases, and some social parasite species, they are especially interesting models to understand social evolution, behavior, and ecology. Reports of many species in decline point to pathogen transmission, habitat loss, pesticide usage, and global climate change, as interconnected causes. These threats to bumblebee diversity make our reliance on a handful of well-studied species for agricultural pollination particularly precarious. To broadly sample bumblebee genomic and phenotypic diversity, we de novo sequenced and assembled the genomes of 17 species, representing all 15 subgenera, producing the first genus-wide quantification of genetic and genomic variation potentially underlying key ecological and behavioral traits. The species phylogeny resolves subgenera relationships while incomplete lineage sorting likely drives high levels of gene tree discordance. Five chromosome-level assemblies show a stable 18-chromosome karyotype, with major rearrangements creating 25 chromosomes in social parasites. Differential transposable element activity drives changes in genome sizes, with putative domestications of repetitive sequences influencing gene coding and regulatory potential. Dynamically evolving gene families and signatures of positive selection point to genus-wide variation in processes linked to foraging, diet and metabolism, immunity and detoxification, as well as adaptations for life at high altitudes. These high-quality genomic resources capture natural genetic and phenotypic variation across bumblebees, offering new opportunities to advance our understanding of their remarkable ecological success and to identify and manage current and future threats.

genomics↗

bioRxiv2020

Molecular Mechanism for Rotational Switching of the Bacterial Flagellar Motor

The bacterial flagellar motor is a remarkable nanomachine that can rapidly rotate in both counter-clockwise (CCW) and clockwise (CW) senses. The transitions between CCW and CW rotation are critical for chemotaxis, and they are controlled by a signaling protein (CheY-P) that interacts with a switch complex at the cytoplasmic side of the flagellar motor. However, the exact molecular mechanism by which CheY-P controls the motor rotational switch remains enigmatic. Here, we use the Lyme disease spirochete, Borrelia burgdorferi, as the model system to dissect the mechanism underlying flagellar rotational switching. We first determined high resolution in situ motor structures in the cheX and cheY3 mutants in which motors are genetically locked in CCW or CW rotation. The structures showed that the CheY3 protein of B. burgdorferi interacts directly with the FliM protein of the switch complex in a phosphorylation-dependent manner. The binding of CheY3-P to FliM induces a major remodeling of the switch protein FliG2 that alters its interaction with the torque generator. Because the remodeling of FliG2 is directly correlated with the rotational direction, our data lead to a model for flagellar function in which the torque generator rotates in response to an inward flow of H+ driven by the proton motive force. Rapid conformational changes of FliG2 allow the switch complex to interact with opposite sides of the rotating torque generator, thereby facilitating rotational switching between CW and CCW.

microbiology↗

bioRxiv2020

Dietary supplementation with products of Citrus reticulata "Chachi" for improving the fecal microbiome of weaned piglets

Nutritional interventions play a critical role in modifying the intestinal microbiome of host animals. This study was conducted to interrogate the physiological effects on fecal microflora of weaned piglets via the dietary supplemented with two types of products of Citrus reticulata "Chachi", respectively. For this purpose, A total of 72 piglets with uniform sizes were randomly assigned to four dietary treatment groups consisted of a negative control group (NCG), a fermented citrus reticulata "Chachi" pulp group (FCRPG), a Citri Reticulatae Pericarpium group (CRPG) and a positive control group (PCG) in a 21-day feeding trial. After the raising experiment, fresh feces of piglets were analyzed systematically using multi-omics technologies. Metagenomics method with high-throughput compositional characterization indicated that the architecture and diversity of fecal microbiome were both influenced by these two additives and compound antibiotics. Metabolite analysis showed that FCRPG have an significant effects on fecal short-chain fatty acids (SCFAs) among four treatment groups. Results of functional proteomics approaches found that FCRPG presented the highest butyrate metabolic level, and CRPG showed the highest flavone and flavonol biosynthesis level in feces. In addition, NCP produced an effective effect on adjusting fecal microbiota profile. Consequently, our findings demonstrate that dietary supplementation with FCRP or CRP modulates the microbial taxa, metabolic and proteomic alterations in fecal microbiota of weaned piglets for health maintaining.

microbiology↗

bioRxiv2020

Magnetic Bead-Quantum Dot (MB-Qdot) CRISPR Assay for Instrument-Free Viral DNA Detection

We have developed a novel detection system which couples CRISPR-Cas recognition of target sequences, Cas mediated nucleic acid probe cleavage, and quantum dots as highly sensitive reporter molecules for instrument-free detection of viral nucleic acid targets. After target recognition and Cas mediated cleavage of biotinylated ssDNA probe molecules, the probe molecules are bound to magnetic particles. A complimentary ssDNA oligonucleotide quantum dot conjugate is then added, which only hybridizes to un-cleaved probes on the magnetic beads. After separation of hybridized from unhybridized quantum dot conjugates by magnetic sequestration, the signal is measured fluorometrically to provide a signal proportional to the cleaved probes and therefore the amount of target nucleic acid. To demonstrate the power of this assay, a 250 bp DNA target sequence matching a portion of the African swine fever virus (ASFV) genome is used and a detection limit of ~0.5 nM is achieved without target amplification using a simple portable ultraviolet flashlight. The positive samples are readily confirmed by visual inspection, completely avoiding the need for complicated devices and instruments. This work establishes the feasibility of a simple, instrument free assay for rapid nucleic acid screening in both hospitals and point-of-care settings.

biochemistry↗

bioRxiv2020

Phylogenomic analyses of non-Dikarya fungi supports horizontal gene transfer driving diversification of secondary metabolism in the amphibian gastrointestinal symbiont, Basidiobolus

Research into secondary metabolism (SM) production by fungi has resulted in the discovery of diverse, biologically active compounds with significant medicinal applications. However, the fungi rich in SM production are taxonomically restricted to Dikarya, two phyla of Kingdom Fungi, Ascomycota and Basidiomycota. Here, we explore the potential for SM production in Mucoromycota and Zoopagomycota, two phyla of nonflagellated fungi that are not members of Dikarya, by predicting and identifying core genes and gene clusters involved in SM. The majority of non-Dikarya have few genes and gene clusters involved in SM production except for the amphibian gut symbionts in the genus Basidiobolus. Basidiobolus genomes exhibit an enrichment of SM genes involved in siderophore, surfactin-like, and terpene cyclase production, all these with evidence of constitutive gene expression. Gene expression and chemical assays confirm that Basidiobolus has significant siderophore activity. The expansion of SMs in Basidiobolus are partially due to horizontal gene transfer from bacteria, likely as a consequence of its ecology as an amphibian gut endosymbiont.

genomics↗

bioRxiv2020

Siderophores provoke extracellular superoxide production by carbon-starving Arthrobacter strains when carbon sources recover

Superoxide and other reactive oxygen species (ROS) in the environment shape microbial communities1 and drive transformation of metals2,3 and inorganic/organic matter4,5. Taxonomically diverse bacteria and phytoplankton can produce extracellular superoxide during laboratory cultivation6-11. Understanding the physiological reasons for extracellular superoxide production by aerobes in the environment is a crucial question yet not fully solved. Here, we showed that iron-starving Arthrobacter sp. QXT-31 (referred to as A. QXT-31 hereafter) secreted a type of siderophore (deferoxamine, DFO), which provoked extracellular superoxide production by carbon-starving A. QXT-31 when carbon sources were recovered. Several other siderophores also demonstrated similar effects. RNA-Seq data hinted that DFO stripped iron from iron-bearing proteins in the electron transfer chain (ETC) of metabolically active A. QXT-31, resulting in electron leakage from the electron-rich (resulting from carbons source metabolism) ETC and superoxide production. Considering that most aerobes secrete siderophore(s)12 and often suffer from carbon starvation in the environment, certain aerobes are expected to produce extracellular superoxide when carbon source(s) recover/fluctuate, thus influencing the microbial community and cycling of many elements. In addition, an artificial iron-chelator (diethylenetriamine pentaacetic acid, DTPA) was widely used in microbial superoxide quantification. Our results showed that DTPA provoked superoxide production by A. QXT-31 and highlighted its potential interference in microbial superoxide quantification.

microbiology↗

bioRxiv2020

Population sequencing enhances understanding of tea plant evolution

Tea is an economically important plant characterized by a large genome size and high heterozygosity and species diversity. In this study, we assembled a 3.26 Gb high-quality chromosome-scale genome for tea using the Longjing 43 cultivar of Camellia sinensis var. sinensis. Population resequencing of 139 tea accessions from around the world was used to investigate the evolution of tea and to reveal the phylogenetic relationships among tea accessions. With the spread of tea cultivation, hybridization has increased the heterozygosity and wide-ranging gene flow among tea populations. Population genetics and transcriptomics analyses revealed that during domestication, the selection for disease resistance and flavor in C. sinensis var. sinensis populations has been stronger than that in C. sinensis var. assamica populations. The data compiled in this study provide new resources for the marker assisted breeding of tea and are a basis for further research on the genetics and evolution of tea.

genomics↗

bioRxiv2020

Plasmin improves oedematous blood-gas barrier by cleaving epithelial sodium channels

Background and PurposeLung oedema in association with suppressed fibrinolysis is a hallmark of lung injury. We aimed to test whether plasmin cleaves epithelial sodium channels (ENaC) to resolve lung oedema fluid. Experimental ApproachesHuman lungs and airway acid-instilled mice were used for analysing fluid resolution. In silico prediction, mutagenesis, Xenopus oocytes, immunoblotting, voltage clamp, mass spectrometry, protein docking, and alveolar fluid clearance were combined for identifying plasmin specific cleavage sites and benefits. Key ResultsPlasmin led to a marked increment in lung fluid resolution in both human lungs ex vivo and injured mice. Plasmin specifically activated {beta}{gamma}ENaC channels in oocytes in a time-dependent manner. Deletion of four consensus proteolysis tracts ({Delta}432-444, {gamma}{Delta}131-138, {gamma}{Delta}178-193, and {gamma}{Delta}410-422) eliminated plasmin-induced activation significantly. Further, immunoblotting assays identified 7 cleavage sites (K126, R135, K136, R153, K168, R178, K179) for plasmin to trim both furin-cleaved C-terminal fragments and full-length human {gamma}ENaC proteins. In addition to confirming the 7 cleavage sites, 9 new sites (R122, R137, R138, K150, K170, R172, R180, K181, K189) in synthesized peptides were found to be cleaved by plasmin with mass spectrometry. These cleavage sites were located in the finger and the thumb, particularly the GRIP domain of human ENaC 3D model composed of two proteolytic centres for plasmin. Novel uncleaved sites beyond the GRIP domain in both and {gamma} subunits were identified to interrupt the plasmin cleavage-induced conformational change in ENaC channel complexes. Additionally, plasmin could regulate ENaC activity via the G protein signal. Conclusion and ImplicationsWe demonstrate that plasmin could cleave ENaC to benefit the blood-gas exchange by resolving oedema fluid as a potent fibrinolytic therapy for oedematous pulmonary diseases. Bullet point summaryO_ST_ABSWhat is already knowC_ST_ABSO_LISerine proteases proteolytically cleave epithelial sodium channels, including plasmin and uPA acutely. C_LIO_LIActivity of epithelial sodium channels is increased post proteolysis. C_LI What this study addsO_LIPlasmin cleaves up to 16 sites composed of two proteolytic centres in both full-length and furin-cleaved human {gamma} subunit of epithelial sodium channels in hours. C_LIO_LINon-proteolytic sites in both and {gamma} subunits interrupt the plasmin cleavage-induced channel gating. C_LIO_LIIntratracheally instilled plasmin facilitates alveolar fluid clearance in normal human and injured mouse lungs. C_LI Clinical significanceO_LIActivation of human lung epithelial sodium channels by plasmin may benefit lung oedema resolution as a novel therapy for ARDS. C_LI

pharmacology and toxicology↗

bioRxiv2020

A single-cell transcriptomic and anatomic atlas of mouse dorsal raphe Pet1 neurons

Among the brainstem raphe nuclei, the dorsal raphe nucleus (DR) contains the greatest number of Pet1-lineage neurons, a predominantly serotonergic group distributed throughout DR subdomains. These neurons collectively regulate diverse physiology and behavior and are often therapeutically targeted to treat affective disorders. Characterizing Pet1 neuron molecular heterogeneity and relating it to anatomy is vital for understanding DR functional organization, with potential to inform therapeutic separability. Here we use high-throughput and DR subdomain-targeted single-cell transcriptomics and intersectional genetic tools to map molecular and anatomical diversity of DR-Pet1 neurons. We describe up to fourteen neuron subtypes, many showing biased cell body distributions across the DR. We further show that P2ry1-Pet1 DR neurons - the most molecularly distinct subtype - possess unique efferent projections and electrophysiological properties. These data complement and extend previous DR characterizations, combining intersectional genetics with multiple transcriptomic modalities to achieve fine-scale molecular and anatomic identification of Pet1 neuron subtypes.

neuroscience↗