Biology subjects

Shen, L.

Publications and source records attributed to Shen, L..

At least 109 records · Page 6Linked to original sources

bioRxiv2023

Multi-omics map revealed PPARalpha activation protecting against myocardial ischemia-reperfusion injury by maintaining cardiac metabolic homeostasis

Timely percutaneous coronary intervention is the most effective initial therapy for the acute myocardial infarction (MI). However, the mechanism in energy metabolism underlying time-dependent coronary reperfusion remains largely unknown. Here, we generated an integrative map of cardiac cells using bulk and single-nucleus RNA-seq combined with metabolomics profiling of hearts with reperfusion at distinct time points post MI in rat. We found early time reperfusion (ETR), but not late time reperfusion (LTR) reduced myocardial injury by maintaining cardiac energy homeostasis. PPAR was identified as a key regulator for maintaining fatty acid metabolism after MI/R injury. Importantly, pretreatment with FDA-approved PPAR agonist, fenofibrate, improved the transcriptional signatures, and ameliorated the function of the MI/R injured hearts, particularly in the ETR. Together, our data not only deciphered the protective effect of ETR by maintaining cardiac energy homeostasis, but also provided insights into the translational potential of PPAR activation in alleviating MI/R injury.

cell biology↗

bioRxiv2023

Molecular dynamics analyses of CLDN15 pore size and charge selectivity

Claudin-15 (CLDN15) molecules form channels that directly regulate cation and water transport. In the gastrointestinal tract, this transport indirectly impacts nutrient absorption. However, the mechanisms governing ion transport through these channels remain poorly understood. We addressed this question by building on our previous cell culture studies and all atom molecular dynamic simulation model of CLDN15. By mutating D55 to a bulkier glutamic acid (E) or neutral amino acid asparagine (N), our in vitro measurements showed that the D55E mutation decreased charge selectivity and favored small ion permeability, while the D55N mutation led to reduced charge selectivity without markedly altering size selectivity. By establishing a simplified (reduced) CLDN15 molecular dynamics model that excludes non-essential transmembrane regions, we were able to probe how D55 modified cation dehydration, charge interaction, and permeability. These results provide novel insight into organization of the CLDN15 selectivity filter and suggests that D55 plays a dual role in shaping both electrostatic and steric properties of the pore, but its electrostatic role is more prominent in determining CLDN15 cation permeability. This knowledge can be used toward the development of effective strategies to modulate CLDN15 function. The experimental approach established can be further extended to study the function of other claudin channels. Together, these advancements will help us to modulate tight junctions to promote human health. SUMMARYCell culture and molecular dynamics simulations reveal the role of the CLDN15-D55 residue in ion size and charge selectivity. A reduced CLDN15 model offers novel insights into ion conductance, providing a valuable tool for therapeutic modulation of tight junctions.

physiology↗

bioRxiv2023

R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription

Here, we construct genome-scale maps for R-loops, three-stranded nucleic acid structures comprised of a DNA/RNA hybrid and a displaced single strand of DNA, in the proliferative and differentiated zones of the human prenatal brain. We show that R-loops are abundant in the progenitor-rich germinal matrix, with preferential formation at promoters slated for upregulated expression at later stages of differentiation, including numerous neurodevelopmental risk genes. RNase H1-mediated contraction of the genomic R-loop space in neural progenitors shifted differentiation toward the neuronal lineage and was associated with transcriptomic alterations and defective functional and structural neuronal connectivity in vivo and in vitro. Therefore, R- loops are important for fine-tuning differentiation-sensitive gene expression programs of neural progenitor cells.

genomics↗

bioRxiv2023

Detection of interactions between genetic marker sets and environment in a genome-wide study of hypertension

As human complex diseases are influenced by the interplay of genes and environment, detecting gene-environment interactions (G x E) can shed light on biological mechanisms of diseases and play an important role in disease risk prediction. Development of powerful quantitative tools to incorporate G x E in complex diseases has potential to facilitate the accurate curation and analysis of large genetic epidemiological studies. However, most of existing methods that interrogate G x E focus on the interaction effects of an environmental factor and genetic variants, exclusively for common or rare variants. In this study, we proposed two tests, MAGEIT_RAN and MAGEIT_FIX, to detect interaction effects of an environmental factor and a set of genetic markers containing both rare and common variants, based on the MinQue for Summary statistics. The genetic main effects in MAGEIT_RAN and MAGEIT_FIX are modeled as random or fixed, respectively. Through simulation studies, we illustrated that both tests had type I error under control and MAGEIT_RAN was overall the most powerful test. We applied MAGEIT to a genome-wide analysis of gene-alcohol interactions on hypertension in the Multi-Ethnic Study of Atherosclerosis. We detected two genes, CCNDBP1 and EPB42, that interact with alcohol usage to influence blood pressure. Pathway analysis identified sixteen significant pathways related to signal transduction and development that were associated with hypertension, and several of them were reported to have an interactive effect with alcohol intake. Our results demonstrated that MAGEIT can detect biologically relevant genes that interact with environmental factors to influence complex traits.

genetics↗

bioRxiv2023

Lipid-anchored Proteasomes Control Membrane Protein Homeostasis

Protein degradation in eukaryotic cells is mainly carried out by the 26S proteasome, a macromolecular complex not only present in the cytosol and nucleus but also associated with various membranes. How proteasomes are anchored to the membrane and the biological meaning thereof have been largely unknown in higher organisms. Here we show that N-myristoylation of the Rpt2 subunit is a general mechanism for proteasome-membrane interaction. Loss of this modification in the Rpt2-G2A mutant cells leads to profound changes in the membrane-associated proteome, perturbs the endomembrane system and undermines critical cellular processes such as cell adhesion, endoplasmic reticulum-associated degradation (ERAD) and membrane protein trafficking. Rpt2G2A/G2A homozygous mutation is embryonic lethal in mice and is sufficient to abolish tumor growth in a nude mice xenograft model. These findings have defined an evolutionarily conserved mechanism for maintaining membrane protein homeostasis and underscored the significance of compartmentalized protein degradation by myristoyl-anchored proteasomes (MAPs) in health and disease.

biochemistry↗

bioRxiv2023

Monomethylation of Lysine 27 at Histone 3 Confers Lifelong Susceptibility to Stress

Histone post-translational modifications are critical for mediating persistent alterations in gene expression. By combining unbiased proteomics profiling, and genome-wide approaches, we uncovered a role for mono-methylation of lysine 27 at histone H3 (H3K27me1) in the enduring effects of stress. Specifically, mice exposed to early life stress (ELS) or to chronic social defeat stress (CSDS) in adulthood displayed increased enrichment of H3K27me1, and transient decreases in H3K27me2, in the nucleus accumbens (NAc), a key brain-reward region. Stress induction of H3K27me1 was mediated by the VEFS domain of SUZ12, a core subunit of the polycomb repressive complex-2, which is induced by chronic stress and controls H3K27 methylation patterns. Overexpression of the VEFS domain led to social, emotional, and cognitive abnormalities, and altered excitability of NAc D1 mediums spiny neurons. Together, we describe a novel function of H3K27me1 in brain and demonstrate its role as a "chromatin scar" that mediates lifelong stress susceptibility.

neuroscience↗

bioRxiv2023

ZBTB7A regulates MDD-specific chromatin signatures and astrocyte-mediated stress vulnerability in orbitofrontal cortex

Hyperexcitability in the orbitofrontal cortex (OFC) is a key clinical feature of anhedonic domains of Major Depressive Disorder (MDD). However, the cellular and molecular substrates underlying this dysfunction remain unknown. Here, cell-population-specific chromatin accessibility profiling in human OFC unexpectedly mapped genetic risk for MDD exclusively to non-neuronal cells, and transcriptomic analyses revealed significant glial dysregulation in this region. Characterization of MDD-specific cis-regulatory elements identified ZBTB7A - a transcriptional regulator of astrocyte reactivity - as an important mediator of MDD-specific chromatin accessibility and gene expression. Genetic manipulations in mouse OFC demonstrated that astrocytic Zbtb7a is both necessary and sufficient to promote behavioral deficits, cell-type-specific transcriptional and chromatin profiles, and OFC neuronal hyperexcitability induced by chronic stress - a major risk factor for MDD. These data thus highlight a critical role for OFC astrocytes in stress vulnerability and pinpoint ZBTB7A as a key dysregulated factor in MDD that mediates maladaptive astrocytic functions driving OFC hyperexcitability.

neuroscience↗

bioRxiv2023

Histone H3 serotonylation dynamics in dorsal raphe nucleus contribute to stress-induced gene expression and behavior

BackgroundMajor depressive disorder (MDD), along with related mood disorders, is a debilitating illness that affects millions of individuals worldwide. While chronic stress increases incidence levels of mood disorders, stress-mediated disruptions in brain function that precipitate these illnesses remain elusive. Serotonin-associated antidepressants (ADs) remain the first line of therapy for many with depressive symptoms, yet low remission rates and delays between treatment and symptomatic alleviation have prompted skepticism regarding precise roles for serotonin in the precipitation of mood disorders. Our group recently demonstrated that serotonin epigenetically modifies histone proteins (H3K4me3Q5ser) to regulate transcriptional permissiveness in brain. However, this phenomenon has not yet been explored following stress and/or AD exposures. MethodsWe employed a combination of genome-wide and biochemical analyses in dorsal raphe nucleus (DRN) of male and female mice exposed to chronic social defeat stress to examine the impact of stress exposures on H3K4me3Q5ser dynamics, as well as associations between the mark and stress-induced gene expression. We additionally assessed stress-induced regulation of H3K4me3Q5ser following AD exposures, and employed viral-mediated gene therapy to reduce H3K4me3Q5ser levels in DRN and examine the impact on stress-associated gene expression and behavior. ResultsWe found that H3K4me3Q5ser plays important roles in stress-mediated transcriptional plasticity. Chronically stressed mice displayed dysregulated H3K4me3Q5ser dynamics in DRN, with both AD- and viral-mediated disruption of these dynamics proving sufficient to rescue stress-mediated gene expression and behavior. ConclusionsThese findings establish a neurotransmission-independent role for serotonin in stress-/AD-associated transcriptional and behavioral plasticity in DRN.

neuroscience↗

bioRxiv2023

Novel discovery of a lymphatic bridge connecting Schlemm's canal to limbal and conjunctival lymphatic pathway

PurposeSchlemms canal (SC) is a critical structure regulating aqueous humor (AH) drainage and intraocular pressure (IOP). It is known that in the conventional outflow pathway, AH flows from SC to episcleral veins. We recently reported a high-resolution three-dimensional (3D) imaging technology for intact eyeballs, SC and ocular surface. Using this advanced technology, we herein report the discovery of a new structure, termed lymphatic bridge, that directly connects SC to the limbal and conjunctival lymphatic pathway. Further investigation on this novel outflow pathway may provide new mechanisms and therapeutic approaches for glaucoma. MethodsAs reported previously, intact eyeballs were harvested from Prox-1-GFP (green fluorescent protein) mice and processed by a tissue clearing technique with CLARITY. Samples were immunolabeled with specific antibodies for CD31 (pan-endothelial marker) and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1) and imaged by light-sheet fluorescent microscopy. The limbal areas were examined to locate connecting channels between SC and limbal and conjunctival lymphatic vessels. Moreover, in vivo anterior chamber dye injection was performed with Texas Red dextran for functional analysis on AH outflow. ResultsA novel lymphatic bridge structure that expressed both Prox-1 and LYVE-1 was discovered between the SC and limbal lymphatic vessels connected with conjunctival lymphatic pathway. Results from the anterior chamber dye injection showed AH drainage into the conjunctival lymphatic outflow pathway. ConclusionsThis study provides the first evidence on the direct connection between SC and limbal and conjunctival lymphatic pathway. This new pathway is distinctive from the conventional episcleral vein pathway and merits further investigation.

physiology↗

bioRxiv2023

The Genetic Heterogeneity of Multimodal Human Brain Age

The complex biological mechanisms underlying human brain aging remain incompletely understood, involving multiple body organs and chronic diseases. In this study, we used multimodal magnetic resonance imaging and artificial intelligence to examine the genetic architecture of the brain age gap (BAG) derived from gray matter volume (GM-BAG, N=31,557 European ancestry), white matter microstructure (WM-BAG, N=31,674), and functional connectivity (FC-BAG, N=32,017). We identified sixteen genomic loci that reached genome-wide significance (P-value<5x10-8). A gene-drug-disease network highlighted genes linked to GM-BAG for treating neurodegenerative and neuropsychiatric disorders and WM-BAG genes for cancer therapy. GM-BAG showed the highest heritability enrichment for genetic variants in conserved regions, whereas WM-BAG exhibited the highest heritability enrichment in the 5 untranslated regions; oligodendrocytes and astrocytes, but not neurons, showed significant heritability enrichment in WM and FC-BAG, respectively. Mendelian randomization identified potential causal effects of several exposure variables on brain aging, such as type 2 diabetes on GM-BAG (odds ratio=1.05 [1.01, 1.09], P-value=1.96x10-2) and AD on WM-BAG (odds ratio=1.04 [1.02, 1.05], P-value=7.18x10-5). Overall, our results provide valuable insights into the genetics of human brain aging, with clinical implications for potential lifestyle and therapeutic interventions. All results are publicly available at the MEDICINE knowledge portal: https://labs.loni.usc.edu/medicine.

genetics↗

bioRxiv2023

scART: recognizing cell clusters and constructing trajectory from single-cell epigenomic data

The development of single-cell assay for transposase-accessible chromatin using sequencing data (scATAC-seq) has allowed the characterization of epigenetic heterogeneity at single-cell resolution. However, the sparse and noisy nature of scATAC-seq data poses unique computational challenges. To address this, we introduce scART, a novel bioinformatics tool specifically designed for scATAC-seq data analysis. scART utilizes analytical methods highly stable for processing sparse and noisy data, such as k-nearest neighbor (KNN) imputation, Term Frequency-Inverse Document Frequency (TF-IDF) weighting scheme, and the cosine similarity metric to identify underlying cellular heterogeneity in scATAC-seq data. It accurately and robustly identifies cell identities, particularly in data with low sequencing depth, and constructs the trajectory of cellular states. As a demonstration of its utility, scART successfully reconstructed the development trajectory of the embryonic mouse forebrain and uncovered the dynamics of layer-specific neurogenesis. scART is available at GitHub.

bioinformatics↗

bioRxiv2023

Phosphorylation of pyruvate dehydrogenase marks the inhibition of in vivo neuronal activity

For decades, the expression of immediate early genes (IEGs) such as c-fos has been the most widely used molecular marker representing neuronal activation. However, to date, there is no equivalent surrogate available for the decrease of neuronal activity (i.e., inhibition). Here, we developed an optogenetic-based biochemical screen in which population neural activities can be controlled by light with single action potential precision, followed by unbiased phosphoproteomic profiling. We identified that the phosphorylation of pyruvate dehydrogenase (pPDH) inversely correlated with the intensity of action potential firing in primary neurons. In in vivo mouse models, monoclonal antibody-based pPDH immunostaining detected neuronal inhibition across the brain induced by a wide range of factors including general anesthesia, sensory experiences, and natural behaviors. Thus, as an in vivo marker for neuronal inhibition, pPDH can be used together with IEGs or other cell-type markers to profile and identify bi-directional neural dynamics induced by experiences or behaviors.

neuroscience↗

bioRxiv2023

Targeted repression of DNA topoisomerase I by CRISPRi reveals a critical function for it in the Chlamydia trachomatis developmental cycle

Chlamydia trachomatis is an obligate intracellular bacterium that is responsible for the most prevalent bacterial sexually transmitted infections. Changes in DNA topology in this pathogen have been linked to its pathogenicity-associated developmental cycle. Here, evidence is provided that the balanced activity of DNA topoisomerases (Topos) contributes to Chlamydia developmental processes. Utilizing catalytically inactivated Cas12 (dCas12) based-clustered regularly interspaced short palindromic repeats interference (CRISPRi) technology, we demonstrate targeted knockdown of chromosomal topA transcription in C. trachomatis without detected toxicity of dCas12. Repression of topA impaired the growth of C. trachomatis mostly through disruption of its differentiation from a replicative form to an infectious form. Consistent with this, expression of late developmental genes of C. trachomatis was downregulated while early genes maintained their expression. Importantly, the growth defect associated with topA knockdown was rescued by overexpressing topA at an appropriate degree and time, directly linking the growth patterns to the levels of topA expression. Interestingly, topA knockdown had pleiotropic effects on DNA gyrase expression, indicating a potential compensatory mechanism for survival to offset TopA deficiency. C. trachomatis with topA knocked down displayed hypersensitivity to moxifloxacin that targets DNA gyrase in comparison with the wild type. These data underscore the requirement of integrated topoisomerase actions to support the essential development and transcriptional processes of C. trachomatis. ImportanceWe used genetic and chemical tools to demonstrate the relationship of topoisomerase activities and their obligatory role for the chlamydial developmental cycle. Successfully targeting the essential gene topA with a CRISPRi approach, using dCas12, in C. trachomatis indicates that this method will facilitate the characterization of the essential genome. These findings have an important impact on our understanding of the mechanism(s) by which well-balanced topoisomerase activities enable C. trachomatis to adapt to unfavorable growth conditions imposed by antibiotics.

microbiology↗

bioRxiv2023

Multi-omics of the gut microbial ecosystem in the immunotherapy resistance in microsatellite instability-high gastrointestinal cancer patients

Despite the encouraging efficacy of anti-PD-1/PD-L1 immunotherapy in microsatellite instability-high/deficient mismatch repair (MSI-H/dMMR) advanced gastrointestinal cancer, many patients exhibit primary or acquired resistance. Using multi-omics approaches, we interrogated gut microbiome, blood metabolome and cytokines/chemokines of MSI-H/dMMR advanced gastrointestinal cancer patients (N=77) and identified a number of microbes (e.g. Alistipes putredinis) and metabolites (e.g. arginine and SCFA) highly associated with primary resistance. Fecal microbiota transplantation of patients stool replicated the clinical responsiveness as well as certain molecular signatures. Based on the clinical microbiome data, we developed a predictive machine learning model for primary resistance and achieved accuracy at 0.83 on an external validation set. Furthermore, several microbes were pinpointed which gradually changed during the process of acquired resistance. In summary, our study demonstrated the essential role of gut microbiome in drug resistance, and this could be utilized as a preventative diagnosis tool as well as therapeutic targets in the future.

cancer biology↗

bioRxiv2023

Integrated Cell Landscape and Dynamics in the Progression of Bone Repair

Bone homeostasis and repair is a systematic progress with spatiotemporal interaction of multiple cell types involved in skeletal and immune system. Precise spatiotemporal regulation of cell type-specific functions in bone repair contributes to further development of tissue engineering and regenerative medicine. Here, we utilized single-cell RNA sequencing to illustrate a map of cell landscape and dynamics in the progression of rodent bone self-healing and a perturbation by lymphoid cell-deficiency. We identified different functions of myeloid cell and lymphoid cell to osteogenesis and angiogenesis during bone repair and their mutual complementation under lymphoid cell-deficient condition. Additionally, we used CD34+ humanized reconstituted mice to reveal further insights into the mechanism of human bone homeostasis and repair. Our integrated cellular analysis of bone repair explores the functional diversity and complementation between myeloid cells and lymphoid cells during bone healing process and provides further therapeutic implications for the treatment of bone disease and degeneration following ageing.

cell biology↗

bioRxiv2023

Porosity-permeability tensor relationship of closely and randomly packed fibrous biomaterials and biological tissues: Application to the brain white matter

The constitutive model for the porosity-permeability relationship is a powerful tool to estimate and design the transport properties of porous materials, and has thus attracted a significant number of attention for the advancement of composite materials. However, in comparison with engineering composite materials, biomaterials, especially natural and artificial tissues, have more complex micro-structures such as high anisotropy, high randomness of cell/fibre dimensions and very low porosity. Consequently, these properties of the biomaterial make the porosity-permeability relationship more difficult to obtain than traditional composites. To fill this gap, we start the mathematical derivation from the fundamental brain white matter (WM) formed by nerve fibres. This is augmented by a numerical characterisation and experimental validations to obtain an anisotropic permeability tensor of the brain WM as a function of the tissue porosity. Moreover, we propose an anisotropic poroelastic model enhanced by the newly defined porosity-permeability tensor relationship which precisely captures the tissues macro-scale permeability changes due to the micro-structural deformation in an infusion scenario. The constitutive model, the theories and protocols established in this study will both provide improved design strategies to tailor the transport properties of fibrous biomaterials and enable the non-invasive characterisation of the transport properties of biological tissues. This will lead to the provision of better patient-specific medical treatments, such as drug delivery.

bioengineering↗

bioRxiv2023

Transcriptional signatures of heroin intake and seeking throughout the brain reward circuit

Opioid use disorder (OUD) looms as one of the most severe medical crises currently facing society. More effective therapeutics for OUD requires in-depth understanding of molecular changes supporting drug-taking and relapse. Recent efforts have helped advance these aims, but studies have been limited in number and scope. Here, we develop a brain reward circuit-wide atlas of opioid-induced transcriptional regulation by combining RNA sequencing (RNAseq) and heroin self-administration in male mice modeling multiple OUD-relevant conditions: acute heroin exposure, chronic heroin intake, context-induced drug-seeking following prolonged abstinence, and heroin-primed drug-seeking (i.e., "relapse"). Bioinformatics analysis of this rich dataset identified numerous patterns of molecular changes, transcriptional regulation, brain-region-specific involvement in various aspects of OUD, and both region-specific and pan-circuit biological domains affected by heroin. Integrating RNAseq data with behavioral outcomes using factor analysis to generate an "addiction index" uncovered novel roles for particular brain regions in promoting addiction-relevant behavior, and implicated multi-regional changes in affected genes and biological processes. Comparisons with RNAseq and genome-wide association studies from humans with OUD reveal convergent molecular regulation that are implicated in drug-taking and relapse, and point to novel gene candidates with high therapeutic potential for OUD. These results outline broad molecular reprogramming that may directly promote the development and maintenance of OUD, and provide a foundational resource to the field for future research into OUD mechanisms and treatment strategies.

neuroscience↗

bioRxiv2023

Comprehensive approach to study branched ubiquitinchains reveals roles for K48-K63 branches inVCP/p97-related processes

Branched ubiquitin (Ub) chains make up a significant proportion of Ub polymers in human cells and are formed when two or more sites on a single Ub molecule are modified with Ub creating bifurcated architectures. Despite their abundance, we have a poor understanding of the cellular functions of branched Ub signals that stems from a lack of facile tools and methods to study them. Here we develop a comprehensive pipeline to define branched Ub function, using K48-K63-branched chains as a case study. We discover branch-specific binders and, by developing a method that monitors cleavage of linkages within complex polyUb, we discover the VCP/p97-associated ATXN3, and MINDY family deubiquitinases to act as debranching enzymes. By engineering and utilizing a branched K48-K63-Ub chain-specific nanobody, we reveal roles for these chains in VCP/p97-related processes. In summary, we provide a blueprint to investigate branched Ub function that can be readily applied to study other branched chain types. HighlightsO_LIAssembly of defined branched ubiquitin chains enables identification of specific binding proteins C_LIO_LIDevelopment of quantitative DUB assay monitoring cleavage of individual Ub linkages within complex ubiquitin chains identifies debranching enzymes C_LIO_LIEngineering specific, high-affinity nanobody against branched K48-K63 ubiquitin reveals roles in VCP/p97 related processes and DNA damage responses C_LIO_LIGeneral blueprint of new methods and tools for in-depth characterization of branched ubiquitin chains and their underlying biology C_LI

biochemistry↗