Biology subjects

Yang, M.

Publications and source records attributed to Yang, M..

At least 271 records · Page 15Linked to original sources

bioRxiv2022

Invigorating human MSCs for transplantation therapy via Nrf2/DKK1 co- stimulation in a mice acute-on-chronic liver failure model

Boosting stem cell resilience against an extrinsically harsh recipient environment is critical to therapeutic efficiency of stem cell-based transplantation innovations in liver disease contexts. We aimed to establish the efficacy of a transient plasmid-based preconditioning strategy to boost mesenchymal stromal cells (MSCs) capacity for anti-inflammation/antioxidant defense and paracrine actions on recipient hepatocytes. In MSCs, the master antioxidant regulator Nrf2 was found to bind directly to the antioxidant response element in the DKK1 promoter region. Activation of Nrf2 and DKK1 enhanced the anti-stress capacities of MSCs in vitro. In an acute-on-chronic liver failure (ACLF) murine model, transient co-overexpression of Nrf2 and DKK1 via plasmid transfection markedly improved MSC resilience against inflammatory and oxidative assaults, boosted MSC transplantation efficacy and promoted recipient liver regeneration because of a shift from the activation of the anti-regenerative IFN-{gamma}/STAT1 pathway to the pro-regenerative IL-6/STAT3 pathway in the liver. Moreover, specific ablation of DKK1 receptor CKAP4 but not LRP6 in recipient hepatocytes nullified therapeutic benefits from MSC transplantation. In long-term observations, tumorigenicity was undetected in mice following transplantation of such transiently preconditioned MCSs. In conclusion, co-stimulation of Nrf2/DKK1 signaling decisively and safely improves the efficacy of human MSC-based therapies in mouse ACLF models through apparently CKAP4-dependent paracrine mechanisms.

molecular biology↗

bioRxiv2022

Orchestration of alternative splicing regulates bone marrow mesenchymal stem cells fate during aging

Senescence and change of differentiation direction in bone marrow stromal cells (BMSCs) are two of the most important causes of age-related bone loss. As an important post-transcriptional regulatory pathway, alternative splicing (AS) regulates diversity of gene expression. However, the role of AS in BMSCs during aging remains poorly defined. Here we identify AS in specific genes disrupt gene expression pattern and result in age-related debility of BMSCs. We demonstrate the deficiency of splicing factor Y-box protein 1 (YBX1) result in mis-splicing in genes such as Fn1, Taz, Sirt2 and Sp7, further contributing to senescence and shift in differentiation direction of BMSCs during aging. Deletion or over-expression of YBX1 in BMSCs accelerate bone loss or stimulate bone formation in mice. Notably, we identify a small compound sciadopitysin which attenuate the degradation of YBX1 and attenuate bone loss in old mice. Our study demonstrates elaborately controlled RNA splicing governs cell fate of BMSCs and provides a potential therapeutic target for age-related osteoporosis. SummaryThis study demonstrates that YBX1 deficiency induces pre-mRNA mis-splicing and causes senescence and shift in differentiation direction of BMSCs and further accelerates aging-related bone loss. This study identifies Sciadopitysin could reverse this process by targeting YBX1.

cell biology↗

bioRxiv2022

Structural basis of a two-antibody cocktail exhibiting highly potent and broadly neutralizing activities against SARS-CoV-2 variants including diverse Omicron sublineages

SARS-CoV-2 variants of concern (VOCs), especially the latest Omicron, have exhibited severe antibody evasion. Broadly neutralizing antibodies with high potency against Omicron are urgently needed for understanding working mechanisms and developing therapeutic agents. In this study, we characterized previously reported F61, which was isolated from convalescent patients infected with prototype SARS-CoV-2, as a broadly neutralizing antibody against all VOCs including Omicron BA.1, BA.1.1, BA.2, BA.3 and BA.4 sublineages by utilizing antigen binding and cell infection assays. We also identified and characterized another broadly neutralizing antibody D2 with epitope distinct from that of F61. More importantly, we showed that a combination of F61 with D2 exhibited synergy in neutralization and protecting mice from SARS-CoV-2 Delta and Omicron BA.1 variants. Cryo-EM structures of the spike-F61 and spike-D2 binary complexes revealed the distinct epitopes of F61 and D2 at atomic level and the structural basis for neutralization. Cryo-EM structure of the Omicron-spike-F61-D2 ternary complex provides further structural insights into the synergy between F61 and D2. These results collectively indicated F61 and F61-D2 cocktail as promising therapeutic antibodies for combating SARS-CoV-2 variants including diverse Omicron sublineages.

biochemistry↗

bioRxiv2022

Loss-of-function mutation in human Oxidation Resistance gene 1 disrupts the spatial-temporal regulation of histone arginine methylation in early brain development

We report a loss-of-function mutation in the TLDc domain of human Oxidation Resistance 1 (OXR1) gene, resulting in early-onset epilepsy, developmental delay, cognitive disabilities, and cerebellar atrophy. Patient lymphoblasts show impaired cell survival, proliferation, and hypersensitivity to oxidative stress. These phenotypes are rescued by TLDc domain replacement. We generated patient derived induced pluripotent stem cells (iPSCs) revealing impaired neural differentiation along with dysregulation of genes essential for neurodevelopment. We identified that OXR1 influences histone arginine methylation by activating protein arginine methyltransferases (PRMTs), suggesting OXR1 dependent mechanisms regulating gene expression during neurodevelopment. We modeled the function of OXR1 in early human brain development using patient derived brain organoids revealing that OXR1 contributes to the spatial-temporal regulation of histone arginine methylation in specific brain regions. Our work provides new insights into pathological features and molecular underpinnings associated with OXR1 deficiency, highlighting the therapeutic potential of OXR1 in numerous neurodegenerative and neurodevelopmental disorders.

pathology↗

bioRxiv2022

Nucleotide resolution genetic mapping in pigs by publicly accessible whole genome imputation

Genetic mapping to identify genes and alleles associated with or causing economically important quantitative trait variation in livestock animals such as pigs is a major goal in the genetic improvement animals. Despite recent advances in high throughput genotyping technologies, resolution of genetic mapping in pigs remains poor due in part to the low density of genotyped variant sites. In this study, we overcame this limitation by developing a reference haplotype panel for pigs based on 2,259 whole genome sequenced animals representing 44 pig breeds. We optimized the imputation procedure to achieve an average concordance rate in excess of 97%, non-reference concordance rate 91%, and r2 0.89. We demonstrated that genotype imputation using this resource can dramatically improve resolution of genetic mapping. Finally, we developed a public web server (swimgeno.org) to allow the pig genetics community to fully utilize this resource. We expect the resource and server to significantly facilitate genetic mapping and accelerate genetic improvement in pigs.

genomics↗

bioRxiv2022

SCExecute: cell barcode-stratified analyses of scRNA-seq data

MotivationIn single-cell RNA-sequencing (scRNA-seq) data, stratification of sequencing reads by cellular barcode is necessary to study cell-specific features. However, apart from gene expression, the analyses of cell-specific features are not supported by available tools that are designed for bulk RNA-Seq data. ResultsWe introduce a tool - SCExecute - which executes a user-provided command on barcode-stratified, extracted on-the-fly, single cell binary alignment map (scBAM) files. SCExecute extracts the cell barcode from aligned, pooled single-cell sequencing data. The user-specified command option executes all the commands defined in the session from monolithic programs and multi-command shell-scripts to complex shell-based pipelines. The execution can be further restricted to barcodes or/and genomic regions of interest. We demonstrate SCExecute with two popular variant callers - GATK and Strelka2 - combined with modules for bam file manipulation and variant filtering, to detect single cell-specific expressed Single Nucleotide Variants (sceSNVs) from droplet scRNA-seq data (10X Genomics Chromium System). ConclusionSCExecute facilitates custom cell-level analyses on barcoded scRNA-seq data using currently available tools and provides an effective solution for studying low (cellular) frequency transcriptome features. AvailabilitySCExecute is implemented in Python3 using the PySAM package and distributed for Linux and Python environments from https://github.com/HorvathLab/NGS/tree/master/SCExecute.

genomics↗

bioRxiv2022

Global Spatial Transcriptome of Macaque Brain at Single-Cell Resolution

Global profile of gene expression at single-cell resolution remains to be determined for primates. Using a recently developed technology ("Stereo-seq"), we have obtained a comprehensive single-cell spatial transcriptome map at the whole-brain level for cynomolgus monkeys, with [~]600 genes per cell for 10 m-thick coronal sections (up to 15 cm2 in size). Large-scale single-nucleus RNA-seq analysis for [~]1 million cells helped to identify cell types corresponding to Stereo-seq gene expression profiles, providing a 3-D cell type atlas of the monkey brain. Quantitative analysis of Stereo-seq data revealed molecular fingerprints that mark distinct neocortical layers and subregions, as well as domains within subcortical structures including hippocampus, thalamus, striatum, cerebellum, hypothalamus and claustrum. Striking whole-brain topography and coordinated patterns were found in the expression of genes encoding receptors and transporters for neurotransmitters and neuromodulators. These results pave the way for cellular and molecular understanding of organizing principles of the primate brain.

cell biology↗

bioRxiv2022

Biochemical analysis challenging Western blot analysis as validation step for antibodies intended for ELISA and immunohistochemistry

The relative contributions of the conformation and primary structure of an epitope to overall antigen-antibody interaction (AAI) at denatured, native or formalin fixed (FF) state were compared using six randomly chosen commercial antibodies using Quantitative Dot Blot (QDB) method. AAIs at native and FF states were found ranged 1.3 [~] 10.2 and 0.5 [~] 45.4 folds, respectively, over those at denatured state in cellular and tissue lysates. Using two antibodies against different epitopes of PYGL protein, we showed that PYGL levels in several types of tissues and cell lines were highly correlated (r=0.99 from Pearson, p<0.0001, n=25) when measured with these two antibodies at native state. Yet, one antibody was found to be nonspecific with one type of these tissues using Western blot analysis. These observations suggested that the conformation of an epitope may serve as dominant contributor of overall AAI at native state in general, regardless of linear or conformational epitopes. In many cases, it would override nonspecific interactions formed at denatured state to challenge Western blot analysis as a validation tool for antibodies intended for immunohistochemistry (IHC) and ELISA.

biochemistry↗

bioRxiv2022

Preclinical Pharmacokinetics and In Vitro Properties of GS-441524, A Potential Oral Drug Candidate for COVID-19 Treatment

Preclinical pharmacokinetics (PK) and in vitro ADME properties of GS-441524, a potential oral agent for the treatment of Covid-19, were studied. GS-441524 was stable in vitro in liver microsomes, cytosols, and hepatocytes of mice, rats, monkeys, dogs, and humans. The plasma free fractions of GS-441524 were 62-78% across all studied species. The in vitro transporter study results showed that GS-441524 was a substrate of MDR1, BCRP, CNT3, ENT1, and ENT2; but not a substrate of CNT1, CNT2, and ENT4. GS-441524 had a low to moderate plasma clearance (CLp), ranging from 4.1 mL/min/kg in dogs to 26 mL/min/kg in mice; the steady state volume distribution (Vdss) ranged from 0.9 L/kg in dogs to 2.2 L/kg in mice after IV administration. Urinary excretion appeared to be the major elimination process for GS-441524. Following oral administration, the oral bioavailability was 8.3% in monkeys, 33% in rats, 39% in mice, and 85% in dogs. The PK and ADME properties of GS-441524 support its further development as an oral drug candidate.

pharmacology and toxicology↗

bioRxiv2022

Generative pretraining from large-scale transcriptomes: Implications for single-cell deciphering and clinical translation

Exponential accumulation of single-cell transcriptomes poses great challenge for efficient assimilation. Here, we present an approach entitled tGPT towards integration of 22.3 million single-cell transcriptomes by modeling gene expression rankings as generative pretraining task. tGPT is conceptually simple in that it autoregressively models the ranking of a gene in the context of its preceding neighbors. We demonstrated the high performance of tGPT on a range of fundamental single-cell analysis tasks and novel applications on bulk tissues. The single-cell clusters and cell lineage trajectories derived from tGPT are highly aligned with known cell labels and states. The feature patterns of tumor bulk tissues learned by tGPT are associated with a wide range of genomic alteration events, prognosis and treatment outcome of immunotherapy. tGPT represents a new analytical paradigm for integrating and deciphering massive amount of transcriptome data and it will facilitate the interpretation and clinical translation of single-cell transcriptomes.

bioinformatics↗

bioRxiv2022

Novel MBLs inhibitors screened from FDA-approved drug library restore the susceptibility of carbapenems to NDM-1-harbouring bacteria

The production of metallo-{beta}-lactamases (MBLs) is one of the major mechanisms adopted by bacterial pathogens to resist carbapenems. One promising strategy to overcome MBLs-mediated carbapenems resistance is to develop effective inhibitors. Repurposing approved drugs to restore the efficacy of carbapenems represents an efficient and cost-effective approach to fight infections caused by carbapenem resistant pathogens. Here, twelve FDA-approved compounds were screened to neutralize the ability of NDM-1. Among these compounds, dexrazoxane, embelin, candesartan cilexetil (CAN) and nordihydroguaiaretic acid (NDGA) were further demonstrated to inhibit all tested MBLs, and showed an in vitro synergistic bactericidal effect with meropenem against MBLs-producing bacteria. Mechanistic studies revealed that dexrazoxane, embelin and CAN are metal ion chelating agents, while the inhibition of NDM-1 by NDGA involves its direct binding with the active region of NDM-1. Furthermore, dexrazoxane, embelin and CAN and NDGA dramatically rescued the treatment efficacy of meropenem in three infection models. Our observations indicated that dexrazoxane, embelin, CAN and NDGA are promising carbapenem adjuvants against MBLs-positive carbapenem resistant bacterial pathogens.

microbiology↗

bioRxiv2022

Barley stripe mosaic virus γb protein targets thioredoxin h-type 1 to dampen SA-mediated antiviral defenses

Salicylic acid (SA) acts as a signaling molecule to perceive and defend against pathogen infections. Accordingly, pathogens evolve versatile strategies to disrupt the SA-mediated signal transduction. However, it is necessary to further characterize how plant viruses manipulate the SA-dependent defense responses. Here, we show that Barley stripe mosaic virus (BSMV) infection activates SA-mediated defense signaling pathway and upregulates the expression of Nicotiana benthamiana thioredoxin h-type 1 (NbTRXh1). The {gamma}b protein interacts directly with NbTRXh1 in vivo and in vitro. Overexpression of NbTRXh1, but not a reductase-defective mutant, impedes BSMV infection, whereas low NbTRXh1 expression level results in increased viral accumulation. Similar with its orthologues in Arabidopsis, NbTRXh1 also plays an essential role in SA signaling transduction in N. benthamiana. To counteract NbTRXh1-mediated defenses, the BSMV {gamma}b protein targets NbTRXh1 to dampen its reductase activity and thereby impairing downstream SA defense genes expression to optimize viral cell-to-cell movement. We also found that NbTRXh1-mediated resistance defends against Lychnis ringspot virus, Beet black scorch virus, and Beet necrotic yellow vein virus. Taken together, our results reveal a novel role for the multifunctional {gamma}b protein in counteracting plant defense responses, and broadens the broad-spectrum antibiotic role of SA signaling pathway. One sentence summaryBSMV {gamma}b protein impairs NbTRXh1 reductase activity and dampen downstream SA-related genes expression to facilitate viral cell-to-cell movement.

microbiology↗

bioRxiv2021

Structural and functional characterizations of altered infectivity and immune evasion of SARS-CoV-2 Omicron variant

The SARS-CoV-2 Omicron with increased fitness is spreading rapidly worldwide. Analysis of cryo-EM structures of the Spike (S) from Omicron reveals amino acid substitutions forging new interactions that stably maintain an "active" conformation for receptor recognition. The relatively more compact domain organization confers improved stability and enhances attachment but compromises the efficiency of viral fusion step. Alterations in local conformation, charge and hydrophobic microenvironments underpin the modulation of the epitopes such that they are not recognized by most NTD- and RBD-antibodies, facilitating viral immune escape. Apart from already existing mutations, we have identified three new immune escape sites: 1) Q493R, 2) G446S and 3) S371L/S373P/S375F that confers greater resistance to five of the six classes of RBD-antibodies. Structure of the Omicron S bound with human ACE2, together with analysis of sequence conservation in ACE2 binding region of 25 sarbecovirus members as well as heatmaps of the immunogenic sites and their corresponding mutational frequencies sheds light on conserved and structurally restrained regions that can be used for the development of broad-spectrum vaccines and therapeutics.

biophysics↗

bioRxiv2021

Fine mapping of goat polledness variant in six Chinese native breeds

BackgroundThe genetic mechanism of goat polledness has been studied for decades, but identifying causative variants and functional genes remains challenging. ResultsUsing a genome-wide association study (GWAS), we identified a significant striking locus for polledness in two different goat breeds. To reduce the linkage disequilibrium among variants for localizing causative variants in the finer region, we sequenced 79 goats from six Chinese native breeds (Jining Gray, Matou, Guizhou black, Yunnan black bone, Chaidamu, and Ujumqin) and identified 483.5 kb CNV (150,334,567-150,818,099) translocated into the previously identified 11.7 kb polled intersex syndrome region, which was consistent with previous research using intersex goat populations. Within the 483.5 kb CNV, a ~322 bp horn-specific element, similar to the superfamily of tRNA-derived families of SINEs, located at the first intron of the ERG gene was identified. The results of the GO enrichment analysis showed that the Horn-SINE element-associated genes were involved in both nervous system and head development. Finally, we used RNA sequencing to investigate gene expression profiles in the horn bud and skin tissues of horned and polled goats. We identified 1077 and 1222 differentially expressed genes between the horn bud and skin tissue in polled and horned goats, respectively. We also identified 367 differentially expressed genes in horn buds between polled and horned animals, and found that the two CNV-related genes, ERG and FOXL2, were upregulated in the horn bud of polled goats. Gene functional enrichment analysis demonstrated that the downregulated genes in the horn bud of polled goats were enriched in skeletal system development, whereas the upregulated genes were significantly overexpressed in muscle tissue development. ConclusionsBroadly, this study describes a novel structural variant responsible for polledness/intersex traits and contributes to the discovery of molecular mechanisms underlying the development and regulation of the polledness trait.

genetics↗

bioRxiv2021

KDM6B sensitizes PARthanatos via suppression of O6-methylguanine DNA methyltransferase repair and sustained checkpoint response

Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA damage sensor and contributes to both DNA repair and cell death processes. However, how PARP-1 signaling is regulated to switch its function from DNA repair to cell death remains largely unknown. Here, we found that PARP-1 plays a central role in alkylating agent-induced PARthanatic cancer cell death. Lysine demethylase 6B (KDM6B) was identified as a key cell death effector in PARthanatos. Knockout of KDM6B or loss of KDM6B demethylase activity conferred cancer cells resistance to PARthanatic cell death in response to alkylating agents. Mechanistically, KDM6B knockout suppressed methylation at the promoter of O6-methylguanine-DNA methyltransferase (MGMT) to enhance MGMT expression and its direct DNA repair function, thereby inhibiting DNA damage-evoked PARP-1 hyperactivation and subsequent cell death. Moreover, KDM6B knockout triggered sustained Chk1 phosphorylation and activated a second repair machinery to fix DNA damage evading from MGMT repair. Inhibition of MGMT or checkpoint response re-sensitized KDM6B deficient cells to PARthanatos induced by alkylating agents. These findings provide new molecular insights into epigenetic regulation of PARP-1 signaling mediating DNA repair or cell death and identify KDM6B as a biomarker for prediction of cancer cell vulnerability to alkylating agent treatment.

molecular biology↗

bioRxiv2021

Biogenesis of a bacterial metabolosome for propanediol utilization

Bacterial metabolosomes are a family of protein organelles in bacteria. Elucidating how thousands of proteins self-assemble to form functional metabolosomes is essential for understanding their significance in cellular metabolism and pathogenesis. Here we investigate the de novo biogenesis of propanediol-utilization (Pdu) metabolosomes and characterize the roles of the key constituents in generation and intracellular positioning of functional metabolosomes. Our results demonstrate that the Pdu metabolosome undertakes both "Shell first" and "Cargo first" assembly pathways, unlike the {beta}-carboxysome structural analog which only involves the "Cargo first" strategy. Shell and cargo assemblies occur independently at the cell poles. The internal cargo core is formed through the ordered assembly of multiple enzyme complexes, and exhibits liquid-like properties within the metabolosome architecture. Our findings provide mechanistic insight into the molecular principles driving bacterial metabolosome assembly and expand our understanding of liquid-like organelle biogenesis.

microbiology↗

bioRxiv2021

Direct Imaging and Identification of Proteoforms up to 70 kDa from Human Tissue

Imaging of proteoforms in human tissues is hindered by low molecular specificity and limited proteome coverage. Here, we introduce proteoform imaging mass spectrometry (PiMS), which increases the size limit for proteoform detection and identification by 4-fold compared to reported methods, and reveals tissue localization of proteoforms at <80 m spatial resolution. PiMS advances proteoform imaging by combining ambient nanospray desorption electrospray ionization (nano-DESI) with ion detection using individual ion mass spectrometry (I2MS). We demonstrate the first proteoform imaging of human kidney, identifying 169 of 400 proteoforms <70 kDa using top-down mass spectrometry and database lookup from the human proteoform atlas, including dozens of key enzymes in primary metabolism. PiMS images reveal distinct spatial localizations of proteoforms to both anatomical structures and cellular neighborhoods in the vasculature, medulla, and cortex regions of the human kidney. The benefits of PiMS are poised to increase proteome coverage for label-free protein imaging of tissues. TeaserNano-DESI combined with individual ion mass spectrometry generates images of proteoforms up to 70 kDa.

biochemistry↗

bioRxiv2021

Fbxo7 promotes Cdk6 activity to inhibit PFKP and glycolysis in T cells.

Deregulated Fbxo7 expression is associated with many pathologies, including anaemia, male sterility, cancer, and Parkinsons disease, demonstrating its critical role in a variety of cell types. Although Fbxo7 is an F-box protein that recruits substrates for SCF-type E3 ubiquitin ligases, it also promotes the formation of cyclin D/Cdk6/p27 complexes in an E3-ligase independent fashion. We discovered PFKP, the major gatekeeper of glycolysis, in a screen for Fbxo7 substrates. PFKP has been previously shown to be a critical substrate of Cdk6 for the viability of T-ALL cells. We investigated the molecular relationships between Fbxo7, Cdk6 and PFKP, and the functional effect Fbxo7 has on T cell metabolism, viability, and activation. Fbxo7 promotes Cdk6-independent ubiquitination and Cdk6-dependent phosphorylation of PFKP. Importantly Fbxo7-deficient cells have reduced Cdk6 activity, and haematopoietic and lymphocytic cell lines show a significant dependency on Fbxo7. Compared to WT cells, CD4+ T cells with reduced Fbxo7 expression show increased glycolysis, despite lower cell viability and activation levels. Metabolomic studies of activated CD4+ T cells confirm increased glycolytic flux in Fbxo7-deficient cells, as well as altered nucleotide biosynthesis and arginine metabolism. We show Fbxo7 expression is glucose-responsive at the mRNA and protein level, and we propose Fbxo7 inhibits PFKP and glycolysis via its activation of Cdk6.

cell biology↗