Biology subjects

Zhou, M.

Publications and source records attributed to Zhou, M..

At least 181 records · Page 10Linked to original sources

bioRxiv2021

Single Cell T Cell Receptor Repertoire Profiling for Dogs

BackgroundSpontaneous cancers in companion dogs are increasingly recognized as robust models of human disease. This recognition has led to translational clinical trials in companion dogs with osteosarcoma, lymphoma, melanoma, squamous cell carcinoma, and soft tissue sarcoma. The ability to precisely track tumor-specific immune responses in such clinical trials would benefit from reagents to perform species-specific single cell T cell receptor sequencing (scTCRseq). This technology defines clones of T cells reacting to immune interventions and can help identify the specific epitope of response. Single cell gene expression data give insights into the activity and polarization of the T cell. To date, scTCRseq has not been demonstrated for canine samples. MethodsSamples from two responding dogs in a trial of an autologous deglycosylated melanoma vaccine were selected to demonstrate applicability of scTCRseq in a cancer immunotherapy setting. A single-cell suspension of cryopreserved peripheral blood mononuclear cells (PBMC) was prepared for 10X single cell sequencing. Full length 10X cDNA was amplified using a custom-designed nested PCR of the alpha/beta V(D)J region. A library made from this enriched product (scTCRseq) and a 10X gene expression (GEX) library (scRNAseq) were sequenced on the NovaSeq 6000. Results1,850-2,172 estimated V(D)J-expressing cells yielded 87-103.7 million reads with 73.8%-75.8% mapped to a V(D)J gene (beta/alpha chains ratio 1.5:1). 43 TRAJ, 29 TRAV, 12 TRBJ, and 22 TRBV gene segments were observed representing 72.9%, 51.8%, 100%, and 62.9% of all known V and J gene segments respectively. A large diversity of clonotypes was captured with 966-1,253 TRA/TRB clonotypes identified. Both dogs also exhibited a small number of highly abundant T cell clonotypes suggesting the presence of an anti-tumor T cell population. GEX enriched libraries successfully defined large clusters of CD8+ and CD4+ T cells that overlapped with V(D)J-expressing cells. DiscussionThe developed reagents successfully generated scTCRseq data, for the first time, which allowed the T cell repertoire to be surveyed in dogs responding to anti-tumor immunotherapy. These reagents will allow longitudinal tracking of anti-tumor T cell dynamics in canine cancer immunotherapy trials.

genomics↗

bioRxiv2021

An Empirical Energy Landscape Reveals Mechanism of Proteasome in Polypeptide Translocation

The ring-like ATPase complexes in the AAA+ family perform diverse cellular functions that require coordination between the conformational transitions of their individual ATPase subunits1,2. How the energy from ATP hydrolysis is captured to perform mechanical work by these coordinated movements is not known. In this study, we developed a novel approach for delineating the nucleotide-dependent free-energy landscape (FEL) of the proteasomes heterohexameric ATPase complex based on complementary structural and kinetic measurements. We used the FEL to simulate the dynamics of the proteasome and quantitatively evaluated the predicted structural and kinetic properties. The FEL model predictions were widely consistent with experimental observations in this and previous studies and suggested novel features of the mechanism of proteasomal ATPase. We find that the cooperative movements of the ATPase subunits result from the design of the ATPase hexamer entailing a unique free-energy minimum for each nucleotide-binding state. ATP hydrolysis dictates the direction of substrate translocation by triggering an energy-dissipating conformational transition of the ATPase complex.

biophysics↗

bioRxiv2021

Stem cell-free therapy for glaucoma to preserve vision

Glaucoma is the leading cause of irreversible blindness with trabecular meshwork (TM) dysfunction resulting in elevated intraocular pressure and retinal ganglion cell (RGC) damage leading to vision loss. In this study, we discovered that secretome, derived from human TM stem cells, via minimal invasive periocular injection, can reduce intraocular pressure, restore TM homeostasis, protect RGC, and restore RGC function in both steroid-induced and genetic myocilin mutant mouse models of glaucoma. The secretome upregulated the COX2-PGE2 axis via mitochondrial TMEM177 and led to activation of endogenous stem cells and TM regeneration. Inhibition of COX2 abolished the protective effect of secretome on TM cells. Secretome treatment also enhanced RGC survival and function. Proteomic analysis revealed that the secretome is enriched with proteins involved in extracellular matrix modulation leading to the remodeling of TM to restore homeostasis. This study highlights the feasibility of stem cell-free therapy for glaucoma with minimal invasive administration and the involvement of multiple novel pathways for a cumulative regenerative effect on the TM to protect RGC. Brief summaryThis study describes a cell-free treatment using stem cell secretome in two animal models of glaucoma and explores the potential mechanisms

cell biology↗

bioRxiv2021

CRISPRa screen on a genetic risk locus shared by multiple autoimmune diseases identifies a dysfunctional enhancer that affects IRF8 expression through cooperative lncRNA and DNA methylation machinery

Dysregulated transcription factors represent a major class of drug targets that mediate the abnormal expression of many critical genes involved in SLE and other autoimmune diseases. Although strong evidence suggests that natural human genetic variation affects basal and inducible gene expression, it is still a considerable challenge to establish a biological link between GWAS-identified non-coding genetic risk variants and their regulated gene targets. Here, we combine genetic data, epigenomic data, and CRISPR activation (CRISPRa) assays to screen for functional variants regulating IRF8 expression. Using CRISPR-mediated deletion and 3D chromatin structure analysis, we demonstrate that the locus containing rs2280381 is a cell-type-specific distal enhancer for IRF8 that spatially interacts with the IRF8 promoter. Further, rs2280381 mediates IRF8 expression through enhancer RNA AC092723.1, which recruits TET1 to the IRF8 promoter to modulate IRF8 expression by affecting methylation levels. The alleles of rs2280381 modulate PU.1 binding and chromatin state to differentially regulate AC092723.1 and IRF8 expression. Our work illustrates a strategy to define the functional genetic variants modulating transcription factor gene expression levels and identifies the biologic mechanism by which autoimmune diseases risk genetic variants contribute to the pathogenesis of disease.

genetics↗

bioRxiv2021

Screening of Botanical Drugs against SARS-CoV-2 Entry

An escalating pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is impacting global health. Specific treatment options for diseases caused by SARS-CoV-2 are largely lacking. Herein, we used a pseudotype virus (pv) bearing the SARS-CoV-2 S glycoprotein to screen a botanical drug library to identify an agent against SARS-CoV-2 entry. All the four hits, including angeloylgomisin O, schisandrin B, procyanidin, and oleanonic acid, were identified for effective inhibition of SARS-CoV-2 S pv entry in the micromolar range. A mechanistic study revealed that these four agents inhibit SARS-CoV-2 S pv entry by blocking S-mediated membrane fusion. Furthermore, angeloylgomisin O, schisandrin B, and oleanonic acid inhibited authentic SARS-CoV-2 with a high selective index (SI). We also showed that all the four hits could also inhibit the entry of pv of Middle East respiratory syndrome coronavirus (MERS-CoV) and newly emerged SARS-CoV-2 variants (D614G, K417N/E484K/N501Y/D614G). In drug combination studies performed in cellular antiviral assays, angeloylgomisin O and schisandrin B displayed synergistic effects in combination with remdesivir. These results indicated that angeloylgomisin O, schisandrin B, procyanidin, and oleanonic acid can inhibit SARS-CoV-2 and that they are potential therapeutic agents for COVID-19.

microbiology↗

bioRxiv2021

RNA-seq and differential expression analysis of the duck transcriptome: The effect of short-term cage-rearing

Ducks are an important source of meat and egg products for human beings. In China, duck breeding has gradually changed from the traditional floor-water combination system to multilayer cage breeding. Therefore, the present study collected the hypothalamus and pituitary of 113-day-old ducks after being caged for 3 days, in order to investigate the effect of cage-rearing on the birds. In addition, the same tissues (hypothalamus and pituitary) were collected from ducks raised in the floor-water combination system, for comparison. Thereafter, the transcriptomes were sequenced and the expression level of genes were compared. The results of sequencing analysis showed that a total of 506 and 342 genes were differentially expressed in the hypothalamus and pituitary, respectively. Additionally, the differentially expressed genes were mainly enriched in signaling pathways involved in processing environmental information, including ECM-receptor interaction, neuroactive ligand-receptor interaction and focal adhesion. The findings also showed that there was a change in the alternative splicing of genes when ducks were transferred into the cage rearing system. However, there was no difference in the expression of some genes although there was a change in the expression of the isoforms of these genes. The findings herein can therefore help in understanding the mechanisms underlying the effect of caging on waterfowl. The results also highlight the gene regulatory networks involved in animal responses to acute stress.

genetics↗

bioRxiv2021

Multi-animal pose estimation and tracking with DeepLabCut

Estimating the pose of multiple animals is a challenging computer vision problem: frequent interactions cause occlusions and complicate the association of detected keypoints to the correct individuals, as well as having extremely similar looking animals that interact more closely than in typical multi-human scenarios. To take up this challenge, we build on DeepLabCut, a popular open source pose estimation toolbox, and provide high-performance animal assembly and tracking--features required for robust multi-animal scenarios. Furthermore, we integrate the ability to predict an animals identity directly to assist tracking (in case of occlusions). We illustrate the power of this framework with four datasets varying in complexity, which we release to serve as a benchmark for future algorithm development.

animal behavior and cognition↗

bioRxiv2021

Pparg drives luminal differentiation and luminal tumor formation in the urothelium

Pparg, a nuclear receptor, is downregulated in basal subtype bladder cancers that tend to be muscle invasive and amplified in luminal subtype bladder cancers that tend to be non-muscle invasive. Bladder cancers derive from the urothelium, one of the most quiescent epithelia in the body which is composed of basal, intermediate, and superficial cells. We find that expression of an activated form of Pparg (VP16;Pparg) in basal progenitors induces formation of superficial cells in situ, that exit the cell cycle, and do not form tumors. Expression in basal progenitors that have been activated by mild injury however, results in luminal tumor formation. We find that tumors are immune deserted, which may be linked to downregulation of Nf-kb, a Pparg target. Interestingly, some luminal tumors begin to shift to basal subtype tumors with time, downregulating Pparg and other luminal markers. Our findings have important implications for treatment and diagnosis bladder cancer.

cancer biology↗

bioRxiv2021

A spatial and cellular distribution of neurotropic virus infection in the mouse brain revealed by fMOST and single cell RNA-seq

Neurotropic virus infection can cause serious damage to the central nervous system (CNS) in both human and animals. The complexity of the CNS poses unique challenges to investigate the infection of these viruses in the brain using traditional techniques. In this study, we explore the use of fluorescence micro-optical sectioning tomography (fMOST) and single cell RNA sequencing (scRNA-seq) to map the spatial and cellular distribution of a representative neurotropic virus, rabies virus (RABV), in the whole brain. Mice were inoculated with a lethal dose of recombinant RABV expressing enhanced green fluorescent protein (EGFP) under different infection routes, and a three-dimensional view of the distribution of RABV in the whole mouse brain was obtained using fMOST. Meanwhile, we pinpointed the cellular distribution of RABV by utilizing scRNA-seq. Our fMOST data provide the first evidence that RABV can infect multiple nuclei related to fear independent of different infection routes. More surprisingly, scRNA-seq data indicate that besides neurons RABV can infect macrophages and NK cells in vivo. Collectively, this study draws a comprehensively spatial and cellular map of RABV infection in the mouse brain, providing a novel and insightful strategy to investigate the pathogenesis of neurotropic viruses.

microbiology↗

bioRxiv2021

Predicting hosts based on early SARS-CoV-2 samples and analyzing later world-wide pandemic in 2020

The SARS-CoV-2 pandemic has raised the concern for identifying hosts of the virus since the early-stage outbreak. To address this problem, we proposed a deep learning method, DeepHoF, based on extracting the viral genomic features automatically, to predict host likelihood scores on five host types, including plant, germ, invertebrate, non-human vertebrate and human, for novel viruses. DeepHoF made up for the lack of an accurate tool applicable to any novel virus and overcame the limitation of the sequence similarity-based methods, reaching a satisfactory AUC of 0.987 on the five-classification. Additionally, to fill the gap in the efficient inference of host species for SARS-CoV-2 using existed tools, we conducted a deep analysis on the host likelihood profile calculated by DeepHoF. Using the isolates sequenced in the earliest stage of COVID-19, we inferred minks, bats, dogs and cats were potential hosts of SARS-CoV-2, while minks might be one of the most noteworthy hosts. Several genes of SARS-CoV-2 demonstrated their significance in determining the host range. Furthermore, the large-scale genome analysis, based on DeepHoFs computation for the later world-wide pandemic in 2020, disclosed the uniformity of host range among SARS-CoV-2 samples and the strong association of SARS-CoV-2 between humans and minks.

bioinformatics↗

bioRxiv2021

Pharmacological targeting of a PWWP domain demonstrates cooperative control of NSD2 localization

NSD2 is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36me2), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two PWWP and five PHD domains believed to serve as chromatin reading modules, but their exact function in the regulation of NSD2 activity remains underexplored. Here we report a first-in-class chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 binds potently (Kd of 91 {+/-} 8 nM) to PWWP1, antagonizes its interaction with nucleosomal H3K36me2, and selectively engages endogenous NSD2 in cells. Crystal structures show that UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1 which is juxtaposed to the DNA-binding surface. In cells, UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 as a result of translocations prevalent in multiple myeloma. Mutation of other NSD2 chromatin reader domains also increases NSD2 nucleolar localization, and enhances the effect of UNC6934. Finally we identified two C-terminal nucleolar localization sequences in NSD2 that appear to drive nucleolar accumulation when one or more chromatin reader domains are disabled. These data support a model in which NSD2 chromatin engagement is achieved in a cooperative manner and subcellular localization is controlled by multiple competitive structural determinants. This chemical probe and the accompanying negative control, UNC7145, will be useful tools in defining NSD2 biology.

cell biology↗

bioRxiv2021

The CLASSY family controls tissue-specific DNA methylation patterns in Arabidopsis

DNA methylation shapes the epigenetic landscape of the genome, plays critical roles in regulating gene expression, and ensures transposon silencing. As evidenced by the numerous defects associated with aberrant DNA methylation landscapes, establishing proper tissue-specific methylation patterns is critical. Yet, how such differences arise remains a largely open question in both plants and animals. Here we demonstrate that CLASSY1-4 (CLSY1-4), four locus-specific regulators of DNA methylation that are differentially expressed during plant development, play major roles in controlling tissue-specific DNA methylation patterns. Depending on the tissue, the genetic requirements for specific CLSYs differ significantly and, on a global scale, certain clsy mutants are sufficient to largely shift the epigenetic landscape between tissues. Together, these findings not only reveal substantial epigenetic diversity between tissues, but assign these changes to specific CLSY proteins, revealing how locus-specific targeting combined with tissue-specific expression enables the CLSYs to generate epigenetic diversity during plant development.

plant biology↗

bioRxiv2021

RNAi screening reveals requirement for PDGFRβ in JEV infection.

Mosquito-borne Japanese encephalitis virus (JEV) causes serious illness worldwide and is associated with high morbidity and mortality. To identify potential host therapeutic targets, a high-throughput receptor tyrosine kinase small interfering RNA library screening was performed with recombinant JEV particles. Platelet-derived growth factor receptor beta (PDGFR{beta}) was identified as a hit after two rounds of screening. Knockdown of PDGFR{beta} blocked JEV infection, and trans-complementation of PDGFR{beta} could partly restore its infectivity. The PDGFR{beta} inhibitor imatinib, which has been approved for the treatment of malignant metastatic cancer, protected mice against JEV-induced lethality by decreasing the viral load in the brain, while abrogating the histopathological changes associated with JEV infection. These findings demonstrated that PDGFR{beta} is important in viral infection and provided evidence for the potential to develop imatinib as a therapeutic intervention against JEV infection.

microbiology↗

bioRxiv2021

High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV2 nsp15 endoribonuclease to identify small-molecule inhibitors of viral activity.

SARS-CoV-2 has caused a global pandemic, and has taken over 1.7 million lives as of mid-December, 2020. Although great progress has been made in the development of effective countermeasures, with several pharmaceutical companies approved or poised to deliver vaccines to market, there is still an unmet need of essential antiviral drugs with therapeutic impact for the treatment of moderate-to-severe COVID-19. Towards this goal, a high-throughput assay was used to screen SARS-CoV-2 nsp15 uracil-dependent endonuclease (endoU) function against 13 thousand compounds from drug and lead repurposing compound libraries. While over 80% of initial hit compounds were pan-assay inhibitory compounds, three hits were confirmed as nsp15 endoU inhibitors in the 1-20 M range in vitro. Furthermore, Exebryl-1, a {beta}-amyloid anti-aggregation molecule for Alzheimers therapy, was shown to have antiviral activity between 10 to 66 M, in VERO, Caco-2, and Calu-3 cells. Although the inhibitory concentrations determined for Exebryl-1 exceed those recommended for therapeutic intervention, our findings show great promise for further optimization of Exebryl-1 as an nsp15 endoU inhibitor and as a SARS-CoV-2 antiviral. Author summaryDrugs to treat COVID-19 are urgently needed. To address this, we searched libraries of drugs and drug-like molecules for inhibitors of an essential enzyme of the virus that causes COVID-19, SARS-CoV-2 nonstructural protein (nsp)15. We found several molecules that inhibited the nsp15 enzyme function and one was shown to be active in inhibiting the SARS-CoV-2 virus. This demonstrates that searching for SARS-CoV-2 nsp15 inhibitors can lead inhibitors of SARS-CoV-2, and thus therapeutics for COVID-19. We are currently working to see if these inhibitors could be turned into a drug to treat COVID-19.

microbiology↗

bioRxiv2021

Enhancing top-down proteomics of brain tissue with FAIMS

Proteomic investigations of Alzheimers and Parkinsons disease have provided valuable insights into neurodegenerative disorders. Thus far, these investigations have largely been restricted to bottom-up approaches, hindering the degree to which one can characterize a proteins "intact" state. Top-down proteomics (TDP) overcomes this limitation, however it is typically limited to observing only the most abundant proteoforms and of a relatively small size. Therefore, offline fractionation techniques are commonly used to reduce sample complexity, limiting throughput. A higher throughput alternative is online fractionation, such as gas phase high-field asymmetric waveform ion mobility spectrometry (FAIMS). Utilizing a high complexity sample derived from Alzheimers disease brain tissue, we describe how the addition of FAIMS to TDP can robustly improve the depth of proteome coverage. For example, implementation of FAIMS at -50 compensation voltage (CV) more than doubled the mean number of non-redundant proteoforms observed (1,833 {+/-} 17, n = 3), compared to without (754 {+/-} 35 proteoforms). We also found FAIMS can influence the transmission of proteoforms and their charge envelopes based on their size. Importantly, FAIMS enabled the identification of intact amyloid beta (A{beta}) proteoforms, including the aggregation-prone A{beta}1-42 variant which is strongly linked to Alzheimers disease.

biochemistry↗

bioRxiv2020

Sterilizing immunity against SARS-CoV-2 in hamsters conferred by a novel recombinant subunit vaccine

A safe and effective SARS-CoV-2 vaccine is essential to avert the on-going COVID-19 pandemic. Here, we developed a subunit vaccine, which is comprised of CHO-expressed spike ectodomain protein (StriFK) and nitrogen bisphosphonates-modified zinc-aluminum hybrid adjuvant (FH002C). This vaccine candidate rapidly elicited the robust humoral response, Th1/Th2 balanced helper CD4 T cell and CD8 T cell immune response in animal models. In mice, hamsters, and non-human primates, 2-shot and 3-shot immunization of StriFK-FH002C generated 28- to 38-fold and 47- to 269-fold higher neutralizing antibody titers than the human COVID-19 convalescent plasmas, respectively. More importantly, the StriFK-FH002C immunization conferred sterilizing immunity to prevent SARS-CoV-2 infection and transmission, which also protected animals from virus-induced weight loss, COVID-19-like symptoms, and pneumonia in hamsters. Vaccine-induced neutralizing and cell-based receptor-blocking antibody titers correlated well with protective efficacy in hamsters, suggesting vaccine-elicited protection is immune-associated. The StriFK-FH002C provided a promising SARS-CoV-2 vaccine candidate for further clinical evaluation.

microbiology↗

bioRxiv2020

Mechanism through which retrocyclin targets flavivirus multiplication

Currently, there are no approved drugs for the treatment of flavivirus infection. Accordingly, we tested the inhibitory effects of the novel {theta}-defensin retrocyclin-101 (RC-101) against flavivirus infection, and investigated the mechanism underlying the potential inhibitory effects. First, RC-101 robustly inhibited both Japanese encephalitis virus (JEV) and Zika virus (ZIKV) infections. RC-101 exerted inhibitory effects on the entry and replication stages. Results also indicated that the non-structural protein NS2B-NS3 serine protease might serve as a potential viral target. Further, RC-101 inhibited protease activity at the micromolar level. We also demonstrated that with respect to the glycoprotein E protein of flavivirus, the DE loop of domain III, which is the receptor-binding domain of the E protein, might serve as another viral target of RC-101. Moreover, a JEV DE mutant exhibited resistance to RC-101, which was associated with deceased binding affinity of RC-101 to DIII. These findings provide a basis for the development of RC-101 as a potential candidate for the treatment of flavivirus infection. ImportanceRC has been reported to have a broad-spectrum antimicrobial activity. In this study, we firstly report that RC-101 could inhibit ZIKV and JEV infections. Moreover, both the NS2B-NS3 serine protease and the DE loop in the E glycoprotein might serve as the viral targets of RC-101.

microbiology↗

bioRxiv2020

Peptide Ancestry Informative Markers in Uterine Neoplasms from Women of European, African and Asian Ancestry

Characterization of ancestry-linked peptide variants in disease-relevant patient tissues represents a foundational step to connect patient ancestry with molecular disease pathogenesis. Nonsynonymous single nucleotide polymorphisms (SNPs) encoding missense substitutions within tryptic peptides exhibiting high allele frequencies in European, African, and East Asian populations, termed peptide ancestry informative markers (pAIMs), were prioritized from 1000 genomes. In silico analysis shows that as few as 20 pAIMs can determine ancestry proportions similarly to >260K SNPs (R2=0.9905). Multiplexed proteomic analysis of >100 human endometrial cancer cell lines and uterine leiomyoma tissues resulted in the quantitation of 62 pAIMs that correlate with self-described race and genotype-confirmed patient ancestry. Candidates include a D451E substitution in GC vitamin D-binding protein previously associated with altered vitamin D levels in African and European populations. These efforts describe a generalized set of markers for proteoancestry assessment that will further support studies investigating the impact of ancestry on the human proteome and how this relates to the pathogenesis of uterine neoplasms.

cancer biology↗