Biology subjects

Qin, D.

Publications and source records attributed to Qin, D..

29 records · Page 2Linked to original sources

bioRxiv2023

A framework for ultra-low input spatial tissue proteomics

Spatial tissue proteomics combining microscopy-based cell phenotyping with ultra-sensitive mass spectrometry (MS)-based proteomics is an emerging and powerful concept for the study of cell function and heterogeneity in health and disease. However, optimized workflows that preserve morphological information for image-based phenotype discovery and maximize proteome coverage of few or even single cells from laser microdissected archival tissue, are currently lacking. Here, we report a robust and scalable workflow for the proteomic analysis of ultra-low input formalin-fixed, paraffin-embedded (FFPE) material. Benchmarking in the murine liver resulted in up to 2,000 quantified proteins from single hepatocyte contours and nearly 5,000 proteins from 50-cell regions with high quantitative reproducibility. Applied to human tonsil, we profiled 146 microregions including spatially defined T and B lymphocyte niches and quantified cell type specific markers, cytokines, immune cell regulators and transcription factors. These rich data also highlighted proteome dynamics in spatially defined zones of activated germinal centers, illuminating sites undergoing active B-cell proliferation and somatic hypermutation. Our results demonstrate the power of spatially-resolved proteomics for tissue phenotyping by integrating high-content imaging, laser microdissection, and ultra-sensitive mass spectrometry. This approach has broad implications for a wide range of biomedical applications, including early disease profiling, drug target discovery and biomarker research.

systems biology↗

bioRxiv2022

Naturally occurring Alzheimer's disease in rhesus monkeys

Alzheimers disease (AD) is the most common neurodegenerative disease. To date, its cause is unclear and there are no effective treatments or preventive measures. Despite there are accumulating evidences for the existence of AD pathological hallmarks in the brain of aging rhesus monkeys, it remains a mainstream notion that monkeys do not develop AD naturally. This is an important issue because it will determine how we use monkeys in AD studies. To settle down this issue, a group (n=10) of aged rhesus monkeys 26 years old or above went through a systematic AD screening procedure in this study. Three of these monkeys showed severe memory impairments (SMI) after evaluated with a classic working memory test. Further behavioral testing revealed that the SMI monkeys also exhibited apathy-like behavior, which is another core AD clinical symptom. In addition to the cognitive deficits, two of the three SMI monkeys developed all of the three AD pathological hallmarks, including neurofibrillary tangles, senile plaques and neuronal loss. According to the diagnostic criteria of human AD, the two SMI monkeys were clearly naturally occurring AD monkeys. These results suggest that AD is not a uniquely human disease and monkeys have great potential for the development of much needed etiological AD models, which are vital for better understanding of developmental process of AD and the base of identification of early diagnostic biomarkers and effective therapeutic targets of AD.

neuroscience↗

bioRxiv2022

The role of GmXTH1, a new xyloglucan endotransglycosylase/hydrolase from soybean, in regulating soybean root growth at seedling stage

Soybean is an important crop for both food and oil, providing high quality protein and oil for humans and animals. Drought stress has an significant effect on the yield and quality of soybean. In the early vegetative stage of soybean, the developed root system can effectively improve the tolerance of soybean plant to drought. Xyloglucan endoglycosylase/hydrolase (XTH) is a cell wall remodeling enzyme involved in cell wall expansion and degradation. However, there are few studies on XTHs regulation of root growth and response to drought stress in soybean seedlings.The objectives of this study were to clone GmXTH1 gene and determine the effects of over and interference-expression of GmXTH1 on soybean root development at seedling stage. A 1,015 bp full-length of the GmXTH1 gene was cloned from soybean (Glycine max L. Merril) cv. RM18 with a 840 bp open reading frame (ORF) of a putative protein. The over and interference expression vectors of GmXTH1 were transformed into soybean using Agrobacterium method. Transformed strains OEA1, OEA3, IEA4, IEA5 were identified by molecular detection methods. The roots of transformed strains OEA1and OEA3 with GmXTH1 gene overexpression are clearly developed and the root phenotypic attributes, including main root length, primary root number, lateral root length, root surface area, root volume, root dry weight, root fresh weight are higher than the control. In contrast, root attributes of IEA4 and IEA5 with GmXTH1 gene interference expression were significantly lower than those of the control, suggesting that the expression of GmXTH1 gene has an important effect on the soybean root growth and development at seedling stage. Under drought stress conditions, the activities of Superoxide dismutase (SOD), Peroxidase(POD)and Catalase(CAT)in the transgenic strains with an overexpression of the GmXTH1 gene were significantly increased, and the root activity, leaf relative water content and total chlorophyll content were ranked in order of OEAs, CK, and IEAs. In conclusion, GmXHT1 plays an important role in the growth of soybean root at seedling stage. Overexpression of GmXTH1 gene can promote root development of soybean seedling. The results of activities of protective enzymes (SOD, POD, CAT), root activity, leaf relative water content and total chlorophyll content under drought stress indicated that transgenic strains OEA1 and OEA3 with overexpressing GmXTH1 gene were resistant to drought stress, suggesting that GmXTH1 may play a positive role in responding to drought stress at soybean seedling stage.

genetics↗

bioRxiv2022

Fate tracing reveals differences between Reelin+ HSCs and Desmin+ HSCs in activation, migration, and proliferation activities

The activation of hepatic stellate cells (HSCs) which comprise distinct clusters, is the main cause of liver fibrogenesis in response to different etiologies of chronic liver injuries. In this study, we constructed a novel ReelinCreERT2 transgenic mouse in which cells expressing Reelin were fully marked and demonstrated that about 50% HSCs were labeled. These ReelinCreERT2-labeled HSCs displayed distinct characteristics in migration, activation and proliferation compared to Desmin+ HSCs (total HSCs) in cholestatic (bile duct ligation; BDL) or hepatotoxic (carbon tetrachloride; CCl4) liver injuries. In BDL-induced fibrotic livers, Desmin+ HSCs were activated with increased proliferation and accumulation activities around the portal triad, but mGFP+ HSCs did not show proliferation or accumulation activity around the portal triad, and only a small part was activated. In CCl4-induced fibrotic livers, most of Desmin+ and mGFP+ HSCs were activated along with proliferation and accumulation potential around the central vein, however fewer mGFP+ HSCs were activated compared to Desmin+ HSCs. Moreover, in the regression of CCl4-induced fibrosis, mGFP+ HSCs were apoptosed whereas Desmin+ HSCs recovered to normal state. Besides, we didnt find evidence that mGFP+ HSCs transdifferentiated into hepatocytes or cholangiocytes through mesenchymal-epithelial transition (MET).

pathology↗

bioRxiv2022

Clustering Deviation Index (CDI): A robust and accurate unsupervised measure for evaluating scRNA-seq data clustering

Single-cell RNA-sequencing (scRNA-seq) technology allows us to explore cellular heterogeneity in the transcriptome. Because most scRNA-seq data analyses begin with cell clustering, its accuracy considerably impacts the validity of downstream analyses. Although many clustering methods have been developed, few tools are available to evaluate the clustering "goodness-of-fit" to the scRNA-seq data. In this paper, we propose a new Clustering Deviation Index (CDI) that measures the deviation of any clustering label set from the observed single-cell data. We conduct in silico and experimental scRNA-seq studies to show that CDI can select the optimal clustering label set. Particularly, CDI also informs the optimal tuning parameters for any given clustering method and the correct number of cluster components.

bioinformatics↗

bioRxiv2021

Transcription factor-like 5 is a potential DNA/RNA-binding protein essential for maintaining male fertility in mice.Running title: Tcfl5 is haploinsufficient in vivo

Tissue-specific transcription factors often play key roles in the development of specific cell lineages. Transcription factor-like 5 (TCFL5) is a testis-specific protein that contains the basic helix-loop-helix domain, although the in vivo functions of TCFL5 remain unknown. Herein, we generated CRISPR/Cas9-mediated knockout mice to dissect the function of TCFL5 in mouse testes. Surprisingly, we found that it was difficult to generate homozygous mice with the Tcfl5 deletion since the heterozygous males (Tcfl5+/-) were infertile. We did, however, observe markedly abnormal phenotypes of spermatids and spermatozoa in the testes and epididymides of Tcfl5+/- mice. Mechanistically, we demonstrated that TCFL5 transcriptionally regulated a set of genes participating in male germ cell development, which we uncovered via RNA-sequencing and TCFL5 ChIP-sequencing. We also found that TCFL5 interacted with RNA-binding proteins (RBPs) that regulated RNA processing, and further identified the fragile X mental retardation gene 1, autosomal homolog (FXR1, a known RBP) as an interacting partner of TCFL5 that may coordinate the transition and localization of TCFL5 in the nucleus. Collectively, we herein report for the first time that Tcfl5 is haploinsufficient in vivo and hypothesize that TCFL5 may be a dual-function protein that mediates DNA and RNA to regulate spermatogenesis.

cell biology↗

bioRxiv2021

Brazil Nut Effect Drives Pattern Formation in Early Mammalian Embryos

The formation of three-dimensional ordered spatial patterns, which is essential for embryonic development, tissue regeneration, and cancer metastasis, is mainly guided by the chemical concentration gradient of morphogens. However, since no chemical concentration gradient has been observed in the early embryonic development (pre-implantation) of mammals, the pattern formation mechanism has been unsolved for a long time. During the second cell fate decision of mouse embryos, the inner cell mass (ICM) segregates into topographically regionalized epiblast (EPI) and primitive endoderm (PrE) layers. Here, we report that the segregation process of PrE/EPI precursors coincides with an emerged periodic expansion-contraction vibration of the blastocyst cavity, which induces phase transition in the ICM compartment to a higher fluidity state and generates directional tissue flows. By experiments and modeling, we demonstrate that the spatial segregation of PrE and EPI precursors is mediated by a "Brazil nut effect"-like viscous segregation mechanism in which PrE precursors with low affinity gradually migrate to the surface of ICM along with the tissue flow, while EPI precursors with high affinity remains inside ICM under cavity vibration. Artificially manipulation of the frequency and amplitude of cavity vibration could control the process of spatial separation as well as lineage specification of PrE/EPI. Furthermore, disruption of the cavity vibration in the initial stage after segregation could reverse the ICM cells back to a mixed state. Therefore, this study reveals a fundamental mechanism that guarantees the robustness of cell segregation and pattern formation without specific morphogens in early mammalian embryos. Our model also emphasizes a conserved function of cavity structure that widely exists in organisms as an energy reservoir and converter between different forms, such as chemical and mechanical energy.

developmental biology↗

bioRxiv2020

Histone demethylase complexes KDM3A and KDM3B cooperate with OCT4/SOX2 to construct pluripotency gene regulatory network

The pluripotency gene regulatory network of porcine-induced pluripotent stem cells (piPSCs), especially in epigenetics, remains elusive. To determine this biological function of epigenetics, we cultured piPSCs in different culture conditions. We found that activation of pluripotent gene- and pluripotency-related pathways requires the erasure of H3K9 methylation modification which was further influenced by mouse embryonic fibroblast (MEF) served feeder. By dissecting the dynamic change of H3K9 methylation during loss of pluripotency, we demonstrated that the H3K9 demethylases KDM3A and KDM3B regulated global H3K9me2/me3 level and that their co-depletion led to the collapse of the pluripotency gene regulatory network. Immunoprecipitation-mass spectrometry (IP-MS) provided evidence that KDM3A and KDM3B formed a complex to perform H3K9 demethylation. The genome-wide regulation analysis revealed that OCT4 (O) and SOX2 (S), the core pluripotency transcriptional activators, maintained the pluripotent state of piPSCs depending on the H3K9 hypomethylation. Further investigation revealed that O/S cooperating with histone demethylase complex containing KDM3A and KDM3B promoted pluripotency genes expression to maintain the pluripotent state of piPSCs. Together, these data offer a unique insight into the epigenetic pluripotency network of piPSCs. SummaryErasure of H3K9 methylation in porcine pluripotent stem cells depends on the complex of transcription factors OCT4/SOX2 and histone demethylase KDM3A/KDM3B.

cell biology↗

bioRxiv2020

Discovery of potential epigenetic inhibitors against histone methyltransferases through molecular docking and molecular dynamics simulations

Histone methyltransferases (HMTases) catalyze histone methylations that are the important epigenetic marks regulating gene expression, cell fate, and disease progression. In this study, we investigated potential epigenetic inhibitors against HMTases through in silico approaches, including ensembled molecular docking and molecular dynamics simulations (MDS).We identified three candidate compounds, including echinomycin, emetine, and streptonigrin, which showed interactions with HMTases. Echinomycin showed similar binding affinity with H3K4-HMTase NSD3 and H3K9-HMTase SETDB1 but streptonigrin and emetine had preferential binding affinity with NSD3 and SETDB1, respectively. The binding of NSD3 to streptonigrin and echinomycin and binding of SETDB1 to emetine and echinomycin were further confirmed by the results of hydrogen bonding profile and MM/PBSA calculations. Together, our results uncover the binding affinities of echinomycin, emetine, and streptonigrin with histone methyltransferases, and suggest that these compounds are potential epigenetic inhibitors regulating cell activities.

molecular biology↗

bioRxiv2020

Structural dynamics of non-synonymous SNPs of histone methyltransferase EZH2 involved in Weaver Syndrome

Enhancer of zeste homolog 2 (EZH2) is a histone H3 lysine 27 methyltransferases. Non-synonymous SNPs (nsSNPs) in the ezh2 gene may cause Weaver Syndrome that is a prominent and rare congenital disorder. Several EZH2 genetic variants have been characterized and reported, but there is no information available on their structural dynamics. Our study employs an in silico approach for structural and functional characterization of EZH2 nsSNPs. We identified 19 EZH2 nsSNPs majorly associated with Weaver Syndrome, among which four SET-domain nsSNPs including V621M, A677T, R679C, and H689Y significantly affected EZH2 structure. We conducted the triplicate of 100 ns of molecular dynamics simulations (MDS) to compare the dynamic interaction behaviors between wild-type and mutants of EZH2 with their substrate, H3K27me0 peptide. The simulation results revealed that the mutants had higher levels of structural variations as evidenced by secondary structure, density, distance plots, and principal component analysis. Compared to EZH2-WT, the mutants A677T and H689Y have shown lower binding energy with H3K27me0 peptide due to the denaturing of 310 helixes as exemplified by MM/PBSA calculations and secondary structure analysis. Our analysis shed light on mechanisms of structural variations of EZH2 nsSNPs and lay a stone to develop mutant-based therapeutic strategies for the design of target-specific scaffolds against Weaver Syndrome.

molecular biology↗

bioRxiv2019

Detection of EGFR deletion using unique RepSeq technology

We are reporting a novel sequencing technology, RepSeq (Repetitive Sequence), that has high sensitivity, specificity and quick turn-around time. This new sequencing technology is developed by modifying traditional Sanger sequencing technology in several aspects. The first, a homopolymer tail is added to the PCR primer(s), which makes interpreting electropherograms a lot easier than that in traditional Sanger sequencing. The second, an indicator nucleotide is added at the 5end of the homopolymer tail. In the presence of a deletion, the position of the indicator nucleotide in relation to the wild type confirms the deletion. At the same time, the indicator of the wild type serves as the internal control. Furthermore, the specific design of the PCR and/or sequencing primers will specifically enrich/select mutant alleles, which increases sensitivity and specificity significantly. Based on serial dilution studies, the analytical lower limit of detection was 1.47 copies. A total of 89 samples were tested for EGFR exon 19 deletion, of which 21 were normal blood samples and 68 were samples previously tested by either pyrosequencing or TruSeq Next Generation Sequencing Cancer Panel. There was 100 % concordance among all the samples tested. RepSeq technology has overcome the shortcomings of Sanger sequencing and offers an easy-to-use novel sequencing method for personalized precision medicine.

genomics↗