Biology subjects

Tang, X.

Publications and source records attributed to Tang, X..

At least 109 records · Page 6Linked to original sources

bioRxiv2023

Functional interrogation of twenty type 2 diabetesassociated genes using isogenic hESC-derived β-like cells

Genetic studies have identified numerous loci associated with type 2 diabetes (T2D), but the functional role of many loci has remained unexplored. In this study, we engineered isogenic knockout human embryonic stem cell (hESC) lines for 20 genes associated with T2D risk. We systematically examined {beta}-cell differentiation, insulin production and secretion, and survival. We performed RNA-seq and ATAC-seq on hESC-{beta} cells from each knockout line. Analyses of T2D GWAS signals overlapping with HNF4A-dependent ATAC peaks identified a specific SNP as a likely causal variant. In addition, we performed integrative association analyses and identified four genes (CP, RNASE1, PCSK1N and GSTA2) associated with insulin production, and two genes (TAGLN3 and DHRS2) associated with sensitivity to lipotoxicity. Finally, we leveraged deep ATAC-seq read coverage to assess allele-specific imbalance at variants heterozygous in the parental hESC line, to identify a single likely functional variant at each of 23 T2D GWAS signals.

genetics↗

bioRxiv2023

Integration of microbial and chemical synthesis for the efficient production of plitidepsin, a promising anticancer and antiviral agent

Plitidepsin, a marine-derived anticancer medicine, is being tested in phase III clinical trials for treating COVID-19. However, the current supply of plitidepsin relies on laborious chemical synthesis processes. Here, we present a new approach that combines microbial and chemical synthesis to produce plitidepsin. We screened a Tistrella strain library to identify a high-yield didemnin B producer, and then introduced a second copy of the didemnin biosynthetic gene cluster into its genome, resulting in the highest yield of didemnin B reported in the literature. Next, we developed two straightforward chemical strategies to convert didemnin B to plitidepsin, one of which involved a one-step synthetic route giving over 90% overall yield. We also synthesized two new didemnin analogues and assessed their anticancer and antiviral activities. Our findings offer a practical and sustainable solution for producing plitidepsin and its derivatives, potentially expediting didemnin drug development.

synthetic biology↗

bioRxiv2023

Broadly effective ACE2 decoy proteins protect mice from lethal SARS-CoV-2 infection

As SARS-CoV-2 variants have been causing increasingly serious drug resistance problem, development of broadly effective and hard-to-escape anti-SARS-CoV-2 agents is in urgent need. Here we describe further development and characterization of two SARS-CoV-2 receptor decoy proteins, ACE2-Ig-95 and ACE2-Ig-105/106. We found that both proteins had potent and robust in vitro neutralization activities against diverse SARS-CoV-2 variants including Omicron, with an average IC50 of up to 37 pM. In a stringent lethal SARS-CoV-2 infection mouse model, both proteins lowered lung viral load by up to [~]1000 fold, prevented the emergence of clinical signs in >75% animals, and increased animal survival rate from 0% (untreated) to >87.5% (treated). These results demonstrate that both proteins are good drug candidates for protecting animals from severe COVID-19. In a head-to-head comparison of these two proteins with five previously-described ACE2-Ig constructs, we found that two of these constructs, each carrying five surface mutations in the ACE2 region, had partial loss of neutralization potency against three SARS-CoV-2 variants. These data suggest that extensively mutating ACE2 residues near the RBD-binding interface should be avoided or performed with extra caution. Further, we found that both ACE2-Ig-95 and ACE2-Ig-105/106 could be produced to gram/liter level, demonstrating the developability of them as biologic drug candidates. Stress-condition stability test of them further suggests that more studies are required in the future to improve the stability of these proteins. These studies provide useful insight into critical factors for engineering and preclinical development of ACE2 decoys as broadly effective therapeutics against diverse ACE2-utilizing coronaviruses. Abstract ImportanceEngineering soluble ACE2 proteins that function as a receptor decoy to block SARS-CoV-2 infection is a very attractive approach to broadly effective and hard-to-escape anti-SARS-CoV-2 agents. This study here describes development of two antibody-like soluble ACE2 proteins that broadly block diverse SARS-CoV-2 variants including Omicron. In a stringent COVID-19 mouse model, both proteins successfully protected >87.5% animals from lethal SARS-CoV-2 infection. In addition, a head-to-head comparison of the two constructs developed in this study with five previously-described ACE2 decoy constructs were performed here. Two previously-described constructs with relatively more ACE2-surface mutations were found with less robust neutralization activities against diverse SARS-CoV-2 variants. Further, the developability of the two proteins as biologic drug candidates was also assessed here. This study provides two broadly anti-SARS-CoV-2 drug candidates and useful insight into critical factors for engineering and preclinical development of ACE2 decoy as broadly effective therapeutics against diverse ACE2-utilizing coronaviruses. TweetTwo antibody-like ACE2 decoy proteins could block diverse SARS-CoV-2 variants and prevent animals from severe COVID-19.

microbiology↗

bioRxiv2023

cGAS-activating lupus autoantibody for cancer immunotherapy

Cytoplasmic DNA triggers a cGAS-mediated signaling cascade that promotes an innate immune response and is potentially actionable in cancer immunotherapy. Here we show that a cytoplasmic-localizing lupus anti-DNA autoantibody activates cGAS and facilitates an immune-mediated prolongation of survival in orthotopic models of glioblastoma (GBM). Mechanistically, cellular penetration and blood-brain barrier crossing by the anti-DNA autoantibody is linked to nucleoside transport. Pulldown, knockdown, signaling, and cytotoxicity assays demonstrate autoantibody association with and activation of cGAS. In orthotopic GBM models, the autoantibody localizes to brain tumor, increases tumor CD8+ T cell content, and prolongs survival in immunocompetent but not immunodeficient mice. This work introduces the new concept of a cGAS-activating anti-DNA autoantibody, which impacts theories on mechanisms of autoimmunity and has translational applications in cancer immunotherapy.

immunology↗

bioRxiv2023

STAT3 protects HSCs from intrinsic interferon signaling and loss of long-term blood-forming activity

STAT3 function in hematopoietic stem and progenitor cells (HSPCs) has been difficult to discern as Stat3 deficiency in the hematopoietic system induces systemic inflammation, which can impact HSPC activity. To address this, we established mixed bone marrow (BM) chimeric mice with CreER-mediated Stat3 deletion in 20% of the hematopoietic compartment. Stat3-deficient HSPCs had impaired hematopoietic activity and failed to undergo expansion in BM in contrast to Stat3-sufficient (CreER) controls. Single-cell RNA sequencing of Lin-ckit+Sca1+ BM cells revealed altered transcriptional responses in Stat3-deficient hematopoietic stem cells (HSCs) and multipotent progenitors, including intrinsic activation of cell cycle, stress response, and interferon signaling pathways. Consistent with their deregulation, Stat3-deficient Lin-ckit+Sca1+ cells accumulated {gamma}H2AX over time. Following secondary BM transplantation, Stat3-deficient HSPCs failed to reconstitute peripheral blood effectively, indicating a severe functional defect in the HSC compartment. Our results reveal essential roles for STAT3 in HSCs and suggest the potential for using targeted synthetic lethal approaches with STAT3 inhibition to remove defective or diseased HSPCs. Key PointsO_LISTAT3 is critical for hematopoietic activity and hematopoietic stem cell maintenance in non-inflammatory conditions C_LIO_LISTAT3 has a cell-intrinsic role in the suppression of interferon signaling and myeloid-skewed transcription in hematopoietic stem cells C_LI

immunology↗

bioRxiv2023

Model-guided Design of Biological Controller for Septic Wound Healing Regulation

Immune response is critical in septic wound healing. The aberrant and imbalanced signaling dynamics primarily cause a dysfunctional innate immune response, exacerbating pathogen invasion of injured tissue and further stalling the healing process. To design biological controllers that regulate the critical divergence of the immune response during septicemia, we need to understand the intricate differences in immune cell dynamics and coordinated molecular signals of healthy and sepsis injury. Here, we deployed an ordinary differential equation (ODE)-based model to capture the hyper and hypo-inflammatory phases of sepsis wound healing. Our results indicate that impaired macrophage polarization leads to a high abundance of monocytes, M1, and M2 macrophage phenotypes, resulting in immune paralysis. Using a model-based analysis framework, we designed a biological controller which successfully regulates macrophage dysregulation observed in septic wounds. Our model describes a systems biology approach to predict and explore critical parameters as potential therapeutic targets capable of transitioning septic wound inflammation toward a healthy, wound-healing state.

systems biology↗

bioRxiv2022

Molecular evolution of protein sequences and codon usage in monkeypox viruses

The monkeypox virus (mpox virus, MPXV) epidemic in 2022 poses a significant public health risk. However, the evolutionary principles of MPXV are largely unknown. Here, we examined the evolutionary patterns of protein sequences and codon usage in MPXV. We first showed the signal of positive selection in OPG027 specifically in Clade I. We then discovered accelerated protein sequence evolution over time in 2022 outbreak-causing variants. We also found strong epistasis between amino acid substitutions located in different genes. Codon adaptation index (CAI) analysis revealed that MPXV tended to use more unpreferred codons than human genes and that the CAI decreased with time and diverged between clades, with Clade I > IIa and IIb-A > IIb-B. Although the decrease in fatality rate in the three groups matched the CAI pattern, it is unclear whether this is a coincidence or if the deoptimization of codon usage in MPXV induced a decrease in fatality. This study sheds new light on the processes influencing the evolution of MPXV in human populations.

evolutionary biology↗

bioRxiv2022

Comparative transcriptome revealed potential genes related with drug resistance in Melanoma

Chemotherapy remains a relatively ineffective and unsatisfactory treatment because of drug resistance in melanoma (whether due to intrinsic resistance or the use of cytostatic drugs). In order to explore the genes and signaling pathways related to melanoma drug resistance, the study presented here obtained drug-resistant melanoma cell lines of A375 and M14 by gradually increasing the concentration of dacarbazine (DTIC), followed by comparative transcriptomics studies (RNA-seq) and real-time quantitative PCR (RT-qPCR) and Western Blotting validation. The results showed that after 8 months of continuous treatment, the IC50 values of A375 and M14 to DTIC were increased by more than 5 folds. Meanwhile, flow cytometry analysis found that drug-resistant melanoma cells have a significant ability to resist apoptosis induced by DTIC. Subsequently, RNA-seq revealed high expression of SENP1 and abnormal activation of the Hippo signaling pathway in drug-resistant cells. Finally, we found that compared with wild-type cells, the expressions of SENP1 and YAP were both up-regulated in drug-resistant cells via RT-qPCR and Western Blotting. Roles of SENP1 in drug resistance was finally verified via its overexpression in normal A375 cell lines. Therefore, this paper infers that there is a positive correlation between the ubiquitin-specific protease SENP1 and the drug resistance of melanoma. Meanwhile, the up-regulation of its expression may lead to changes in the Hippo signaling pathway and increase the resistance of melanoma to DTIC.

cancer biology↗

bioRxiv2022

Single-cell gene expression and chromatin accessibility profiling of human pancreatic islets at basal and stimulatory conditions nominates mechanisms of type 1 diabetes genetic risk

Type 1 diabetes (T1D) is a complex autoimmune disease characterized by the loss of pancreatic islet beta cells. The mechanisms of T1D genetic risk remain poorly understood. Here, we present a multi-omic integrative study of single-cell/nucleus molecular profiles of gene expression and chromatin accessibility in the same biological samples from healthy and beta cell autoantibody+ (AAB+) human pancreatic islets to characterize mechanisms of islet-mediated T1D genetic risk. We additionally performed single-cell/nucleus multi-omic profiling of healthy islets under two stimulatory conditions used as in vitro models of T1D (cytokine cocktail and CVB4 infection) to evaluate how environmental exposures recapitulate multi-omic signatures of T1D. In total, we analyzed 121,272 cells/nuclei across 34 libraries, identifying 10 distinct cell types. We identified cell-type-specific and disease-associated cis-regulatory elements and nominated likely target genes. We provide evidence that T1D genetic risk is mediated through multiple pancreatic cell populations, including islet endocrine cells (beta, alpha, gamma, and delta), exocrine acinar and ductal cells, and immune cells. Finally, we identified three independent T1D risk variants acting through pancreatic islet endocrine cells at the TOX, RASGRP1, and DLK1/MEG3 loci. Together, this work improves our understanding of how non-coding genetic variants encode T1D risk through a complex interplay of different cell types in the pancreas.

genomics↗

bioRxiv2022

The entry of pinewood nematode is linked to programmed tracheal development of vector beetles

The transmission of some pathogens by insect vectors often requires fine-tuned biological synchronization and communication between the two parties. However, the role of tissue development in mediating this pathogen-vector coordination remains elusive. Here we investigated the association between tracheal development of Monochamus alternatus beetle and a notorious plant parasitic pine wood nematode Bursaphelenchus xylophilus, which enter and inhabit inside the tracheal system after adult beetle eclosion and then were transmitted to pine trees (Pinus spp.) by the beetle. We found that the tracheal systems of newly emerged adult beetles underwent morphological changes during the adult sclerotization, characterized by a remarkable increase of diameter of thoracic tracheal dorsal tubes. Consistently, comparative transcriptomics further revealed dramatic changes in gene expression occurring within five days after eclosion, demonstrating sequential regulation of tracheal genes. Genes controlling primary branching were up-regulated soon after eclosion, whereas those controlling terminal branching and branch fusion were up-regulaed at the later stage. Interestingly, during tracheal maturation, genes involved in biosynthesis of Juvenile Hormone (JH) and ecdysteroids were activated. Nematode loading assay further revealed that the entry of nematodes to beetle tracheae was initiated earliest after three days post eclosion, implying that nematode entry is reliant on the formation of the vectors primary tracheal branches and is perhaps stimulated by production of insect hormones or their precursors. In addition, specific regulation on secreted proteins, such as Defensin-1-like and membrane proteins, at later stages of tracheal maturation may further facilitate the entry of nematodes by improving their retention. Therefore, this study highlights vital role of programed tracheal development on nematode entry into its vector beetle, and suggests a potential roles of insect genes and metabolites in manipulation of interspecific biological developmental synchronization between parasites and their insect vectors.

ecology↗

bioRxiv2022

Development of an affinity-enhanced clinical candidate TCR targeting NY-ESO-1 with optimal potency and high specificity

The clinical success of T-cell receptor (TCR)-based immunotherapy depends on the efficacy and specificity of TCRs. Naturally occurring TCRs have limited anti-tumor potency due to their low affinity for tumor antigens. Affinity enhancement is a promising strategy to generate highly potent TCRs. However, it is concerned that affinity-enhanced TCRs are prone to lose specificity. We isolated low affinity TCRs specific for NY-ESO-1157-165/HLA-A*02:01 from peripheral blood mononuclear cells of healthy donors. An affinity-enhanced TCR candidate with optimal affinity and specificity was generated using phage display and an extensive set of in vitro and in vivo assays. Alanine scanning mutagenesis showed that the TCR candidate retained specificity by making extensive contacts to the side chains of NY-ESO-1157-165 peptide. Adoptive transfer of T cells engineered with this candidate (termed TAEST16001) significantly inhibited tumor growth in subcutaneous, metastatic, and patient-derived xenograft (PDX) mouse tumor models. This study demonstrates that sophisticated engineering and screening techniques can be utilized to generate a clinical candidate TCR with potent anti-tumor activity without losing specificity. TAEST16001 was approved by the Center for Drug Evaluation (CDE) as the first TCR-based immunotherapy clinical trial in China (ClinicalTrials.gov Identifier: NCT03159585).

immunology↗

bioRxiv2022

Improved in situ sequencing for high-resolution targeted spatial transcriptomic analysis in tissue sections

Spatial transcriptomics enables the study of localization-indexed gene expression activity in tissues, providing the transcriptional landscape that in turn indicates the potential regulatory networks of gene expression. In situ sequencing (ISS) is a targeted spatial transcriptomic technique, which is based on padlock probe and rolling circle amplification combined with next-generation sequencing chemistry, for highly multiplexed in situ gene expression profiling. Here, we present improved in situ sequencing (IISS) that exploits a new probing and barcoding approach, combined with advanced image analysis pipeline for high-resolution targeted spatial gene expression profiling. We developed an improved combinatorial probe anchor ligation chemistry using a 2-base encoding strategy for barcode interrogation. The new encoding strategy resulted in higher signal intensity as well as improved specificity for in situ sequencing, while maintaining a streamlined analysis pipeline for targeted spatial transcriptomics. We showed that IISS can be applied to both fresh frozen tissue and formalin-fixed paraffin embedded tissue sections for single cell level spatial gene expression analysis, based on which the developmental trajectory and cell-cell communication networks can also be constructed. In conclusion, our method is a versatile molecular tool for targeted spatial transcriptomic analysis.

molecular biology↗

bioRxiv2022

Spatiotemporally resolved transcriptomics reveals subcellular RNA kinetic landscape

Spatiotemporal regulation of the cellular transcriptome is crucial for proper protein expression and cellular function1. However, the intricate subcellular dynamics of RNA synthesis, decay, export, and translocation remain obscured due to the limitations of existing transcriptomics methods2-8. Here, we report a spatiotemporally resolved RNA mapping method (TEMPOmap) to uncover subcellular RNA profiles across time and space at the single-cell level in heterogeneous cell populations. TEMPOmap integrates pulse-chase metabolic labeling of the transcriptome with highly multiplexed three-dimensional (3D) in situ sequencing to simultaneously profile the age and location of individual RNA molecules. Using TEMPOmap, we constructed the subcellular RNA kinetic landscape of 991 genes in human HeLa cells from upstream transcription to downstream subcellular translocation. Clustering analysis of critical RNA kinetic parameters across single cells revealed kinetic gene clusters whose expression patterns were shaped by multi-step kinetic sculpting. Importantly, these kinetic gene clusters are functionally segregated, suggesting that subcellular RNA kinetics are differentially regulated to serve molecular and cellular functions in cell-cycle dependent manner. Together, these single-cell spatiotemporally resolved transcriptomics measurements provide us the gateway to uncover new gene regulation principles and understand how kinetic strategies enable precise RNA expression in time and space.

genomics↗

bioRxiv2022

Optimization and deoptimization of codons in SARS-CoV-2 and the implications for vaccine development

The spread of Coronavirus Disease 2019 (COVID-19), caused by the SARS-CoV-2 coronavirus, has progressed into a global pandemic. To date, thousands of genetic variants have been identified across SARS-CoV-2 isolates from patients. Sequence analysis reveals that the codon usage of viral sequences decreased over time but fluctuated from time to time. In this study, through evolution modeling, we found that this phenomenon might result from the virus preference for mutations during transmission. Using dual luciferase assays, we further discovered that the deoptimization of codons on viruses might weaken protein expression during the virus evolution, indicating that the choice of codon usage might play important role in virus fitness. Finally, given the importance of codon usage in protein expression and particularly for mRNA vaccine, we designed several omicron BA.2.12.1 and BA.4/5 spike mRNA vaccine candidates based on codon optimization, and experimentally validated their high levels of expression. Our study highlights the importance of codon usage in virus evolution and mRNA vaccine development.

evolutionary biology↗

bioRxiv2022

PLK1 O-GlcNAcylation is essential for dividing mammalian cells and inhibits uterine carcinoma

The O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) mediates intracellular O-GlcNAcylation modification, whose function and substrates have entranced biologists and chemists alike. O-GlcNAcylation occurs on Ser/Thr residues and takes part in a vast array of physiological processes. OGT is essential for dividing mammalian cells, and it underscores many human diseases. Yet many of its fundamental substrates in the cell division process remains to be unveiled. Here we focus on its effect on Polo-like kinase 1 (PLK1), a mitotic master kinase that governs DNA replication, mitotic entry, chromosome segregation and mitotic exit. We found that PLK1 interacts with OGT and is O-GlcNAcylated. By utilizing stepped collisional energy/higher-energy collisional dissociation (sceHCD) mass spectrometry (MS) and mutagenesis studies, the critical O-GlcNAc site is located to be Thr291. Interestingly, T291N is a uterine carcinoma mutant in the TCGA database. Biochemical assays show that T291A and T291N both increase PLK1 stability. Using stable H2B-GFP cells, we show that PLK1-T291A and -T291N mutants display chromosome segregation defects, and result in misaligned and lagging chromosomes. In mouse xenograft models, we demonstrate that the O-GlcNAc-deficient PLK1-T291A and -T291N mutants would enhance uterine carcinoma in animals. Hence, we propose that OGT partially exerts its mitotic function through O-GlcNAcylation of PLK1, and sceHCD MS might be a new method to reveal many more O-GlcNAcylation substrates.

cancer biology↗

bioRxiv2022

Spatial Atlas of the Mouse Central Nervous System at Molecular Resolution

Spatially charting molecular cell types at single-cell resolution across the three-dimensional (3D) volume of the brain is critical for illustrating the molecular basis of the brain anatomy and functions. Single-cell RNA sequencing (scRNA-seq) has profiled molecular cell types in the mouse brain1, 2, but cannot capture their spatial organization. Here, we employed an in situ sequencing technique, STARmap PLUS3, 4, to map more than one million high-quality cells across the whole adult mouse brain and the spinal cord, profiling 1,022 genes at subcellular resolution with a voxel size of 194 X 194 X 345 nm in 3D. We developed computational pipelines to segment, cluster, and annotate 231 molecularly defined cell types and 64 tissue regions with single-cell resolution. To create a transcriptome-wide spatial atlas, we further integrated the STARmap PLUS measurements with a published scRNA-seq atlas1, imputing 11,844 genes at the single-cell level. Finally, we engineered a highly expressed RNA barcoding system to delineate the tropism of a brain-wide transgene delivery tool, AAV-PHP.eB5, 6, revealing its single-cell resolved transduction efficiency across the molecular cell types and tissue regions of the whole mouse brain. Together, our datasets and annotations provide a comprehensive, high-resolution single-cell resource that integrates a spatial molecular atlas, cell taxonomy, brain anatomy, and genetic manipulation accessibility of the mammalian central nervous system (CNS).

neuroscience↗

bioRxiv2022

Multi-task learning for single-cell multi-modality biology

Current biotechnologies can simultaneously measure multi-modality high-dimensional information from the same cell and tissue samples. To analyze the multi-modality data, common tasks such as joint data analysis and cross-modal prediction have been developed. However, current analytical methods are generally designed to process multi-modality data for one specific task without considering the underlying connections between tasks. Here, we present UnitedNet, a multi-task deep neural network that integrates the tasks of joint group identification and cross-modal prediction to analyze multi-modality data. We have found that multi-task learning for joint group identification and cross-modal prediction significantly improves the performance of each task. When applied to various single-cell multi-modality datasets, UnitedNet shows superior performance in each task, achieving better unsupervised and supervised joint group identification and cross-modal prediction performances compared with state-of-the-art methods. Furthermore, by considering the spatial information of cells as one modality, UnitedNet substantially improves the accuracy of tissue region identification and enables spatially resolved cross-modal prediction.

genomics↗

bioRxiv2022

Integration of multi-omics data shows downregulation of mismatch repair, purin, and tublin pathways in AR-negative triple-negative chemotherapy-resistant breast tumors

Triple negative breast cancer (TNBC) patients who fail to achieve a pathological complete response to neoadjuvant chemotherapy (NAC) will likely experience recurrence of the disease within 3-4 years. Prognostic assessment of early recurrence could facilitate clinical decisions and impact disease survival. This study investigated pre- and post-NAC multi-omics data from both an in-house and a public clinical study to identify biomarkers of NAC response. We observed significant transcriptional differences associated with response in the residual disease (post-NAC biopsies), which did not exist in the pre-NAC biopsies. We further refined the post-NAC transcriptional changes to a 17-gene signature and machine learning models were applied to evaluate the signatures diagnostic potential (area under the curve [≥] 0.8). Interestingly, the signature was enriched with down-regulated immune genes and several of these genes demonstrated prognostic potential in basal TNBC tumors. Our 17-gene signature provides additional insight into TNBC recurrence, which warrants further investigation.

cancer biology↗