Biology subjects

Ding, H.

Publications and source records attributed to Ding, H..

84 records · Page 5Linked to original sources

bioRxiv2020

SLE non-coding Genetic Risk Variant Determines the Epigenetic Dysfunction of an Immune Cell Specific Enhancer that Controls Disease-critical microRNA Expression

The human genome contains millions of putative regulatory elements, which regulate gene expression. We are just beginning to understand the functional consequences of genetic variation within these regulatory elements. Since the bulk of common genetic variation impacting polygenic disease phenotypes localizes to these non-coding regions of the genome, understanding the consequences will improve our understanding of the mechanisms mediating genetic risk in human disease. Here, we define the systemic lupus erythematosus (SLE) risk variant rs2431369 as likely causal for SLE and show that it is located in a functional regulatory element that modulates miR-146a expression. We use epigenomic analysis and genome-editing to show that the rs2431697-containing region is a distal enhancer that specifically regulates miR-146a expression in a cell-type dependent manner. 3D chromatin structure analysis demonstrates physical interaction between the rs2431697-containing region and the miR-146a promoter. Further, our data show that NF-kB binds the disease protective allele in a sequence-specific manner, leading to increased expression of this immunoregulatory microRNA. Our work provides a strategy for using disease-associated variants to define the functional regulatory elements of non-coding RNA molecules such as miR-146a and provides mechanistic links between autoimmune disease risk genetic variation and disease etiology.

genetics↗

bioRxiv2020

The PK/PD Integration and Resistance of Ti lmi cosin Against Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is the major pathogen causing enzootic pneumonia in pigs. M. hyopneumoniae infection can lead to considerable economic losses in the pig-breeding industry. Here, this study established a first-order absorption, one-compartment model to study the relationship between the pharmacokinetics/pharmacodynamics (PK/PD) index of tilmicosin against M. hyopneumoniae in vitro. We simulated the drug concentration of timicosin in the fluid lining the lung epithelia of pigs. The minimum inhibitory concentration (MIC) of tilmicosin against M. hyopneumoniae with an inoculum of 106 CFU/mL was 1.6 g/mL using the microdilution method. Static time-kill curves showed that, if the drug concentration >1 MIC, the antibacterial effect showed different degrees of inhibition. At 32 MIC, the amount of bacteria decreased by 3.16 log10 CFU/mL, thereby achieving a mycoplasmacidal effect. The M. hyopneumoniae count was reduced from 3.61 to 5.11 log10 CFU/mL upon incubation for 96 h in a dynamic model with a dose of 40-200 mg, thereby achieving mycoplasmacidal activity. The peak concentration by MIC (Cmax/MIC) and the area under the concentration-time curve over 96 h divided by the MIC (AUC0-96 h/MIC) were the best-fit PK/PD parameters for predicting the antibacterial activity of tilmicosin against M. hyopneumoniae (R2 = 0.99), suggesting that tilmicosin had concentration-dependent activity. The estimated value for Cmax/MIC and AUC0-96 h/MIC for 2log10 (CFU/mL) reduction and 3log10 (CFU/mL) reduction from baseline was 1.44 and 1.91, and 70.55 h and 96.72 h, respectively. Four M. hyopneumoniae strains (M1-M4) with reduced sensitivity to tilmicosin were isolated from the four dose groups. The susceptibility of these strains to tylosin, erythromycin and lincomycin was also reduced significantly. For sequencing analyses of 23S rRNA, an acquired A2058G transition in region V was found only in resistant M. hyopneumoniae strains (M3, M4). In conclusion, in an in vitro model, the effect of tilmicosin against M. hyopneumoniae was concentration-dependent and had a therapeutic effect. These results will help to design the optimal dosing regimen for tilmicosin in M. hyopneumoniae infection, and minimize the emergence of resistant bacteria.

pharmacology and toxicology↗

bioRxiv2020

Evolutionary context-integrated deep sequence modeling for protein engineering

Protein engineering seeks to design proteins with improved or novel functions. Compared to rational design and directed evolution approaches, machine learning-guided approaches traverse the fitness landscape more effectively and hold the promise for accelerating engineering and reducing the experimental cost and effort. A critical challenge here is whether we are capable of predicting the function or fitness of unseen protein variants. By learning from the sequence and large-scale screening data of characterized variants, machine learning models predict functional fitness of sequences and prioritize new variants that are very likely to demonstrate enhanced functional properties, thereby guiding and accelerating rational design and directed evolution. While existing generative models and language models have been developed to predict the effects of mutation and assist protein engineering, the accuracy of these models is limited due to their unsupervised nature of the general sequence contexts they captured that is not specific to the protein being engineered. In this work, we propose ECNet, a deep-learning algorithm to exploit evolutionary contexts to predict functional fitness for protein engineering. Our method integrated local evolutionary context from homologous sequences that explicitly model residue-residue epistasis for the protein of interest, as well as the global evolutionary context that encodes rich semantic and structural features from the enormous protein sequence universe. This biologically motivated sequence modeling approach enables accurate mapping from sequence to function and provides generalization from low-order mutants to higher-orders. Through extensive benchmark experiments, we showed that our method outperforms existing methods on [~]50 deep mutagenesis scanning and random mutagenesis datasets, demonstrating its potential of guiding and expediting protein engineering.

bioinformatics↗

bioRxiv2019

AFF3 and BACH2 are master regulators of metabolic inflexibility, β/α-cell transition, and dedifferentiation in type 2 diabetes

Type 2 Diabetes is associated with defective insulin secretion, reduced {beta}-cell mass, and increased glucagon production. Cell lineage-tracing in rodents and human autopsy surveys support the notion of {beta}-cell dedifferentiation as a unifying mechanism for these abnormalities. Yet, mechanistic determinants of human {beta}-cell failure remain elusive. Using regulatory-network-based single-cell analysis of human islets, we identify aberrant, diabetes-enriched transitional states characterized by metabolic inflexibility, /{beta}-transition, and endocrine progenitor/stem cell features. A coordinated transcription factor hierarchy mediating cell state transition emerged and was validated using barcoded guide-based, single-cell gene transfer and calcium flux measures in primary human islet cells. Specifically, two master regulators and associated epigenetic drivers emerged, one (AFF3) controlling {beta}- to -like-cell reprogramming, the other (BACH2) transition to a dedifferentiated endocrine progenitor-like cell. The findings provide mechanistic insight into diabetic islet cell dysfunction and suggest actionable pathways for pharmacological intervention.

physiology↗

bioRxiv2019

Analysis of the Mutant Selection Window and Killing of Mycoplasma hyopneumoniae for Doxycycline, Tylosin, Danofloxacin, Tiamulin, and Valnemulin

Mycoplasma hyopneumoniae is the major pathogenic microorganism causing enzootic pneumonia in pigs. With increasing resistance of M. hyopneumoniae to conventional antibiotics, treatment is becoming complicated. Herein, we investigated the mutant selection window (MSW) of doxycycline, tylosin, danofloxacin, tiamulin, and valnemulin for treating M. hyopneumoniae strain (ATCC 25934) to determine the likelihood of promoting resistance with continued use of these antibiotics. Minimum inhibitory concentration (MIC) values against M. hyopneumoniae were determined for each antimicrobial agent and ranged from 105 colony-forming units (CFU)/mL to 109 CFU/mL based on microdilution broth and agar dilution methods. The minimal concentration inhibiting colony formation by 99% (MIC99) and the mutant prevention concentration (MPC) were determined by the agar dilution method with three inoculum sizes. Antimicrobial killing was determined based on MIC99 and MPC values for all five agents. MIC values ranged from 0.001 to 0.25 g/mL based on the microdilution broth method, and from 0.008 to 1.0 g/mL based on the agar dilution method. MPC values ranged from 0.0016 to 10.24 g/mL. MPC/MIC99 values were ordered tylosin >doxycycline >danofloxacin >tiamulin >valnemulin. MPC achieved better bactericidal action than MIC99. Based on pharmacodynamic analyses, danofloxacin, tylosin, and doxycycline are more likely to select resistant mutants than tiamulin and valnemulin.

pharmacology and toxicology↗

bioRxiv2019

Single-cell based elucidation of molecularly-distinct glioblastoma states and drug sensitivity

Glioblastoma heterogeneity and plasticity remain controversial, with proposed subtypes representing the average of highly heterogeneous admixtures of independent transcriptional states. Single-cell, protein-activity-based analysis allowed full quantification of >6,000 regulatory and signaling proteins, thus providing a previously unattainable single-cell characterization level. This helped identify four novel, molecularly distinct subtypes that successfully harmonize across multiple GBM datasets, including previously published bulk and single-cell profiles and single cell profiles from seven orthotopic PDX models, representative of prior subtype diversity. GBM is thus characterized by the plastic coexistence of single cells in two mutually-exclusive developmental lineages, with additional stratification provided by their proliferative potential. Consistently, all previous subtypes could be recapitulated by single-cell mixtures drawn from newly identified states. Critically, drug sensitivity was predicted and validated as highly state-dependent, both in single-cell assays from patient-derived explants and in PDX models, suggesting that successful treatment requires combinations of multiple drugs targeting these distinct tumor states.\n\nSignificanceWe propose a new, 4-subtype GBM classification, which harmonizes across bulk and single-cell datasets. Single-cell mixtures from these subtypes effectively recapitulate all prior classifications, suggesting that the latter are a byproduct of GBM heterogeneity. Finally, we predict single-cell level activity of three clinically-relevant drugs, and validate them in patient-derived explant.

systems biology↗

bioRxiv2019

Cross-Species Alignment of Single Cell States with Biological Process Activity

The maintenance and transition of cellular states are controlled by biological processes. Here we present a gene set-based transformation of single cell RNA-Seq data into biological process activities that provides a robust description of cellular states. Moreover, as these activities represent species-independent descriptors, they facilitate the alignment of single cell states across different organisms.

systems biology↗

bioRxiv2018

Nano immunoconjugates crossing blood-brain barrier activate local brain tumor immune system for glioma treatment

Treatment of brain gliomas with checkpoint inhibitor antibodies to cytotoxic T-lymphocyte-associated antigen 4 (a-CTLA-4) and programmed cell death-1 (a-PD-1) was largely unsuccessful due to their inability to cross the blood-brain barrier (BBB). We describe a new generation of nano immunoconjugates (NICs) developed on natural biopolymer scaffold, poly({beta}-L-malic acid), with covalently attached a-CTLA-4 and/or a-PD-1 for delivery across the BBB and activation of local brain anti-tumor immune response in glioma-bearing mice. NIC treatment of mice bearing intracranial GL261 glioblastoma (GBM) resulted in an increase of CD8+ T-cells with a decrease of T regulatory cells (Tregs) in the brain tumor area. Survival of GBM-bearing mice treated with combination of NICs was significantly longer compared to animals treated by single checkpoint inhibitor-bearing NICs or free a-CTLA-4 and a-PD-1. Our study demonstrates trans-BBB delivery of nanopolymer-conjugated checkpoint inhibitors as an effective treatment of GBM via activation of both systemic and local brain tumor immune response.

immunology↗

bioRxiv2018

Differential gene expression, including Sjfs800, in Schistosoma japonicum females before, during, and after male-female pairing

Schistosomiasis is a prevalent but neglected tropical disease caused by parasitic trematodes of the genus Schistosoma, with the primary disease-causing species being S. haematobium, S. mansoni, and S. japonicum. Male-female pairing of schistosomes is necessary for sexual maturity and the production of a large number of eggs, which are primarily responsible for schistosomiasis dissemination and pathology. Here, we used microarray hybridization, bioinformatics, quantitative PCR, in situ hybridization, and gene silencing assays to identify genes that play critical roles in S. japonicum reproduction biology, particularly in vitellarium development, a process that affects male-female pairing, sexual maturation, and subsequent egg production. Microarray hybridization analyses generated a comprehensive set of genes differentially transcribed before and after male-female pairing. Although the transcript profiles of females were similar 16 and 18 days after host infection, marked gene expression changes were observed at 24 days. The 30 most abundantly transcribed genes on day 24 included those associated with vitellarium development. Among these, genes for female-specific 800 (fs800), eggshell precursor protein, and superoxide dismutase (cu-zn-SOD) were substantially upregulated. Our in situ hybridization results in female S. japonicum indicated that cu-zn-SOD mRNA was highest in the ovary and vitellarium, eggshell precursor protein mRNA was expressed in the ovary, ootype, and vitellarium, and Sjfs800 mRNA was observed only in the vitellarium, localized in mature vitelline cells. Knocking down the Sjfs800 gene in female S. japonicum by approximately 60% reduced the number of mature vitelline cells, decreased rates of pairing and oviposition, and decreased the number of eggs produced in each male-female pairing by about 50%. These results indicate that Sjfs800 is essential for vitellarium development and egg production in S. japonicum and suggest that Sjfs800 regulation may provide a novel approach for the prevention or treatment of schistosomiasis.\n\nAuthor SummarySchistosomiasis is a common but largely unstudied tropical disease caused by parasitic trematodes of the genus Schistosoma. The eggs of schistosomes are responsible for schistosomiasis transmission and pathology, and the production of these eggs is dependent on the pairing of females and males. In this study, we determined which genes in Schistosoma japonicum females were differentially expressed before and after pairing with males, identifying the 30 most abundantly expressed of these genes. Among these 30 genes, we further characterized those in female S. japonicum that were upregulated after pairing and that were related to reproduction and vitellarium development, a process that affects male-female pairing, sexual maturation, and subsequent egg production. We identified three such genes, S. japonicum female-specific 800 (Sjfs800), eggshell precursor protein, and superoxide dismutase, and confirmed that the mRNAs for these genes were primarily localized in reproductive structures. By using gene silencing techniques to reduce the amount of Sjfs800 mRNA in females by about 60%, we determined that Sjfs800 plays a key role in development of the vitellarium and egg production. This finding suggests that regulation of Sjfs800 may provide a novel approach to reduce egg counts and thus aid in the prevention or treatment of schistosomiasis.

genomics↗

bioRxiv2018

Structural insights into the conformational plasticity of the full-length trimeric HIV-1 envelope glycoprotein precursor

The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer mediates viral entry into cells and is the major target for the host antibody response. In infected cells, the mature Env [(gp120/gp41)3] is produced by cleavage of a trimeric gp160 precursor. Proteolytic cleavage decreases Env conformational flexibility, allowing the mature Env to resist antibody binding to conserved elements. The conformational plasticity of the Env precursor skews the humoral immune response towards the elicitation of ineffectual antibodies, contributing to HIV-1 persistence in the infected host. The structural basis for the plasticity of the Env precursor remains elusive. Here we use cryo-electron microscopy to visualize two coexisting conformational states of the full-length Env precursor at nominal resolutions of 5.5 and 8.0 [A]. The State-P2 conformation features a three-helix bundle of the gp41 heptad repeat region in the core, but has disordered membrane-interactive regions. State-P1 trimers lack the three-helix bundle and instead retain ordered transmembrane and membrane-proximal external regions embracing a central cavity. Our structural data shed light on the unusual plasticity of the Env precursor and provide new clues to Env immunogen discovery.

microbiology↗

bioRxiv2018

Quantitative Assessment of Protein Activity in Orphan Tissues and Single Cells Using the metaVIPER Algorithm

We and others have shown that transition and maintenance of biological states is controlled by master regulator protein, which can be inferred by interrogating tissue-specific regulatory models (interactomes) with transcriptional signatures, using the VIPER algorithm. Yet, some tissues may lack molecular profiles necessary for interactome inference (orphan tissues), or, as for single cells isolated from heterogeneous samples, their tissue context may be undetermined. To address this problem, we introduce metaVIPER, a novel algorithm designed to assess protein activity in tissue-independent by integrative analysis of multiple, non-tissue-matched interactomes. This assumes that transcriptional targets of each protein will be recapitulated by one or more available interactome. We confirmed the algorithms value in assessing protein dysregulation induced by somatic mutations, as well as in assessing protein activity in orphan tissues and, most critically, in single cells, thus allowing transformation of noisy and potentially biased RNA-Seq signatures into reproducible protein-activity signatures.

systems biology↗

bioRxiv2017

Overlooked polyploidies in lycophytes generalize their roles during the evolution of vascular plants

Seed plants and lycophytes constitute the extant vascular plants. As a model lycophyte, Selaginalla moellendroffii was deciphered its genome, previously proposed to have avoided polyploidies, as key events contributing to the origination and fast expansion of seed plants. Here, using a gold-standard streamline recently proposed to deconvolute complex genomes, we reanalyzed the S. moellendroffii genome. To our surprise, we found clear evidence of multiple paleo-polyploidies, with one being recent (~ 13-15 millions of years ago or Mya), another one occurring about ~125-142 Mya, during the evolution of lycophytes, and at least 2 or 3 events being more ancient. Besides, comparison of reconstructed ancestral genomes of lycophytes and angiosperms shows that lycophytes were likely much more affected by paleo-polyploidies than seed plants. The present analysis here provides clear and solid evidence that polyploidies have contributed the successful establishment of all vascular plants on earth.

evolutionary biology↗