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Fan, R.

Publications and source records attributed to Fan, R..

At least 91 records · Page 5Linked to original sources
bioRxiv2022

Recipient LAG3 deficiency results in antibody-mediated rejection of mouse renal allografts

Lymphocyte activation gene-3 (LAG3) is a coinhibitory receptor expressed by a range of immune cells. While immunomodulatory potential of LAG3 is being actively explored in cancer and autoimmunity fields, there is no information on how this pathway affects alloreactive immune responses following organ transplantation. The goal of this study was to investigate the functions of recipient LAG3 in a mouse model of renal allograft rejection. We found that mice deficient in LAG3 expression have elevated heterologous immunity against a panel of alloantigens prior to transplantation. Recipient LAG3 deficiency results in rapid rejection of MHC-mismatched renal allografts that are spontaneously accepted by WT recipients, with graft histology characteristic of antibody mediated rejection (ABMR). Depletion of recipient B cells but not CD8+ T cells significantly extended kidney allograft survival in LAG3-/- recipients further supporting ABMR as the main mechanism of graft loss. Treatment of WT recipients with an antagonistic LAG3 antibody enhanced anti-donor immune responses and induced kidney damage associated with chronic rejection. The experiments using conditional LAG3 knockout recipients demonstrated that LAG3 expression on either T or B cells is sufficient to regulate anti-donor humoral immunity. These results are the first to identify LAG3 as a regulator of both T and B cell responses to kidney allografts and a potential therapeutic target for ABMR prevention and treatment.

immunology↗
bioRxiv2022

In vivo correction of cystic fibrosis mediated by PNA nanoparticles

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We sought to correct the multiple organ dysfunction of the F508del CF-causing mutation using systemic delivery of peptide nucleic acid gene editing technology mediated by biocompatible polymeric nanoparticles. We confirmed phenotypic and genotypic modification in vitro in primary nasal epithelial cells from F508del mice grown at air-liquid interface and in vivo in F508del mice following intravenous delivery. In vivo treatment resulted in a partial gain of CFTR function in epithelia as measured by in situ potential differences and Ussing chamber assays and correction of CFTR in both airway and GI tissues with no off-target effects above background. This is the first report of systemic gene editing for CF. Our data suggest that systemic delivery of PNA NPs designed to correct CF-causing mutations is a viable option to ameliorate the disease in multiple affected organs.

bioengineering↗
bioRxiv2021

CXCL8/CXCR2 signaling mediates bone marrow fibrosis and represents a therapeutic target in myelofibrosis

Pro-inflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution remain yet to be fully elucidated. Previously we identified a critical role for combined constitutive JAK/STAT and aberrant NF-{kappa}B pro-inflammatory signaling in myelofibrosis development. Using single-cell transcriptional and cytokine-secretion studies of primary MF patient cells and two separate murine models of myelofibrosis, we extend this previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. MF patient hematopoietic stem/progenitor cells are enriched in a CXCL8/CXCR2 gene signature and display dose-dependent proliferation and fitness in response to exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway in MF patients at risk for continued fibrotic progression.

cancer biology↗
bioRxiv2021

Secretory MPP3 reinforce myeloid differentiation trajectory and amplify myeloid cell production

Recent lineage tracing analyses revealed multipotent progenitors (MPP) to be major functional contributors to steady-state hematopoiesis (1-6). However, we are still lacking a precise resolution of myeloid differentiation trajectories and cellular heterogeneity in MPPs. Here, we found that myeloid-biased MPP3 (2, 3) are functionally and molecularly heterogeneous, with a distinct subset of myeloid-primed secretory cells with high endoplasmic reticulum (ER) volume and Fc{gamma}R expression. We show that Fc{gamma}R+/ERhigh MPP3 are a transitional population for rapid production of granulocyte/macrophage progenitors (GMP), which directly amplify myelopoiesis through inflammation-triggered secretion of cytokines in the local bone marrow (BM) microenvironment. Our results identify a novel regulatory function for a subset of secretory MPP3 that controls myeloid differentiation through lineage-priming and cytokine production, and act as a self-reinforcing amplification compartment in stress and disease conditions. One-Sentence SummaryA secretory subset of multipotent hematopoietic progenitors augment myelopoiesis in stress and diseases conditions.

immunology↗
bioRxiv2021

Spatial-ATAC-seq: spatially resolved chromatin accessibility profiling of tissues at genome scale and cellular level

Cellular function in tissue is dependent upon the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping, but it remains elusive to capture spatial epigenetic information of tissue at cellular level and genome scale. Here we report on spatial-ATAC-seq: spatially resolved chromatin accessibility profiling of tissue section via next-generation sequencing by combining in situ Tn5 transposition chemistry and microfluidic deterministic barcoding. Spatial chromatin accessibility profiling of mouse embryos delineated tissue region-specific epigenetic landscapes and identified gene regulators implicated in the central nerve system development. Mapping the accessible genome in human tonsil tissue with 20m pixel size revealed spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology takes spatial biology to a new realm by enabling spatially resolved epigenomics to improve our understanding of cell identity, state, and fate decision in relation to epigenetic underpinnings in development and disease.

genomics↗
bioRxiv2021

Toward comprehensive functional analysis of gene lists weighted by gene essentiality scores

Gene functional enrichment analysis represents one of the most popular bioinformatics methods for annotating the pathways and function categories of a given gene list. Current algorithms for enrichment computation such as Fishers exact test and hypergeometric test totally depend on the category count numbers of the gene list and one gene set. In this case, whatever the genes are, they were treated equally. However, actually genes show different scores in their essentiality in a gene list and in a gene set. It is thus hypothesized that the essentiality scores could be important and should be considered in gene functional analysis. For this purpose, here we proposed WEAT (https://www.cuilab.cn/weat/), a weighted gene set enrichment algorithm and online tool by weighting genes using essentiality scores. We confirmed the usefulness of WEAT using two case studies, the functional analysis of one aging-related gene list and one gene list involved in Lung Squamous Cell Carcinoma (LUSC). Finally, we believe that the WEAT method and tool could provide more possibilities for further exploring the functions of given gene lists.

bioinformatics↗
bioRxiv2021

Single-cell antigen-specific activation landscape of CAR T infusion product identifies determinants of CD19 positive relapse in patients with ALL

Chimeric antigen receptor-modified (CAR) T cells targeting CD19 have mediated dramatic responses in relapsed/refractory acute lymphoblastic leukemia (ALL), yet a notable number of patients have CD19-positive relapse within one year of treatment. It remains unclear if the long-term response is associated with the characteristics of CAR T cells in infusion products, hindering the identification of biomarkers to predict therapeutic outcomes prior to treatment. Herein we present 101,326 single cell transcriptomes and surface protein landscape from the CAR T infusion products of 12 pediatric ALL patients upon CAR antigen-specific stimulation in comparison with TCR-mediated activation and controls. We observed substantial heterogeneity in the antigen-specific activation states, among which a deficiency of Th2 function was associated with CD19-positive relapsed patients (median remission 9.6 months) compared with very durable responders (remission>54 months). Proteomic profiles also revealed that the frequency of early memory T cell subsets, rather than activation or co-inhibitory signatures could distinguish CD19-positive relapse. Additionally, a deficit of type 1 helper and cytotoxic effector function and an enrichment for terminally differentiated CD8+ T cells exhibiting low cytokine polyfunctionality was associated with initial non-responders. By contrast, the single-cell transcriptomic data of unstimulated or TCR-activated CAR T cells failed to predict clinical responses. In aggregate, our results dissect the landscape of CAR-specific activation states in infusion products that can identify patients who do not develop a durable response to the therapy, and unveil the molecular mechanisms that may inform strategies to boost specific T cell function to maintain long term remission.

immunology↗
bioRxiv2021

Genome-wide survey of the F-box/Kelch (FBK) members and molecular identification of a novel FBK gene TaAFR in wheat

F-box proteins play critical roles in plant responses to biotic/abiotic stresses. In the present study, a total of 68 wheat F-box/Kelch (TaFBK) gene sequences encoding for 74 proteins were obtained in a genome-wide survey against EnsemblPlants. The 74 TaFBK proteins were divided into 5 categories based on their domain structures. The FBK proteins from wheat, Arabidopsis, and three other cereal species were grouped into 7 clades, and the number of Kelch domains present was their key clustering criterion. Sixty-eight TaFBK genes were unevenly distributed on 21 chromosomes. Most of TaFBKs were predicted to localize in the nucleus and cytoplasm. In silico analysis of a digital PCR revealed that TaFBKs were expressed at multiple developmental stages and tissues, and in response to drought and/or heat stresses. The TaFBK19 gene, a homologous to the Attenuated Far-Red Response (AFR) genes in other plant species, and hence named TaAFR, was selected for further analysis. The gene was isolated from the wheat line TcLr15 and its expression evaluated by quantitative real-time PCR. TaAFR transcripts were primarily detected in wheat leaves, and its expression was found to be regulated by various abiotic and biotic stresses as well as plant signaling hormones. Of particular interest, TaAFR expression was differentially regulated in the compatible vs incompatible wheat leaf rust reaction. Subcellular localization studies showed that TaAFR accumulates in the nucleus and cytoplasm. Three TaAFR-interacting proteins were identified experimentally: Skp1/ASK1-like protein (Skp1), ADP-ribosylation factor 2-like isoform X1 (ARL2) and phenylalanine ammonia-lyase (PAL). Further analysis revealed that the Skp1 protein interacted specifically with the F-box domain of TaAFR, while ARL2 and PAL were recognized by the Kelch domain. The data presented herein provides a solid foundation from which the function and metabolic network of TaAFR and other wheat FBKs can be further explored.

molecular biology↗
bioRxiv2021

Spatial Epigenome Sequencing at Tissue Scale and Cellular Level

Spatial biology is emerging as a new frontier of biomedical research in development and disease, but currently limited to transcriptome and a panel of proteins. Here we present spatial epigenome profiling for three histone modifications (H3K27me3, H3K4me3, H3K27ac) via next-generation sequencing by combining in-tissue CUT&Tag chemistry and microfluidic deterministic barcoding. Spatial chromatin states in mouse embryos or olfactory bulbs revealed tissue type-specific epigenetic regulations, in concordance with ENCODE reference data, but providing spatially resolved genome-wide profiles at tissue scale. Using fluorescence imaging to identify the tissue pixels (20m) each containing one nucleus allowed us to extract single-cell epigenomes in situ. Spatial chromatin state profiling in tissue may enable unprecedented opportunities to study epigenetic regulation, cell function and fate decision in normal physiology and pathogenesis.

genomics↗
bioRxiv2020

Schizophrenia and autism-associated mutations and disrupted m6A signal by YTHDF1 cause defects in microtubule function and neurodevelopment

Subcellular mRNA localization and local translation are crucial for spatially regulated gene expression in neurons. However, the precise mechanisms governing the selective transport and translation of mRNAs contributing to processes such as axonal growth and branching are only partially understood. Here, we present evidence of N6-methyladenosine (m6A)-mediated translational control of the RNA-binding protein, APC, influencing cytoskeletal dynamics at the growth cone. Our findings demonstrate that m6A modifications occur on Apc mRNA, facilitating its recognition by YTHDF1 to promote APC translation in neuronal somata. Additionally, we observe that disrupting the m6A pathway impairs the transport and local translation of {beta}-actin mRNA in the axon and growth cones, a deficiency that can be rescued by the exogenous expression of APC protein in cultured neurons. Furthermore, we establish the essential role of YTHDF1 in axon development, particularly in callosal projection neurons during cortical development. Our findings suggest a novel mechanism involving m6A-mediated regulation of APC protein translation, linking epitranscriptomics to axonal mRNA targeting, cytoskeletal dynamics, and axon development.

neuroscience↗
bioRxiv2020

DeepDrug: A general graph-based deep learning framework for drug relation prediction

Computational approaches for accurate prediction of drug interactions, such as drug-drug interactions (DDIs) and drug-target interactions (DTIs), are highly demanded for biochemical researchers due to the efficiency and cost-effectiveness. Despite the fact that many methods have been proposed and developed to predict DDIs and DTIs respectively, their success is still limited due to a lack of systematic evaluation of the intrinsic properties embedded in the corresponding chemical structure. In this paper, we develop a deep learning framework, named DeepDrug, to overcome the above limitation by using residual graph convolutional networks (RGCNs) and convolutional networks (CNNs) to learn the comprehensive structural and sequential representations of drugs and proteins in order to boost the DDIs and DTIs prediction accuracy. We benchmark our methods in a series of systematic experiments, including binary-class DDIs, multi-class/multi-label DDIs, binary-class DTIs classification and DTIs regression tasks using several datasets. We then demonstrate that DeepDrug outperforms state-of-the-art methods in terms of both accuracy and robustness in predicting DDIs and DTIs with multiple experimental settings. Furthermore, we visualize the structural features learned by DeepDrug RGCN module, which displays compatible and accordant patterns in chemical properties and drug categories, providing additional evidence to support the strong predictive power of DeepDrug. Ultimately, we apply DeepDrug to perform drug repositioning on the whole DrugBank database to discover the potential drug candidates against SARS-CoV-2, where 3 out of 5 top-ranked drugs are reported to be repurposed to potentially treat COVID-19. To sum up, we believe that DeepDrug is an efficient tool in accurate prediction of DDIs and DTIs and provides a promising insight in understanding the underlying mechanism of these biochemical relations. The source code of the DeepDrug can be freely downloaded from https://github.com/wanwenzeng/deepdrug.

bioinformatics↗
bioRxiv2020

Spatial transcriptome sequencing of FFPE tissues at the cellular level

Formalin-fixed paraffin-embedded (FFPE) tissues are the most abundant archivable specimens in clinical tissue banks, but unfortunately incompatible with single-cell level whole transcriptome sequencing due to RNA degradation in storage and RNA damage in extraction. We developed an in-tissue barcoding approach namely DBiT-seq for spatially revolved whole transcriptome sequencing at cellular level, which required no tissue dissociation or RNA exaction, thus potentially more suited for FFPE samples. Herein, we demonstrated spatial transcriptome sequencing of embryonic and adult mouse FFPE tissue sections at cellular level (25m pixel size) with high coverage (>1,000 genes per pixel). Spatial transcriptome of an E10.5 mouse embryo identified all major anatomical features in the brain and abdominal region. Integration with singlecell RNA-seq data for cell type identification indicated that most tissue pixels were dominated by single-cell transcriptional phenotype. Spatial mapping of adult mouse aorta, atrium, and ventricle tissues identified the spatial distribution of a variety of cell types. Spatial transcriptome sequencing of FFPE samples at cellular level may provide enormous opportunities in a wide range of biomedical research. It may allow us to exploit the huge resource of clinical tissue specimens to study human disease mechanisms and discover tissue biomarkers or therapeutic targets.

genomics↗
bioRxiv2020

Identification and verification of 3 key genes associated with survival and prognosis of patients with colon adenocarcinoma via integrated bioinformatics analysis

BackgroundColorectal cancer (CRC) is the third most lethal malignancy in the world, wherein colon adenocarcinoma (COAD) is the most prevalent type of CRC. Exploring biomarkers is important for the diagnosis, treatment, and prevention of COAD. MethodsWe used GEO2R and Venn online software for differential gene screening analysis. Hub genes were screened via STRING and Cytoscape, following Gene Ontology and KEGG enrichment analysis. Finally, survival analysis and expression validation were performed via UALCAN online software, real-time PCR and immunohistochemistry. ResultsIn this study, we screened 323 common differentially expressed genes from four GSE datasets. Furthermore, four hub genes were selected for survival correlation analysis and expression level verification, three of which were shown to be statistically significant. ConclusionOur study suggests that SERPINE1, SPP1 and TIMP1 may be biomarkers closely related to the prognosis of CRC patients.

bioinformatics↗
bioRxiv2020

Multidimensional Chromatin Regulation of Cell Lineage Differentiation in a Metazoan Embryo

How chromatin dictates cell differentiation is an intriguing question in developmental biology. Here, a reporter gene integrated throughout the genome was used as a sensor to map the chromatin activity landscape in lineage-resolved cells during C. elegans embryogenesis. Single-cell analysis of chromatin dynamics across critical dimensions of cell differentiation was performed, including lineage, tissue, and symmetry. During lineage progression, chromatin gradually diversifies in general and exhibits switch-like changes following specific cell division, which is predictive of anterior-posterior fate asymmetry. Upon tissue differentiation, chromatin of cells from distinct lineages converge to tissue-specific states but retain "memory" of each cells lineage history, which contributes to intra-tissue heterogeneity. However, cells with a morphologically left-right symmetric organization utilize a predetermination chromatin strategy to program analogous regulatory states in early progenitor cells. Additionally, chromatin co-regulation drives the functional coordination of the genome. Collectively, this work reveals the role of multidimensional chromatin regulation in cell differentiation.

developmental biology↗
bioRxiv2019

High-Spatial-Resolution Multi-Omics Atlas Sequencing of Mouse Embryos via Deterministic Barcoding in Tissue

We present DBiT-seq -Deterministic Barcoding in Tissue for spatial omics sequencing - for co-mapping of mRNAs and proteins in a formaldehyde-fixed tissue slide via NGS sequencing. Parallel microfluidic channels were used to deliver DNA barcodes to the surface of a tissue slide and crossflow of two sets of barcodes A1-50 and B1-50 followed by ligation in situ yielded a 2D mosaic of tissue pixels, each containing a unique full barcode AB. Application to mouse embryos revealed major tissue types in early organogenesis as well as fine features like microvasculature in a brain and pigmented epithelium in an eye field. Gene expression profiles in 10m pixels conformed into the clusters of single-cell transcriptomes, allowing for rapid identification of cell types and spatial distributions. DBiT-seq can be adopted by researchers with no experience in microfluidics and may find applications in a range of fields including developmental biology, cancer biology, neuroscience, and clinical pathology. In BriefMicrofluidic deterministic barcoding of mRNAs and proteins in tissue slides followed by high-throughput sequencing enables the construction of a high-spatial-resolution multi-omics atlas at the genome scale. Application to mouse embryos (E10-12) identified major tissue types in early organogenesis and revealed fine tissue features such as retinal pigmented epithelium and endothelial microvasculature at the cellular level. HighlightsO_LIDeterministic barcoding in tissue enables NGS-based spatial multi-omics mapping. C_LIO_LIDBiT-seq identified spatial patterning of major tissue types in mouse embryos. C_LIO_LIDBiT-seq revealed fine features such as retinal pigmented epithelium and microvascular endothelium at the cellular level. C_LIO_LIDirect integration with scRNA-seq data allows for rapid cell type identification. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/788992v2_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@41d937org.highwire.dtl.DTLVardef@11bdc46org.highwire.dtl.DTLVardef@1aa143org.highwire.dtl.DTLVardef@51755c_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗
bioRxiv2019

Detecting GPCR Complexes in Postmortem Human Brain with Proximity Ligation Assay and a Bayesian Classifier

Despite the general controversy regarding the existence and physiological relevance of Class A GPCR dimerization, there is substantial evidence for functional interactions between dopamine D2 receptor (D2R) and adenosine A2A receptor (A2AR). A2AR-D2R complexes have been detected in rodent brains by proximity ligation assay (PLA), but their existence in the human brain is yet to be demonstrated. In this study, we used brightfield PLA, combined with a systematic sampling and a parameter-free naive Bayesian classifier, and demonstrated proximity between D2R and A2AR in the adult human ventral striatum, consistent with their colocalization within complexes and the possible existence of D2R-A2AR heteromers. These methods are applicable to the quantitative analysis of proximity of two proteins and the expression of individual proteins.\n\nMethod SummaryBrightfield proximity ligation assay (PLA) was used to assess the expression of G protein-coupled receptors and their proximity in postmortem adult human brains. A novel automated machine learning method (Bayesian Optimized PLA Signal Sorting) was developed to automatically quantify brightfield PLA data.

neuroscience↗
bioRxiv2019

Behavioural responses to video and live presentations of females reveal a dissociation between performance and motivational aspects of birdsong

Understanding the regulation of social behavioural expression requires insight into motivational and performance aspects of social behaviours. While a number of studies have independently investigated motivational or performance aspects of social behaviours, few have examined how these aspects relate to each other. By comparing behavioural variation in response to live or video presentations of conspecific females, we analysed how variation in the motivation to produce courtship song covaries with variation in performance aspects of courtship song in male zebra finches (Taeniopygia guttata). Consistent with previous reports, we observed that male zebra finches were less motivated to produce courtship songs to videos of females than to live presentations of females. However, we found that acoustic features that reflect song performance were indistinguishable between songs produced to videos of females and songs produced to live presentations of females. For example, songs directed at video presentations of females were just as fast and stereotyped as songs directed at live females. These experimental manipulations and correlational analyses reveal a dissociation between motivational and performance aspects of birdsong and suggest a refinement of neural models of song production and control. In addition, they support the efficacy of videos to study both motivational and performance aspects of social behaviours.

animal behavior and cognition↗