Biology subjects

Han, H.

Publications and source records attributed to Han, H..

At least 91 records · Page 5Linked to original sources

bioRxiv2022

Vomocytosis of Cryptococcus neoformans cells from murine, bone marrow-derived dendritic cells

Cryptococcus neoformans (CN) cells survive within the acidic phagolysosome of macrophages for extended times, then escape without impacting the viability of the host cell via a phenomenon that has been coined vomocytosis. Through this mechanism, CN disseminate throughout the body, sometimes resulting in a potentially fatal condition - Cryptococcal Meningitis (CM). Justifiably, vomocytosis studies have focused primarily on macrophages, as alveolar macrophages within the lung act as first responders that ultimately expel this fungal pathogen. Herein, we hypothesize that dendritic cells (DCs), an innate immune cell with attributes that include phagocytosis and antigen presentation, can also act as vomocytes. Presciently, this report shows that vomocytosis of CN indeed occurs from DCs. Primarily through time-lapse microscopy imaging, we show that rates of vomocytosis events from DCs are comparable to those seen from macrophages and further, are independent of the presence of the CN capsule and infection ratios. Moreover, phagosome-altering drugs such as chloroquine and bafilomycin A, as well as the actin-modifying drug, cytochalasin B inhibit this phenomenon from DCs. Although DC immunophenotype does not affect the total number of vomocytic events, we observed differences in the numbers of CN per phagosome and expulsion times. Interestingly, these observations were similar in primary, murine macrophages. Understanding the vomocytic behavior of different phagocytes and their phenotypic subtypes is needed to help elucidate the full picture of the dynamic interplay between CN and the immune system. Critically, deeper insight into vomocytosis could reveal novel approaches to treat CM, as well as other immune-related conditions.

immunology↗

bioRxiv2022

Multi-level cellular and functional annotation of single-cell transcriptomes

Single-cell RNA-sequencing (scRNA-seq) offers unprecedented insight into heterogenous biology, allowing for the interrogation of cellular populations and gene expression programs at single-cell resolution. Here, we introduce scPipeline, a single-cell analytic toolbox that offers modular workflows for multi-level cellular annotation and user-friendly analysis reports. Novel methods that are introduced to facilitate scRNA-seq annotation include: (i) co-dependency index (CDI)-based differential expression; (ii) cluster resolution optimization using a marker-specificity criterion; (iii) marker-based cell-type annotation with Miko scoring; and (iv) gene program discovery using scale-free shared nearest neighbor network (SSN) analysis. Our unsupervised and supervised procedures were validated using a diverse collection of scRNA-seq datasets and we provide illustrative examples of cellular and transcriptomic annotation of developmental and immunological scRNA-seq atlases. Overall, scPipeline provides a flexible computational framework for in-depth scRNA-seq analysis.

bioinformatics↗

bioRxiv2022

Domain fusion TLR2-4 enhances the autophagy-dependent clearance of Staphylococcus aureus in the genetic engineering goat generated by CRISPR/Cas9

Staphylococcus aureus infections pose a potential threat to livestock production and public health. A novel strategy is needed to control S. aureus infection due to its adaptive evolution to antibiotics. Autophagy plays a key role in degrading bacteria for innate immune cells. In order to promote S. aureus clearance via TLR induced autophagy pathway, the domain fusion TLR2-4 with the extracellular domain of TLR2, specific recognizing S. aureus, and transmembrane and intracellular domains of TLR4 is assembled, then the goats expressing TLR2-4 is generated. TLR2-4 substantially augments the removal of S. aureus within macrophages by elevating autophagy level. Phosphorylated JNK/ERK1/2 promote LC3- puncta in TLR2-4 macrophages during S. aureus-induced autophagy via MyD88-mediated the TAK1 signaling cascade. Meantime, the TRIF-dependent TBK1-TFEB-OPTN signaling is involved in TLR2-4-triggered autophagy after S. aureus challenge. Moreover, the transcript of ATG5 and ATG12 is significantly increased via cAMP-PKA-NF-{kappa}B signaling, which facilitates S. aureus-induced autophagy in TLR2-4 macrophages. Overall, the novel receptor TLR2-4 enhances the autophagy-dependent clearance of S. aureus in macrophages via TAK1/TBK1-JNK/ERK, TBK1-TFEB-OPTN and cAMP-PKA-NF-{kappa}B-ATGs signaling pathways, which provide an alternative approach to resistant against S. aureus infection.

immunology↗

bioRxiv2022

Characterization of the minimal residual disease state reveals distinct evolutionary trajectories of human glioblastoma

Recurrence of solid tumors renders patients vulnerable to a distinctly advanced, highly treatment-refractory disease state that has an increased mutational burden and novel oncogenic drivers not detected at initial diagnosis. Improving outcomes for recurrent cancers requires a better understanding of cancer cell populations that expand from the post-therapy, minimal residual disease (MRD) state. We profiled barcoded tumor stem cell populations through therapy at tumor initiation/engraftment, MRD and recurrence in our therapy-adapted, patient-derived xenograft models of glioblastoma (GBM). Tumors showed distinct patterns of recurrence in which clonal populations exhibited either an a priori, pre-existing fitness advantage, or a priori equipotency fitness acquired through therapy. Characterization of the MRD state by single-cell and bulk RNA sequencing revealed a tumor-intrinsic immunomodulatory signature with strong prognostic significance at the transcriptomic level and in proteomic analysis of cerebrospinal fluid (CSF) collected from GBM patients at all stages of disease. Our results provide insight into the innate and therapy-driven dynamics of human GBM, and the prognostic value of interrogating the MRD state in solid cancers.

cancer biology↗

bioRxiv2022

Explainable t-SNE for single-cell RNA-seq data analysis

BackgroundSingle-cell RNA (scRNA-seq) sequencing technologies trigger the study of individual cell gene expression and reveal the diversity within cell populations. To measure cell-to-cell similarity based on their transcription and gene expression, many dimension reduction methods are employed to retrieve corresponding low-dimensional embeddings of input scRNA-seq data to conduct clustering. However, the methods lack explainability and may not perform well with scRNA-seq data because they are not customized for high-dimensional sparse scRNA-seq data. ResultsIn this study, we propose an explainable t-SNE: cell-driven t-SNE (c-TSNE) that fuses cell differences reflected from biologically meaningful distance metrics for input data. Our study shows that the proposed method not only enhances the interpretation of the original t-SNE visualization but also demonstrates favorable single cell segregation performance on benchmark datasets compared to state-of-the-art peers. The robustness analysis shows that the proposed cell-driven t-SNE demonstrates robustness to dropout and noise in clustering. It provides a novel and practical way to investigate the interpretability of t-SNE in scRNA-seq data analysis. Unlike the general assumption that the explainability of a machine learning method may need to compromise with learning efficiency, the proposed explainable t-SNE improves both clustering efficiency and explainability. More importantly, our work suggests that widely used t-SNE can be easily misused in existing scRNA-seq analysis, because its default Euclidean distance can bring biases or meaningless results in cell difference evaluation for high-dimensional sparse scRNA-seq data. To the best of our knowledge, it is the first explainable t-SNE proposed in scRNA-seq analysis and will inspire other explainable machine learning method development in the field. ConclusionThe proposed explainable t-SNE outperforms classic t-SNE and its peers in meaningful visualization and segregation. The poor performance of the classic t-SNE highlights the importance of developing explainable machine learning methods in scRNA-seq analysis. The explainable t-SNE is a data-centric customized ML enhance efficiency in data analysis through bringing more biological insights and interpretations.

bioinformatics↗

bioRxiv2021

Eukaryotic translation initiation factor eIF4E-5 is required for spermiogenesis in Drosophila melanogaster

Drosophila sperm development is characterized by extensive post-transcriptional regulation whereby thousands of transcripts are preserved for translation during later stages. A key step in translation initiation is the binding of eukaryotic initiation factor 4E (eIF4E) to the 5 mRNA cap. Drosophila has multiple paralogs of eIF4E, including four (eIF4E-3, -4, -5, and -7) that are highly expressed in the testis. Other than eIF4E-3, none of these has been characterized genetically. Here, using CRISPR/Cas9 mutagenesis, we determined that eIF4E-5 is essential for male fertility. eIF4E-5 mutants exhibit defects during post-meiotic stages, including a fully penetrant defect in individualization, resulting in failure to produce mature sperm. eIF4E-5 protein localizes to the distal ends of elongated spermatid cysts, where it regulates non-apoptotic caspase activity during individualization by promoting local accumulation of the E3 ubiquitin ligase inhibitor Soti. eIF4E-5 mutants also have mild defects in spermatid cyst polarization, similar to mutants affecting the cytoplasmic polyadenylation-element binding protein Orb2 and atypical protein kinase C (aPKC). Our results further extend the diversity of non-canonical eIF4Es that carry out distinct spatiotemporal roles during spermatogenesis. Summary StatementThe testis-enriched translation initiation factor eIF4E-5 is needed for spermatid cyst polarization, individualization of mature sperm and male fertility in Drosophila.

developmental biology↗

bioRxiv2021

Proteome-wide cellular thermal shift assay reveals novel crosstalk between brassinosteroid and auxin signaling

Despite the growing interest in using chemical genetics in plant research, small-molecule target identification remains a major challenge. The cellular thermal shift assay coupled with high-resolution mass-spectrometry (CETSA MS) that monitors changes in the thermal stability of proteins caused by their interactions with small molecules, other proteins, or post-translational modifications allows the identification of drug targets, or the study of protein-metabolite and protein-protein interactions mainly in mammalian cells. To showcase the applicability of this method in plants, we applied CETSA MS to intact Arabidopsis thaliana cells and identified the thermal proteome of the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, bikinin. A comparison between the thermal- and the phospho-proteomes of bikinin revealed the auxin efflux carrier PIN-FORMED1 (PIN1) as a novel substrate of the Arabidopsis GSK3s that negatively regulate the brassinosteroid signaling. We established that PIN1 phosphorylation by the GSK3s is essential for maintaining its intracellular polarity that is required for auxin-mediated regulation of vascular patterning in the leaf thus, revealing a novel crosstalk between brassinosteroid and auxin signaling. Significance StatementChemical genetics, which investigates the biological processes using small molecules, is gaining interest in plant research. However, a major challenge is to uncover the mode of action of the small molecule. Here, we applied the cellular thermal shift assay coupled with mass spectrometry (CETSA MS) to intact Arabidopsis cells and showed that bikinin, the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, changed the thermal stability of some of its direct targets and putative GSK3 interacting proteins. In combination with phosphoproteomics, we also revealed that GSK3s phosphorylate the auxin carrier PIN-FORMED1 (PIN1) and regulated its polarity that is required for the vascular patterning in the leaf.

plant biology↗

bioRxiv2021

SARS-CoV-2 papain-like protease PLpro in complex with natural compounds reveal allosteric sites for antiviral drug design

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in evading the hosts innate immune responses. We established a high-throughput X-ray screening to identify inhibitors by elucidating the native PLpro structure refined to 1.42 [A] and performing co-crystallization utilizing a diverse library of selected natural compounds. We identified three phenolic compounds as potential inhibitors. Crystal structures of PLpro inhibitor complexes, obtained to resolutions between 1.7-1.9 [A], show that all three compounds bind at the ISG15/Ub-S2 allosteric binding site, preventing the essential ISG15-PLpro molecular interactions. All compounds demonstrate clear inhibition in a deISGylation assay, two exhibit distinct antiviral activity and one inhibited a cytopathic effect in a non-cytotoxic concentration range. These results highlight the druggability of the rarely explored ISG15/Ub-S2 PLpro allosteric binding site to identify new and effective antiviral compounds. Importantly, in the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

biochemistry↗

bioRxiv2021

Whole-genome sequencing of Chinese native goat offers biological insights into cashmere fiber formation

Cashmere evolved naturally in the goat, and almost all breeds of goat can produce more or less cashmere fibers. However, the genetic alterations underlying cashmere trait selection are still unclear.We sequenced 120 Chinese native goat including two cashmere goat breeds (Ujumain, Chaidamu) and six ordinary goat breeds (Jining Gray, Matou, Guizhou Black, Jintang Black, Yunnan Black Bone, Chengdu Brown). The genome-wide selective sweep of cashmere goat and ordinary goat revealed a novel set of candidate genes as well as pathways, such as Nuclear factor kappa-B and Wnt Signaling pathways. Of them, the LHX2 gene regulating hair follicle development, was evident from the strongest selection signal when comparing the Uhumqin cashmere goat and ordinary goat. Interestingly, we identified a 582bp deletion at 367 kb upstream of LHX2 with higher frequency in cashmere goats and their ancient relatives. This mutation probably rises along the breeding procedures, and is putatively responsible for cashmere production and diameter, as revealed by association studies. Luciferase assay shows that the deletion, which acts as an insulator, restrains the expression of LHX2 by interfering its upstream enhancers.Our study discovers a novel insulator of the LHX2 involved in regulating cashmere production and diameter, which would be beneficial to understanding hair follicle development and regeneration. Our findings also provide new insights into the genetic formation of cashmere, and facilitate subsequent molecular breeding for cashmere goat improvement.

genomics↗

bioRxiv2021

Eicosanoids in the pancreatic tumor microenvironment: a multicellular, multifaceted progression

Eicosanoids, oxidized fatty acids that serve as cell-signaling molecules, have been broadly implicated in tumorigenesis. To identify eicosanoids relevant to pancreatic tumorigenesis, we profiled normal pancreas and pancreatic ductal adenocarcinoma (PDAC) in mouse models and patient samples using mass spectrometry. We interrogated RNA sequencing datasets for eicosanoid synthase or receptor expression. Findings were confirmed by immunostaining. In murine models, we identified elevated levels of PGD2, prostacyclin, and thromboxanes in neoplasia while PGE2, 12-HHTre, HETEs, and HDoHEs are elevated specifically in tumors. Analysis of scRNA-seq datasets suggests that PGE2 and prostacyclins are derived from fibroblasts, PGD2 and thromboxanes from myeloid cells, and PGD2 and 5-HETE from tuft cells. In patient samples, we identified a transition from PGD2 to PGE2-producing enzymes in the epithelium during the transition to PDAC, fibroblast/tumor expression of PTGIS, and myeloid/tumor cell expression of TBXAS1. Altogether, our analyses identify key changes in eicosanoid species during pancreatic tumorigenesis and the cell types responsible for their synthesis.

cancer biology↗

bioRxiv2021

Synaptic and mitochondrial plasticity associated with fear memory revealed by deep learning-based 3D reconstruction

Serial section electron microscopy (ssEM) can provide comprehensive 3D ultrastructural information of the brain with exceptional computational cost. Targeted reconstruction of subcellular structures from ssEM datasets is less computationally demanding but still highly informative. We thus developed a Region-CNN-based deep learning method to identify, segment, and reconstruct synapses and mitochondria to explore the structural plasticity of synapses and mitochondria in the auditory cortex of mice subjected to fear conditioning. Upon reconstructing over 135,000 mitochondria and 160,000 synapses, we found that fear conditioning significantly increases the number of mitochondria but decreases their size, and promotes the formation of multi-contact synapses comprising a single axonal bouton and multiple postsynaptic sites from different dendrites. Modeling indicates that such multi-contact configuration increases the information storage capacity of new synapses by over 50%. With high accuracy and speed in reconstruction, our method yields structural and functional insight into cellular plasticity associated with fear learning.

neuroscience↗

bioRxiv2021

Spinal ganglia control nociceptive input to the central nervous system

Accumulating observations suggest that peripheral somatosensory ganglia may regulate nociceptive transmission, yet direct evidence is sparse. Here we show that the peripheral afferent nociceptive information undergoes dynamic filtering within the dorsal root ganglion (DRG) and suggest that this filtering occurs at the axonal bifurcations (t-junctions). Using synchronous in vivo electrophysiological recordings from the peripheral and central processes of sensory neurons (in the spinal nerve and dorsal root), ganglionic transplantation of GABAergic progenitor cells, and optogenetics we demonstrate existence of tonic and dynamic filtering of action potentials traveling through the DRG. Filtering induced by focal application of GABA or optogenetic GABA release from the DRG-transplanted GABAergic progenitor cells was specific to nociceptive fibers. Light-sheet imaging and computer modeling demonstrated that, compared to other somatosensory fiber types, nociceptors have shorter stem axons, making somatic control over t-junctional filtering more efficient. Optogenetically-induced GABA release within DRG from the transplanted GABAergic cells enhanced filtering and alleviated hypersensitivity to noxious stimulation produced by chronic inflammation and neuropathic injury in vivo. These findings support gating of pain information by DRGs and suggest new therapeutic approaches for pain relief.

neuroscience↗

bioRxiv2021

Endogenous RNA editing of a nuclear gene BOSS triggers flowering in tomato

RNA editing is defined as the production of transcripts with RNA sequences different from those of the DNA template. Most of previous RNA editing studies have focused on organelles, while RNA editing of nuclear transcripts is largely unknown. Here, we describe the first example of nuclear transcript RNA editing in plant, the BOSS RNA editing regulates tomato flowering. The SNP (SNP1) located in the 5UTR of BOSS gene (the Balancer of SP and SFT) is associated with tomato early flowering, and two transcripts of BOSS produced by SNP1 associated RNA editing show functional differentiation, where BOSS-{beta} transcript promotes flowering while BOSS- does not. Furthermore, these two transcripts of BOSS are shown to regulate SP (anti-florigen) pathway at transcription level in the shoot apical meristem (SAM). Our findings reveal a new layer of complexity in the control of plant stem cell proliferation and provide the evidence of RNA-editing of a single gene for flowering, suggesting that molecular breeding programs to increase the RNA-editing efficiency may improve the productivity of tomatoes and other agricultural organisms.

plant biology↗

bioRxiv2021

Transcriptional Inactivation of TP53 and the BMP Pathway Mediates Therapy-induced Dedifferentiation and Metastasis in Prostate Cancer

Unsupervised clustering and deconvolution analysis identifies a novel subtype of M-CRPC endowed with hybrid epithelial/mesenchymal (E/M) and luminal progenitor-like traits (Mesenchymal and Stem-like PC, MSPC). Analysis of patient datasets and mechanistic studies indicate that MSPC arises as a consequence of therapy-induced lineage plasticity. AR blockade instigates two separate and complementary processes: 1) transcriptional silencing of TP53 and hence acquisition of hybrid E/M and stem-like traits; and 2) inhibition of the BMP signaling, which promotes resistance to the pro-apoptotic and anti-proliferative effects of AR inhibition. The drug-tolerant prostate cancer cells generated through reprogramming are rescued by neuregulin and generate metastases in mice. Combined inhibition of HER2/3 and AR or mTORC1 exhibit efficacy in preclinical models of mixed ARPC/MSPC or MSPC, respectively. These results identify a novel subtype of M-CRPC, trace its origin to therapy-induced lineage plasticity, and reveal its dependency on HER2/3 signaling.

cancer biology↗

bioRxiv2021

Rapid auxin-mediated phosphorylation of Myosin regulates trafficking and polarity in Arabidopsis

The signaling molecule auxin controls plant development through a well-known transcriptional mechanism that regulates many genes. However, auxin also triggers cellular responses within seconds or minutes, and mechanisms mediating such fast responses have remained elusive. Here, we identified an ultrafast auxin-mediated protein phosphorylation response in Arabidopsis roots that is largely independent of the canonical TIR1/AFB receptors. Among targets of this novel response are Myosin XI and its adaptor protein MadB2. We show that their auxin-mediated phosphorylation regulates trafficking and polar, subcellular distribution of PIN auxin transporters. This phosphorylation-based auxin signaling module is indispensable during developmental processes that rely on auxin-mediated PIN repolarization, such as termination of shoot gravitropic bending or vasculature formation and regeneration. Hence, we identified a fast, non-canonical auxin response targeting multiple cellular processes and revealed auxin-triggered phosphorylation of a myosin complex as the mechanism for feedback regulation of directional auxin transport, a central component of auxin canalization, which underlies self-organizing plant development.

plant biology↗

bioRxiv2021

The Hepcidin Regulator Erythroferrone is a New Member of the Erythropoiesis-Iron-Bone Circuitry

Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis. We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe-/- mice display low-bone-mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe-/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP-mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe-/- mice. Importantly, Erfe loss in {beta}-thalassemic (Hbbth3/+) mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss. Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in {beta}-thalassemia.

physiology↗

bioRxiv2021

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

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

microbiology↗

bioRxiv2021

Combined Inhibition of SHP2 and CXCR1/2 Promotes Anti-Tumor T Cell Response in NSCLC

Clinical trials of SHP2 inhibitors (SHP2i) alone and in various combinations are ongoing for multiple tumors with over-activation of the RAS/ERK pathway. SHP2 plays critical roles in normal cell signaling; hence, SHP2is could influence the tumor microenvironment. We found that SHP2i treatment depleted alveolar and M2-like macrophages and promoted B and T lymphocyte infiltration in Kras- and Egfr-mutant non-small cell lung cancer (NSCLC). However, treatment also increased intratumor gMDSCs via tumor-intrinsic, NF-kB-dependent production of CXCR2 ligands. Other RAS/ERK pathway inhibitors also induced CXCR2 ligands and gMDSC influx in mice, and CXCR2 ligands were induced in tumors from patients on KRASG12C-inhibitor trials. Combined SHP2(SHP099)/CXCR1/2(SX682) inhibition depleted a specific cluster of S100a8/9high gMDSCs, generated Klrg1+ CD8+ effector T cells with a strong cytotoxic phenotype but expressing the checkpoint receptor NKG2A, and enhanced survival in Kras-and Egfr-mutant models. Our results argue for testing RAS/ERK pathway/CXCR1/2/NKG2A inhibitor combinations in NSCLC patients. Statement of SignificanceOur study shows that inhibiting the SHP2/RAS/ERK pathway triggers NF-kB-dependent up-regulation of CXCR2 ligands and recruitment of S100A8high gMDSCs, which suppress T cells in NSCLC. Combining SHP2 and CXCR2 inhibitors blocks this gMDSC immigration, resulting in enhanced Th1 polarization, induction of CD8+ KLRG1+ effector T cells with high cytotoxic activity and improved survival in multiple NSCLC models.

cancer biology↗