Biology subjects

Dong, H.

Publications and source records attributed to Dong, H..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

Immunopipe: A comprehensive and flexible scRNA-seq and scTCR-seq data analysis pipeline

Single-cell sequencing technologies provide us with information at the level of individual cells. The combination of single-cell RNA-seq and single-cell TCR-seq profiling enables the exploration of cell heterogeneity and T-cell receptor repertoires simultaneously. The integration of both types of data can play a crucial role in enhancing our understanding of T-cell-mediated immunity and, in turn, facilitate the advancement of immunotherapy. Here, we present immunopipe, a comprehensive and flexible pipeline to perform integrated analysis of scRNA-seq and scTCR-seq data. In addition to the command line tool, we provide a user-friendly web interface for pipeline configuration and execution monitoring, benefiting researchers without extensive programming experience. With its comprehensive functionality and ease of use, immunopipe empowers researchers to uncover valuable insights from scRNA-seq and scTCR-seq data, ultimately advancing the understanding of immune responses and immunotherapy development.

bioinformatics↗

bioRxiv2024

Gossamer: Scaling Image Processing and Reconstruction to Whole Brains

Neuronal reconstruction-a process that transforms image volumes into 3D geometries and skeletons of cells- bottlenecks the study of brain function, connectomics and pathology. Domain scientists need exact and complete segmentations to study subtle topological differences. Existing methods are diskbound, dense-access, coupled, single-threaded, algorithmically unscalable and require manual cropping of small windows and proofreading of skeletons due to low topological accuracy. Designing a data-intensive parallel solution suited to a neurons shape, topology and far-ranging connectivity is particularly challenging due to I/O and load-balance, yet by abstracting these vision tasks into strategically ordered specializations of search, we progressively lower memory by 4 orders of magnitude. This enables 1 mouse brain to be fully processed in-memory on a single server, at 67x the scale with 870x less memory while having 78% higher automated yield than APP2, the previous state of the art in performant reconstruction.

neuroscience↗

bioRxiv2024

Advancing CAR T-Cell Therapy: Simultaneously Attack Tumor and Immunosuppressive Cells in the Tumor Microenvironment

Chimeric antigen receptor (CAR) T-cell therapy has encountered limited success in solid tumors. The lack of dependable antigens and the immunosuppressive tumor microenvironment (TME) are major challenges. Within the TME, tumor cells along with immunosuppressive cells employ an immune-evasion mechanism that upregulates programmed death ligand 1 (PD-L1) to deactivate effector T cells; this makes PD-L1 a reliable, universal target for solid tumors. We developed a novel PD-L1 CAR (MC9999) using our humanized anti-PD-L1 monoclonal antibody, designed to simultaneously target tumor and immunosuppressive cells. The antigen-specific antitumor effects of MC9999 CAR T-cells were observed consistently across four solid tumor models: breast cancer, lung cancer, melanoma, and glioblastoma multiforme (GBM). Notably, intravenous administration of MC9999 CAR T-cells eradicated intracranially established LN229 GBM tumors, suggesting penetration of the blood-brain barrier. The proof-of-concept data demonstrate the cytolytic effect of MC9999 CAR T-cells against immunosuppressive cells, including microglia HMC3 cells and M2 macrophages. Furthermore, MC9999 CAR T-cells elicited cytotoxicity against primary tumor-associated macrophages within GBM tumors. The concept of targeting both tumor and immunosuppressive cells with MC9999 was further validated using CAR T-cells derived from cancer patients. These findings establish MC9999 as a foundation for the development of effective CAR T-cell therapies against solid tumors.

cancer biology↗

bioRxiv2024

YAP promotes chromosomal instability by transcriptionally activating AJUBA through its super enhancer in breast cancer

Chromosomal instability (CIN) has been proven to be a fundamental property of cancer and a key underlying mechanism of tumorigenesis and progression. Still, relatively few studies have revealed the mechanism of this process. In this research, we identified the core Hippo component, YAP, played a crucial role in CIN formation in breast cancer. Using public ChIP-seq data and bioinformatic analysis, we found YAP could transcriptionally induce spindle assembly checkpoint, AJUBA, via TEAD, then activate AJUBA-AURKA signaling. Abolish AJUBA-AURKA axis could reverse YAP-induced CIN formation and aberrant mitosis. Mechanically, YAP/TEAD complex could accumulate to the super enhancer region of AJUBA, thus significantly induced its expression, and subsequently promoted AURKA auto-phosphorylating. These findings revealed a novel cross-talk between Hippo and AJUBA-AURKA signaling, and provided a new insight in CIN formation in breast cancer.

cancer biology↗

bioRxiv2024

LncRNA-CIR6 Mediates Repair of Infarcted Hearts by Inducing MSCs Differentiation into Cardiogenic Cells through CDK1

PurposeThis study aims to investigate the induction effect of LncRNA-CIR6 on MSCs differentiation into Cardiogenic Cells in vitro and in vivo. MethodsIn addition to pretreatment with the Ro-3306 (CDK1 inhibitor), LncRNA-CIR6 was transfected into BMSCs and hUCMSCs using jetPRIME. LncRNA-CIR6 was transfected into C57BL/6 mice heart by 100 L of AAV9-cTnT-LncRNA-CIR6-ZsGreen i.v. After 3 weeks of transfection followed by AMI surgery, hUCMSCs (5x105/100 L) were injected by i.v 1 week later. Cardiac function was evaluated using VEVO 2100 and electric mapping 9 days after cell injection. IF, Evans blue-TTC, Masson staining, FACS, and WB were used to determine relevant indicators. ResultsLncRNA-CIR6 induced a significant percentage of differentiation in BMSCs (83.00{+/-}0.58)% and hUCMSCs (95.43{+/-}2.13)% into cardiogenic cells, as determined by the expression of cTnT. Compared with MI group, cardiac contraction and conduction function in MI heart treated by LncRNA-CIR6 or combined with MSCs injection groups were significantly increased as well as the area of MI and fibrosis were significantly lower. The transcriptional expression region of LncRNA-CIR6 was in Chr17 from 80209290 to 80209536. The functional region of LncRNA-CIR6 was located at nucleotides 0-50/190-255 in the sequence. CDK1 is a protein found to be related to the proliferation and differentiation of cardiomyocytes is which located in the functional region of LncRNA-CIR6 secondary structure (from 0 to 17). Ro-3306 impeded the differentiation of MSCs into cardiogenic cells, while MSCs transfected with LncRNA-CIR6 showed high expression of CDK1. LncRNA-CIR6 mediates repair of infarcted hearts by inducing MSCs differentiation into cardiogenic cells through CDK1. ConclusionsLncRNA-CIR6 mediates repair of infarcted hearts by inducing MSCs differentiation into cardiogenic cells through CDK1.

molecular biology↗

bioRxiv2023

Inhibition of O-GlcNAc transferase activates type I interferon-dependent antitumor immunity by bridging cGAS-STING pathway

The O-GlcNAc transferase (OGT) is an essential enzyme that mediates protein O-GlcNAcylation, a unique form of posttranslational modification of many nuclear and cytosolic proteins. Recent studies observed increased OGT and O-GlcNAcylation levels in a broad range of human cancer tissues compared to adjacent normal tissues, indicating a universal effect of OGT in promoting tumorigenesis. Here, we show that OGT is essential for tumor growth in immunocompetent hosts by repressing the cyclic GMP-AMP synthase (cGAS)-dependent DNA sensing pathway. We found that deletion of OGT (Ogt-/-) caused a marked reduction in tumor growth in both syngeneic tumor models and a genetic colorectal cancer (CRC) model induced by mutation of the Apc gene (Apcmin). Pharmacological inhibition or genetic deletion of OGT induced a robust genomic instability (GIN), leading to cGAS-dependent production of the type I interferon (IFN-I) and IFN-stimulated genes (ISGs). As a result, deletion of Cgas or Sting from Ogt-/- cancer cells restored tumor growth, and this correlated with impaired CD8+ T cell-mediated antitumor immunity. Mechanistically, we found that OGT-dependent cleavage of host cell factor C1 (HCF-1) is required for the avoidance of GIN and IFN-I production in tumors. In summary, our results identify OGT-mediated genomic stability and activate cGAS-STING pathway as an important tumor cell-intrinsic mechanism to repress antitumor immunity.

cancer biology↗

bioRxiv2023

Well-Paired-Seq2: High-Throughput and High-Sensitivity Strategy for Characterizing Low RNA-Content Cell/Nucleus Transcriptomes

High-throughput single-cell RNA sequencing (scRNA-seq) is recognized as a powerful technology for disentangling the heterogeneity of cellular states. However, the Poisson-dependent cell capture and low sensitivity in scRNA-seq methods pose challenges for throughput and for samples with low RNA-content. Herein, to address these challenges, we developed Well-Paired-Seq2 (WPS2) based on size-exclusion and locally quasi-static hydrodynamic principles to realize high efficiency of cell utilization, single cell/bead pairing, and cell-free RNA removal. WPS2 exploits molecular crowding effect, tailing activity enhancement in reverse transcription, and homogeneous enzymatic reaction in the initial bead-based amplification to achieve 3116 genes and 8447 transcripts. With an average of [~]20,000 reads per cell, WPS2 detected 1420 more genes and 4864 more transcripts than our previous Well-Paired-Seq. Using WPS2, we overcame the Poisson limit for the capture of both cells and beads and accurately characterized transcriptomes of low RNA-content single cells and nuclei with high sensitivity. WPS2 was further applied to comprehensively profile transcriptomes from frozen clinical samples. We found that clear cell renal cell carcinoma (ccRCC) has a complex microenvironment, and that chromophobe renal cell carcinoma (chRCC) exhibits abundant copy number variations (CNVs). In addition, metanephric adenoma (MA) was characterized at single-cell level for the first time and some potentially specific markers were revealed. With the advantages of high sensitivity, high throughput, and high fidelity, we anticipate that WPS2 will be broadly applicable in basic and clinical research.

genetics↗

bioRxiv2023

A soybean rust effector protease suppresses host immunity and cleaves a 3-deoxy-7-phosphoheptulonate synthase

The devastating soybean rust (SBR) pathogen, Phakopsora pachyrhizi, encodes many secreted proteins, but only two have been functionally characterized for their roles in rust virulence. Here, we demonstrate that transient expression of P. pachyrhizi effector candidate 15 (PpEC15), an aspartic protease, leads to enhanced bacterial growth in planta, suppression of callose deposition, reduced expression of plant defense-related marker genes and suppression of pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species (ROS). Stable expression of PpEC15 in soybean suppresses PAMP-induced ROS production and enhances bacterial growth, indicating that, collectively, PpEC15 suppresses host and non-host innate immune responses. Yeast-two-hybrid and proximity labeling identified putative PpEC15 interacting partners including a peptide-chain release factor (PCRF), a NAC83 (NAM, ATAF, and CUC) transcription factor, and a DAHP (3-deoxy-7-phosphoheptulonate) synthase. We further show that PpEC15 can cleave DAHP but does not cleave PCRF or NAC83. Virus-induced gene silencing of NAC83, PCRF and DAHP altered PAMP-induced ROS production and salicylic acid production, indicating that these proteins may be involved in immune signaling. Collectively, our data show that PpEC15 is conserved across P. pachyrhizi isolates and other economically important rust species and is involved in the suppression of plant basal defense responses. Understanding the role of PpEC15 in P. pachyrhizi virulence will provide a foundation for designing targeted intervention strategies to generate rust-resistant crops.

plant biology↗

bioRxiv2023

Improved dual-color GRAB sensors for monitoring dopaminergic activity in vivo

Dopamine (DA) plays multiple roles in a wide range of physiological and pathological processes via a vast network of dopaminergic projections. To fully dissect the spatiotemporal dynamics of DA release in both dense and sparsely innervated brain regions, we developed a series of green and red fluorescent GPCR activation-based DA (GRABDA) sensors using a variety of DA receptor subtypes. These sensors have high sensitivity, selectivity, and signal-to-noise properties with subsecond response kinetics and the ability to detect a wide range of DA concentrations. We then used these sensors in freely moving mice to measure both optogenetically evoked and behaviorally relevant DA release while measuring neurochemical signaling in the nucleus accumbens, amygdala, and cortex. Using these sensors, we also detected spatially resolved heterogeneous cortical DA release in mice performing various behaviors. These next-generation GRABDA sensors provide a robust set of tools for imaging dopaminergic activity under a variety of physiological and pathological conditions.

neuroscience↗

bioRxiv2023

Neuronal Connectivity as a Determinant of Cell Types and Subtypes

Classifications of single neurons at brain-wide scale is a powerful way to characterize the structural and functional organization of a brain. We acquired and standardized a large morphology database of 20,158 mouse neurons, and generated a whole-brain scale potential connectivity map of single neurons based on their dendritic and axonal arbors. With such an anatomy-morphology-connectivity mapping, we defined neuron connectivity types and subtypes (both called "c-types" for simplicity) for neurons in 31 brain regions. We found that neuronal subtypes defined by connectivity in the same regions may share statistically higher correlation in their dendritic and axonal features than neurons having contrary connectivity patterns. Subtypes defined by connectivity show distinct separation with each other, which cannot be recapitulated by morphology features, population projections, transcriptomic, and electrophysiological data produced to date. Within this paradigm, we were able to characterize the diversity in secondary motor cortical neurons, and subtype connectivity patterns in thalamocortical pathways. Our finding underscores the importance of connectivity in characterizing the modularity of brain anatomy, as well as the cell types and their subtypes. These results highlight that c-types supplement conventionally recognized transcriptional cell types (t-types), electrophysiological cell types (e-types), and morphological cell types (m-types) as an important determinant of cell classes and their identities.

neuroscience↗

bioRxiv2023

High red/far-red ratio promotes root colonization of Serratia plymuthica A21-4 in tomato by root exudates-stimulated chemotaxis and biofilm formation

Effective colonization on plant roots is a prerequisite for plant growth promoting rhizobacterias (PGPR) to exert beneficial activities. Light is essential for plant growth, development and stress response. However, how light modulates root colonization of PGPR remains unclear. Here, we found that high red/far red(R/FR) light promoted and low R/FR light inhibited the colonization and growth enhancement of Serratia plymuthica A21-4 on tomato. Non-targeted metabolomic analysis of root exudates collected from different R/FR ratio treated tomato seedlings with A21-4 inoculation by ultra performance liquid chromatography-tandem mass spectrometry showed that 64 primary metabolites including amino acids, sugars and organic acids in high R/FR light-grown plants significantly increased compared with those determined for low R/FR light-grown plants. Among them, 7 amino acids, 1 organic acid and 1 sugar obviously induced the chemotaxis and biofilm formation of A21-4 compared to the control. Furthermore, exogenous addition of five artificial root exudate compontents (leucine, methionine, glutamine, 6-aminocaproic acid and melezitose) regained and further increased the colonization and growth promoting ability of A21-4 in tomato under low R/FR light and high R/FR light, respectively, indicating their involvement in high R/FR light-regulated the interaction of tomato root and A21-4. Taken together, our results, for the first time, clearly demonstrate that high R/FR light-induced root exudates play a key role in chemotaxis, biofilm formation and root colonization of A21-4. This study provides new insights into the interactions of plant-PGPR under different light conditions and can help promote the combined application of light supplementation and PGPR to facilitate crop growth and health in green agricultural production.

microbiology↗

bioRxiv2023

Monitoring norepinephrine release in vivo using next-generation GRABNE sensors

Norepinephrine (NE) is an essential biogenic monoamine neurotransmitter, yet researches using prototype NE sensors were limited by their low sensitivities. Here, we developed next-generation versions of GPCR activation-based NE sensors (GRABNE2m and GRABNE2h) with a superior response, high sensitivity and selectivity to NE both in vitro and in vivo. Notably, these sensors can detect NE release triggered by either optogenetic or behavioral stimuli in freely moving mice, producing robust signals in the locus coeruleus and hypothalamus. With the development of a novel transgenic mouse line, we recorded both NE release and calcium dynamics with dual-color fiber photometry throughout the sleep-wake cycle; moreover, dual-color mesoscopic imaging revealed cell type-specific spatiotemporal dynamics of NE and calcium during sensory processing and locomotion. Thus, these new GRABNE sensors are valuable tools for monitoring the precise spatiotemporal release of NE in vivo, providing new insights into the physiological and pathophysiological roles of NE.

neuroscience↗

bioRxiv2023

The Toxoplasma monocarboxylate transporters are involved in the metabolism within the apicoplast and are linked to parasite survival

The apicoplast is a four-membrane plastid found in the apicomplexans, which harbors biosynthesis and organelle housekeeping activities in the matrix. However, the mechanism driving the flux of metabolites, in and out, remains unknown. Here we used TurboID and genome engineering to identify apicoplast transporters in Toxoplasma gondii. Among the many novel transporters, we show that one pair of apicomplexan monocarboxylate transporters (AMTs) appears to have evolved from a putative host cell that engulfed a red alga. Protein depletion showed that AMT1 and AMT2 are critical for parasite growth. Metabolite analyses supported the notion that AMT1 and AMT2 are associated with biosynthesis of isoprenoids and fatty acids. However, stronger phenotypic defects were observed for AMT2, including in the inability to establish T. gondii parasite virulence in mice. This study clarifies, significantly, the mystery of apicoplast transporter composition and reveals the importance of the pair of AMTs in maintaining the apicoplast activity in apicomplexans.

microbiology↗

bioRxiv2023

Effect of Aging, Sex, and Gene (Fbln5) on Arterial Stiffness of Mice: 20 Weeks Adult Fbln5-knockout Mice Have Older Arteries than 100 Weeks Wild-Type Mice

The arterial stiffening is a strong independent predictor of cardiovascular risk and has been used to characterize the biological age of arteries ( arterial age). Here we revealed that the Fbln5 gene knockout (Fbln5-/-) significantly increases the arterial stiffening for both male and female mice. We also showed that the arterial stiffening increases with natural aging, but the stiffening effect of Fbln5-/- is much more severe than aging. The arterial stiffening of 20 weeks old mice with Fbln5-/- is much higher than that at 100 weeks in wild-type (Fbln5+/+) mice, which indicates that 20 weeks mice (equivalent to [~]26 years old humans) with Fbln5-/- have older arteries than 100 weeks wild-type mice (equivalent to [~]77 years humans). Histological microstructure changes of elastic fibers in the arterial tissue elucidate the underlying mechanism of the increase of arterial stiffening due to Fbln5-knockout and aging. These findings provide new insights to reverse arterial age due to abnormal mutations of Fbln5 gene and natural aging. This work is based on a total of 128 biaxial testing samples of mouse arteries and our recently developed unified-fiber-distribution (UFD) model. The UFD model considers the fibers in the arterial tissue as a unified distribution, which is more physically consistent with the real fiber distribution of arterial tissues than the popular fiber-family-based models (e.g., the well-know Gasser-Ogden-Holzapfel [GOH] model) that separate the fiber distribution into several fiber families. Thus, the UFD model achieves better accuracies with less material parameters. To our best knowledge, the UFD model is the only existing accurate model that could capture the property/stiffness differences between different groups of the experimental data discussed here.

bioengineering↗

bioRxiv2023

Dual-color GRAB sensors for monitoring spatiotemporal serotonin release in vivo

The serotonergic system plays important roles in both physiological and pathological processes, and is a widely used therapeutic target for many psychiatric disorders. Although several genetically encoded GFP-based serotonin (5-HT) sensors were recently developed, their sensitivities and spectral profiles are relatively limited. To overcome these limitations, we optimized green fluorescent G-protein-coupled receptor (GPCR)-activation-based 5-HT (GRAB5-HT) sensors and developed a new red fluorescent GRAB5-HT sensor. These sensors have excellent cell surface trafficking, high specificity, sensitivity, and spatiotemporal resolution, making them suitable for monitoring 5-HT dynamics in vivo. Besides recording subcortical 5-HT release in freely moving mice, we observed both uniform and gradient 5-HT release in the mouse dorsal cortex with mesoscopic imaging. Finally, we performed dual-color imaging and observed seizure-induced waves of 5-HT release throughout the cortex following calcium and endocannabinoid waves. In summary, these 5-HT sensors can offer valuable insights regarding the serotonergic system in both physiological and pathological states.

neuroscience↗

bioRxiv2023

Noncanonical TRAIL Signaling Facilitates Tumor Immunosuppression and Cholangiocarcinoma Growth via Myeloid-Derived Suppressor Cells

Proapoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling as a cause of cancer cell death is a well-established mechanism. However, TRAIL-receptor (TRAIL-R) agonists have had very limited anticancer activity in humans, challenging the concept of TRAIL as a potent anticancer agent. Herein, we demonstrate that TRAIL+ cancer cells can leverage noncanonical TRAIL signaling in myeloid-derived suppressor cells (MDSCs) promoting their abundance in murine cholangiocarcinoma (CCA). In multiple immunocompetent syngeneic, orthotopic murine models of CCA, implantation of TRAIL+ murine cancer cells into Trail-r-/-mice resulted in a significant reduction in tumor volumes compared to wild type mice. Tumor bearing Trail-r-/- mice had a significant decrease in the abundance of MDSCs due to attenuation of MDSC proliferation. Noncanonical TRAIL signaling with consequent NF-{kappa}B activation in MDSCs facilitated enhanced MDSC proliferation. Single cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq) of CD45+ cells in murine tumors from three distinct immunocompetent CCA models demonstrated a significant enrichment of an NF-{kappa}B activation signature in MDSCs. Moreover, MDSCs were resistant to TRAIL-mediated apoptosis due to enhanced expression of cellular FLICE inhibitory protein (cFLIP), an inhibitor of proapoptotic TRAIL signaling. Accordingly, cFLIP knockdown sensitized murine MDSCs to TRAIL-mediated apoptosis. Finally, cancer cell-restricted deletion of Trail significantly reduced MDSC abundance and murine tumor burden. In summary, our findings define a noncanonical TRAIL signal in MDSCs and highlight the therapeutic potential of targeting TRAIL+ cancer cells for the treatment of a poorly immunogenic cancer.

cancer biology↗

bioRxiv2023

TREM2 mediates MHCII-associated CD4+ T cell response against gliomas

Triggering receptor expressed on myeloid cells 2 (TREM2) was recently highlighted as a novel immune suppressive marker in peripheral tumors. The aim of this study was to characterize TREM2 expression in gliomas and investigate its contribution in glioma progression by using Trem2-/-mouse line. Our results showed that higher TREM2 expression was correlated with poor prognosis in glioma patients. Unexpectedly, TREM2 deficiency did not have a beneficial effect in a pre-clinical model of glioma. The increased TREM2 expression in glioma was likely due to increased myeloid cell infiltration, as evidenced by our single-cell analysis showing that almost all microglia and macrophages in gliomas were TREM2+. Furthermore, we found that deficiency of TREM2 impaired tumor-myeloid phagocytosis and MHCII presentation, and significantly reduced CD4+ T cells in tumor hemispheres. Our results revealed a previously unrecognized protective role of tumor-myeloid TREM2 in promoting MHCII-associated CD4+ T cell response against gliomas. SUMMARYAuthors found that although higher TREM2 expression is correlated with poor prognosis in glioma patients, its absence has no beneficial effect in a pre-clinical model of glioma. Deficiency of TREM2 impairs myeloid cell phagocytosis of tumor debris, leading to a reduction in MHCII-dependent CD4+ anti-glioma immunity.

neuroscience↗

bioRxiv2023

Spatial and single-cell transcriptomics reveal neuron-astrocyte interplay in long-term memory

Memory encodes past experiences, thereby enabling future plans. The basolateral amygdala (BLA) is a center of salience networks that underlie emotional experience and thus plays a key role in long-term fear memory formation1, 2. Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory. We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks. These transcriptional signatures implicate neuropeptide signaling, mitogen-activated protein kinase (MAPK), brain-derived neurotrophic factor (BDNF), cAMP response element-binding protein (CREB), ubiquitination pathways, and synaptic connectivity in long-term memory. We also discovered that a neuronal sub-population, defined by increased Penk expression and decreased Tac expression, constitutes the most prominent component of the BLAs memory engram. These transcriptional changes were observed both with single-cell RNAseq and with single-molecule spatial transcriptomics in intact slices, thereby providing a rich spatial map of a memory engram. The spatial data enabled us to show that this neuronal subpopulation further interacts with spatially related astrocytes that are essential for memory consolidation, indicating that neurons require interactions with astrocytes to encode long term memory.

neuroscience↗