Biology subjects

Cheng, X.

Publications and source records attributed to Cheng, X..

At least 163 records · Page 9Linked to original sources

bioRxiv2023

Optimizing Cell Therapy by Sorting Cells with High Extracellular Vesicle Secretion

Critical challenges remain in clinical translation of extracellular vesicle (EV)-based therapeutics due to the absence of methods to enrich cells with high EV secretion. Current cell sorting methods are limited to surface markers that are uncorrelated to EV secretion or therapeutic potential. We developed a nanovial technology for enrichment of millions of single cells based on EV secretion. This approach was applied to select mesenchymal stem cells (MSCs) with high EV secretion as therapeutic cells for improving treatment. The selected MSCs exhibited distinct transcriptional profiles associated with EV biogenesis and vascular regeneration and maintained high levels of EV secretion after sorting and regrowth. In a mouse model of myocardial infarction, treatment with high-secreting MSCs improved heart functions compared to treatment with low-secreting MSCs. These findings highlight the therapeutic importance of EV secretion in regenerative cell therapies and suggest that selecting cells based on EV secretion could enhance therapeutic efficacy.

bioengineering↗

bioRxiv2023

Marginated aberrant red blood cells induce pathologic vascular stress fluctuations in a computational model of hematologic disorders

Red blood cell (RBC) disorders affect billions worldwide. While alterations in the physical properties of aberrant RBCs and associated hemodynamic changes are readily observed, in conditions such as sickle cell disease and iron deficiency, RBC disorders can also be associated with vascular dysfunction. The mechanisms of vasculopathy in those diseases remain unclear and scant research has explored whether biophysical alterations of RBCs can directly affect vascular function. Here we hypothesize that the purely physical interactions between aberrant RBCs and endothelial cells, due to the margination of stiff aberrant RBCs, play a key role in this phenomenon for a range of disorders. This hypothesis is tested by direct simulations of a cellular scale computational model of blood flow in sickle cell disease, iron deficiency anemia, COVID-19, and spherocytosis. We characterize cell distributions for normal and aberrant RBC mixtures in straight and curved tubes, the latter to address issues of geometric complexity that arise in the microcirculation. In all cases aberrant RBCs strongly localize near the vessel walls (margination) due to contrasts in cell size, shape, and deformability from the normal cells. In the curved channel, the distribution of marginated cells is very heterogeneous, indicating a key role for vascular geometry. Finally, we characterize the shear stresses on the vessel walls; consistent with our hypothesis, the marginated aberrant cells generate large transient stress fluctuations due to the high velocity gradients induced by their near-wall motions. The anomalous stress fluctuations experienced by endothelial cells may be responsible for the observed vascular inflammation. Significance StatementA common and potentially life-threatening complication of blood cell disorders is inflammation and dysfunction of the vascular wall, for reasons that remain unclear. To address this issue, we explore a purely biophysical hypothesis involving red blood cells using detailed computational simulations. Our results show that red blood cells that are pathologically altered in cell shape, size, and stiffness, which occurs in various blood disorders, strongly marginate, residing primarily in the cell-free layer near blood vessel walls, generating large shear stress fluctuations at the vessel wall that may be responsible for endothelial damage and inflammation.

biophysics↗

bioRxiv2023

Endogenous fine-mapping of functional regulatory elements in complex genetic loci

The vast majority of genetic loci associated with polygenic complex traits are located in non-coding regions of the human genome. However, many of these regions exhibit high- order gene regulatory relationships and complicated linkage disequilibrium (LD) configurations, which bring challenges to accurately identify causal variants and their target genes controlling specific molecular processes or traits. We employed multiplexed single-cell CRISPR interference and activation perturbations to explore the links between cis-regulatory element (CRE) and target gene expression within tight LD in the endogenous chromatin context. We validated the prevalence of multiple causality in perfect LD (pLD) for independent expression quantitative trait locus (eQTL), and revealed fine-grained genetic effects on gene expression within pLD. These effects are difficult to decipher using conventional eQTL fine-mapping or to predict via existing computational methods. We found that nearly half of the casual CREs lack classical epigenetic markers, potentially affecting gene expression through hidden regulatory mechanisms. Integrative analysis on different types of perturbation effects suggested a high regulatory plasticity of the human genome. These findings will propel further in-depth exploration of functional genomic elements, facilitating a more comprehensive understanding of gene expression regulatory patterns and the development of complex traits.

genomics↗

bioRxiv2023

Metabolic Reprogramming via targeting ACOD1 promotes polarization and anti-tumor activity of human CAR-iMACs in solid tumors

The pro-inflammatory state of macrophages is crucial in conferring its role in combating tumor cells. That state is closely associated with metabolic reprogramming. Here we identified key metabolic genes regulating macrophage pro-inflammatory activation in a pooled metabolic gene knockout CRISPR screen. We found that KEAP1 and ACOD1 are strong regulators of the pro-inflammatory state, and therefore developed human ACOD1 knockout macrophages with our induced pluripotent stem cell-derived CAR-macrophage (CAR-iMAC) platform. The engineered iMACs showed stronger and more persistent polarization toward the pro-inflammatory state, more ROS production, and more potent phagocytosis and cytotoxic functions against cancer cells in vitro. Upon transplantation to ovarian or pancreatic cancer mouse models, ACOD1 depleted CAR-iMACs exhibited enhanced capacity in repressing tumors in vivo and prolonged the lifespan of mice. In addition, combining ACOD1-depleted CAR-iMACs with immune check point inhibitors (ICIs), such as the anti-CD47 antibody or anti-PD1 antibody resulted in stronger tumor suppressing effect. Mechanistically, the depletion of ACOD1 reduced the immunometabolite itaconate, allowing KEAP1 to prevent NRF2 from entering the nucleus to activate the anti-inflammatory program. This study demonstrates that ACOD1 is a new myeloid target for cancer immunotherapy and metabolically engineered human iPSC-derived CAR-iMACs exhibit enhanced polarization and anti-tumor functions in adoptive cell transfer therapies.

immunology↗

bioRxiv2023

S2' cleavage site plays a decisive role in expansion of Vero cell tropism by infectious bronchitis virus HV80, with Q855H promoting cell-to-cell fusion

Infectious bronchitis virus (IBV) has restricted cell tropism. Apart from the Beaudette strain, other IBVs cannot infect mammalian cell lines. The limited cell tropism of other IBVs has hindered the development of IBV vaccines and research on mechanisms of IBV infection. In a previous study, a new Vero-cell-adapted strain HV80 was obtained via serial chicken embryo and cell passaging of strain H120 and 17 mutations leading to amino acid substitutions occurred in replication gene 1a, S gene and E gene. This study, we constructed recombinants that expressed chimeric S glycoprotein, S1 or S2 subunit of strain H120, and demonstrated that mutations in S2 subunit were related to the Vero cell adaption of strain HV80. With a genome backbone of strain HV80 or H120, and expression of chimeric S2' cleavage site of H120 or HV80, two recombinants demonstrated that the RRRR690/S motif at the S2' cleavage site played a key role in Vero cell adaption of strain HV80. Another six amino acid substitutions in the S2 subunit of the recombinants showed that F692V enhanced the capability of invasion of HV80 strain, and Q855H induced the formation of syncytia. A transient transfection assay demonstrated different mechanisms for virus-to-cell fusion and cell-to-cell fusion induced by S glycoprotein. The PRRR690/S motif at the S2' cleavage site could be activated by proteases in the process of cell-to-cell fusion, while H855Q substitution did not affect the cell invasion of HV80, but hindered the cell-to-cell fusion by blocking activation of the S2' cleavage site. IMPORTANCEInfectious bronchitis is an acute respiratory disease that has caused large economic losses to the poultry industry. As a member of the gamma-coronaviruses, the restricted cell tropism of infectious bronchitis virus (IBV) limits the development of cellular vaccines and research on infection mechanisms. As a strain that can replicate effectively in mammalian cell lines, studies of HV80s adaptive mechanisms point a way for engineering other IBVs for adaptation in mammalian cell lines. In our study, different recombinants were constructed by reverse genetic techniques, and demonstrated the different mechanism between virus-to-cell and cell-to-cell fusion induced by HV80 S glycoprotein. The acquisition of a highly efficient S2' cleavage site enabled the virus to invade Vero cells. The Q855H substitution played a key role in cell-to-cell fusion, and provided a more efficient model of infection in Vero cells. Our study provides new theoretical insights into mechanisms of IBV adaptation in mammalian cell lines.

microbiology↗

bioRxiv2023

Measuring human cerebral blood flow and brain function with fiber-based speckle contrast optical spectroscopy system

Cerebral blood flow (CBF) is an important indicator of brain health and function. Diffuse correlation spectroscopy (DCS) is an optical technique that enables non-invasive and continuous bedside monitoring of human CBF. However, traditional DCS consisting of a few channels has relatively low signal-to-noise ratio (SNR), preventing measurements at long source detector separations (SDS) with increased sensitivity to cerebral rather than extracerebral blood flow. Here we developed a fiber-based speckle contrast optical spectroscopy (SCOS) system and the corresponding data analysis pipeline to measure CBF variations. We show that SCOS outperforms traditional DCS by more than an order of magnitude in SNR with comparable financial cost. We also demonstrated human brain function measurements during a cognitive task at an SDS of 33 mm. This technology will establish the foundation for devices that use spatial speckle statistics to non-invasively monitor human CBF, leading to a new functional neuroimaging approach for cognitive neuroscience.

bioengineering↗

bioRxiv2023

Reply to Barton et al: signatures of natural selection during the Black Death

Barton et al.1 raise several statistical concerns regarding our original analyses2 that highlight the challenge of inferring natural selection using ancient genomic data. We show here that these concerns have limited impact on our original conclusions. Specifically, we recover the same signature of enrichment for high FST values at the immune loci relative to putatively neutral sites after switching the allele frequency estimation method to a maximum likelihood approach, filtering to only consider known human variants, and down-sampling our data to the same mean coverage across sites. Furthermore, using permutations, we show that the rs2549794 variant near ERAP2 continues to emerge as the strongest candidate for selection (p = 1.2x10-5), falling below the Bonferroni-corrected significance threshold recommended by Barton et al. Importantly, the evidence for selection on ERAP2 is further supported by functional data demonstrating the impact of the ERAP2 genotype on the immune response to Y. pestis and by epidemiological data from an independent group showing that the putatively selected allele during the Black Death protects against severe respiratory infection in contemporary populations.

genomics↗

bioRxiv2023

Membrane-induced 2D phase separation of focal adhesion proteins

Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. Made up of hundreds of proteins, focal adhesions are dynamic structures which can assemble and disassemble quickly, withstand strong actomyosin-applied forces, and form highly stable complexes. How they achieve these flexible characteristics is not well understood. Here, we use recombinant focal adhesion proteins to reconstitute the core structural machinery in vitro, with the goal of understanding the underlying protein dynamics and interactions. We observe liquid-liquid phase separation of the core focal adhesion proteins talin and vinculin for a spectrum of conditions and in combination with several interaction partners. Intriguingly, we show that membrane binding triggers phase separation of these proteins on the membrane, which in turn induces the enrichment of integrin in the clusters. We also introduce a novel experimental setup to probe talin-membrane interactions down to the single protein level. Our results suggest that membrane composition triggers condensate assembly at the membrane, a regulatory mechanism which could widely apply to membrane-localized biomolecular condensates and provide a pathway of how spatial organization of lipids within the membrane can couple into the cytosol.

cell biology↗

bioRxiv2023

xTrimoGene: An Efficient and Scalable Representation Learner for Single-Cell RNA-Seq Data

AO_SCPLOWBSTRACTC_SCPLOWThe advances in high-throughput sequencing technology have led to significant progress in measuring gene expressions in single-cell level. The amount of publicly available single-cell RNA-seq (scRNA-seq) data is already surpassing 50M records for human with each record measuring 20,000 genes. This highlights the need for unsupervised representation learning to fully ingest these data, yet classical transformer architectures are prohibitive to train on such data in terms of both computation and memory. To address this challenge, we propose a novel asymmetric encoder-decoder transformer for scRNA-seq data, called xTrimoGene, which leverages the sparse characteristic of the data to scale up the pre-training. This scalable design of xTrimoGene reduces FLOPs by one to two orders of magnitude compared to classical transformers while maintaining high accuracy, enabling us to train the largest transformer models over the largest scRNA-seq dataset today. Our experiments also show that the performance of xTrimoGene improves as we increase the model sizes, and it also leads to SOTA performance over various downstream tasks, such as cell classification, perturb-seq effect prediction, and drug combination prediction.

bioinformatics↗

bioRxiv2023

Identification of a carbohydrate-recognition motif of purinergic receptors

As a major class of biomolecules, carbohydrates play indispensable roles in various biological processes. However, it remains largely unknown how carbohydrates directly modulate important drug targets, such as G-protein coupled receptors (GPCRs). Here, we employed P2Y purinoceptor 14 (P2Y14), a drug target for inflammation and immune responses, to uncover the sugar nucleotide activation of GPCRs. Integrating molecular dynamics simulation with functional study, we identified the uridine diphosphate (UDP)-sugar-binding site on P2Y14, and revealed that a UDP-glucose might activate the receptor by bridging the transmembrane helices (TM) 2 and 7. Between TM2 and TM7 of P2Y14, a conserved salt bridging chain (K2.60-D2.64-K7.35-E7.36, KDKE) was identified to distinguish different UDP-sugars, including UDP-glucose, UDP-galactose, UDP-glucuronic acid and UDP-N-acetylglucosamine. We identified the KDKE chain as a conserved functional motif of sugar binding for both P2Y14 and P2Y purinoceptor 12 (P2Y12), and then designed three sugar nucleotides as agonists of P2Y12. These results not only expand our understanding for activation of purinergic receptors but also provide insights for the carbohydrate drug development for GPCRs.

molecular biology↗

bioRxiv2023

xTrimoDock: Rigid Protein Docking via Cross-Modal Representation Learning and Spectral Algorithm

Protein-protein interactions are the basis for the formation of protein complexes which are essential for almost all cellular processes. Knowledge of the structures of protein complexes is of major importance for understanding the biological function of these protein-protein interactions and designing protein drugs. Here we address the problem of rigid protein docking which assumes no deformation of the involved proteins during interactions. We develop a method called, xTrimoDock, which leverages a cross-modal representation learning to predict the protein distance map, and then uses a spectral initialization and gradient descent to obtain the roto-translation transformation for docking. We show that, on antibody heavy-chain and light-chain docking, and antibody-antigen docking, xTrimoDock consistently outperforms the state-of-the-art such as AlphaFold-Multimer and HDock, and can lead to as much as a 10% improvement in DockQ metric. xTrimoDock has been applied as a useful tool in protein drug design at BioMap.

bioinformatics↗

bioRxiv2023

Single-cell transcriptomic landscape deciphers novel olfactory neuroblastoma subtypes and intratumoral heterogeneity

Olfactory neuroblastoma (ONB) is an uncommon malignant tumor known to originate from the olfactory epithelial. The complex tumor ecosystem of this pathology remained unclear. Here, we explored the cellular components with ONB tumors based on scRNA profiles of 96,325 single-cells derived from 10 tumors and 1 olfactory mucosa sample. We discovered 11 major cell types, including 6 immune cell, 3 stromal cell subtypes and epithelial cluster in the tumor microenvironment (TME), and identified 5 common expression programs from malignant epithelial cells. We analyzed subclusters of TME and the interactions among different cell types in the TME. An innovative three-classification of ONB was established via scRNA analysis. Markers for categorizing tumor samples into new subtypes were elucidated. Different responses towards certain chemotherapy regimens could be inferred according to the molecular features of three tumor types. Relative abundance of immunosuppressive TAMs indicated the benefits of immunotherapies targeting myeloid cells.

cancer biology↗

bioRxiv2023

Structural basis of peptide recognition and activation of endothelin receptors

Endothelin system comprises three endogenous 21-amino-acid peptide ligands endothelin-1, -2, and -3 (ET-1/2/3), and two G protein-coupled receptor (GPCR) subtypes--endothelin receptor A (ETAR) and B (ETBR). Since ET-1, the first endothelin, was identified in 1988 as one of the most potent endothelial cell-derived vasoconstrictor peptides with long-lasting actions, the endothelin system has attracted extensive attention due to its critical role in vasoregulation and close relevance in cardiovascular-related diseases. Here we present three cryo-electron microscopy structures of ETAR and ETBR bound to ET-1 and ETBR bound to the selective peptide IRL1620. These structures reveal a highly conserved recognition mode of ET-1 and characterize the ligand selectivity by ETRs. They also present several conformation features of the active ETRs, thus revealing a specific activation mechanism. Together, these findings deepen our understanding of endothelin system regulation and offer an opportunity to design selective drugs targeting specific ETR subtypes.

molecular biology↗

bioRxiv2023

Illumination of the complement receptors C3aR and C5aR signaling by anaphylatoxins

The complement receptors C3aR and C5aR, whose signaling are selectively activated by anaphylatoxins C3a and C5a, are important regulators of both innate and adaptive immune responses. Dysregulations of C3aR and C5aR signaling lead to multiple inflammatory disorders, including sepsis, asthma, and acute respiratory distress syndrome (ARDS). The mechanism underlying endogenous anaphylatoxin recognition and activation of C3aR and C5aR remains elusive. Here we reported the structures of C3a-bound C3aR and C5a-bound C5aR1 as well as an apo C3aR structure. These structures, combined with mutagenesis analysis, reveal a conserved recognition pattern of anaphylatoxins to the complement receptors that is different from chemokine receptors, unique pocket topologies of C3aR and C5aR1 that mediate ligand selectivity, and a common mechanism of receptor activation. These results provide crucial insights into the molecular understandings of C3aR and C5aR1 signaling and structural templates for rational drug design for treating inflammation disorders.

biochemistry↗

bioRxiv2023

Leafhopper transmits soybean stay-green associated virus to legume plants

A novel geminivirus, soybean stay-green associated virus (SoSGV), was previously shown to cause soybean delayed senescence and is associated with the incidences of soybean stay-green syndrome. The transmission methods of SoSGV were not yet understood. We captured insects belonging to 24 distinct species in a soybean field with the SoSGV outbreak and detected the presence of SoSGV only in leafhoppers and bean bugs (Riptortus pedestris). Caged feeding experiments using captured leafhoppers and bean bugs from soybean fields showed that leafhoppers, but not bean bugs, are vectors transmitting SoSGV. The common brown leafhopper (Orosius orientalis) is identified as the dominant leafhopper species and can establish colonies feeding on soybean plants in experimental conditions. An investigation of SoSGV defective DNA revealed that soybean genomic DNA could be inserted into the SoSGV genome, while sequences from wild soybean, red bean, and cowpea were also identified. We further showed that the common brown leafhopper could transmit SoSGV to wild soybean and red bean plants, emphasizing the vector role of the leafhopper in the transmission of SoSGV in the field.

microbiology↗

bioRxiv2022

PRE-INJURY MECHANORECEPTOR ABLATION REDUCES NOCICEPTOR-DRIVEN SPINAL CORD INJURY-INDUCED NEUROPATHIC PAIN

Evidence from previous studies supports the concept that spinal cord injury (SCI) induced neuropathic pain (NP) has its neural roots in the peripheral nervous system. There is uncertainty about how and to which degree nociceptors and mechanoreceptors contribute. Sensorimotor activation-based interventions (e.g. treadmill training) have been shown to reduce NP following experimental SCI, suggesting transmission of pain-alleviating signals through mechanoreceptors. At the same time, nociceptors have been shown to become hyperexcitable early after SCI and peptidergic axons sprout into deeper laminae of the below injury level dorsal horn. The aim of the present study is to comprehensively understand the relative contribution of each pathway in respect to NP presentation in a moderate mouse contusion SCI model. After genetic ablation of tropomyosin receptor kinase B (TrkB) expressing mechanoreceptors before SCI mechanical allodynia was reduced. The identical genetic ablation after SCI did not yield any change in pain behavior. CGRP sprouting into lamina III/IV below injury level as a consequence of SCI was not altered by either mechanoreceptor ablation. Moreover, detection of hyperexcitability in nociceptors, not in mechanoreceptors, in skin-nerve preparations of contusion SCI mice 7 days after injury makes a substantial direct contribution of mechanoreceptors to NP maintenance unlikely. SNS reporter mice allowing specific visualization of the entire nociceptor population confirmed significant sprouting of respective neurons into laminae III/IV as early as 5 days post-injury. Genetic ablation of SNS-Cre mice severely affected their overall health condition, which precluded them to undergo experimental SCI and subsequent further analysis. Complementing animal data, quantitative sensory testing in human SCI subjects indicated reduced mechanical pain thresholds, whereas the mechanical detection threshold was not altered. Taken together, early mechanoreceptor ablation modulates pain behavior, most likely through indirect mechanisms. Hyperexcitable nociceptors with consecutive peptidergic fiber sprouting in the dorsal horn are confirmed as the likely main driver of SCI-induced NP. Future studies need to focus on injury-derived factors triggering early onset nociceptor hyperexcitability, which could serve as targets for more effective therapeutic interventions.

neuroscience↗

bioRxiv2022

Monopogen: single nucleotide variant calling from single cell sequencing

Distinguishing how genetics impact cellular processes can improve our understanding of variable risk for diseases. Although single-cell omics have provided molecular characterization of cell types and states on diverse tissue samples, their genetic ancestry and effects on cellular molecular traits are largely understudied. Here, we developed Monopogen, a computational tool enabling researchers to detect single nucleotide variants (SNVs) from a variety of single cell transcriptomic and epigenomic sequencing data. It leverages linkage disequilibrium from external reference panels to identify germline SNVs from sparse sequencing data and uses Monovar to identify novel SNVs at cluster (or cell type) levels. Monopogen can identify 100K~3M germline SNVs from various single cell sequencing platforms (scRNA-seq, snRNA-seq, snATAC-seq etc), with genotyping accuracy higher than 95%, when compared against matched whole genome sequencing data. We applied Monopogen on human retina, normal breast and Asian immune diversity atlases, showing that that derived genotypes enable accurate global and local ancestry inference and identification of admixed samples from ancestrally diverse donors. In addition, we applied Monopogen on ~4M cells from 65 human heart left ventricle single cell samples and identified novel variants associated with cardiomyocyte metabolic levels and epigenomic programs. In summary, Monopogen provides a novel computational framework that brings together population genetics and single cell omics to uncover genetic determinants of cellular quantitative traits.

bioinformatics↗

bioRxiv2022

A multi-omics atlas of the human retina at single-cell resolution

Cell classes in the human retina are highly heterogeneous with their abundance varying by several orders of magnitude. Although previous studies reported the profiles of the retinal cell types as the transcriptome level, there is no study regarding the open-chromatin profiles at a similar resolution. Here, we generated and integrated a multi-omics single-cell atlas of the adult human retina, including over 250K nuclei for single-nuclei RNA-seq and 137K nuclei for single-nuclei ATAC-seq. Through enrichment of rare cell types, this single cell multiome atlas is more comprehensive than previous human retina studies. Cross species comparison of the retina atlas among human, monkey, mice, and chicken revealed relatively conserved and non-conserved types. Interestingly, the overall cell heterogeneity in primate retina decreases compared to that of rodent and chicken retina. Furthermore, integrative analysis of the single cell multi-omics data identified 35k distal cis-element-gene pairs with most of these cis-elements being cell type specific. We also showed that the cis-element-gene relationship in different cell types within the same class could be highly heterogenous. Moreover, we constructed transcription factor (TF)-target regulons for over 200 TFs, partitioned the TFs into distinct co-active modules, and annotated each module based on their cell-type specificity. Taken together, we present the most comprehensive single-cell multi-omics atlas of the human retina as a valuable resource that enables systematic in-depth molecular characterization at individual cell type resolution.

genomics↗