Biology subjects

Chi, H.

Publications and source records attributed to Chi, H..

34 records · Page 2Linked to original sources

bioRxiv2024

Metabolic remodelling in hiPSC-derived myofibres carrying the m.3243A>G mutation

Mutations in mitochondrial DNA cause severe multisystem disease, frequently associated with muscle weakness. The m.3243A>G mutation is the major cause of Mitochondrial Encephalomyopathy Lactic Acidosis and Stroke Like episodes (MELAS). Experimental models that recapitulate the disease phenotype in vitro for disease modelling or drug screening are very limited. We have therefore generated hiPSC-derived muscle fibres with variable heteroplasmic mtDNA mutation load without significantly affecting muscle differentiation potential. The cells are excitable and show physiological characteristics of muscle fibres and show well organised myofibrillar structure. In cells carrying the m.3243A>G, the mitochondrial membrane potential and oxygen consumption were reduced in relation to the mutant load. We have shown through proteomic, phosphoproteomic, and metabolomic analyses that the m.3243A>G mutation variably affects the cell phenotype in relation to the mutant load. This variation is reflected by an increase in the NADH/NAD+ ratio, which in turns influences key nutrient-sensing pathways in the myofibres. This model enables detailed study of the impact of the mutation on cellular bioenergetics and on muscle physiology with the potential to provide a platform for drug screening.

cell biology↗

bioRxiv2024

RNA processing by the CRISPR-associated NYN ribonuclease

CRISPR-Cas systems confer adaptive immunity in prokaryotes, facilitating the recognition and destruction of invasive nucleic acids. Type III CRISPR systems comprise large, multisubunit ribonucleoprotein complexes with a catalytic Cas10 subunit. When activated by the detection of foreign RNA, Cas10 generates nucleotide signalling molecules that elicit an immune response by activating ancillary effector proteins. Among these systems, the Bacteroides fragilis type III CRISPR system was recently shown to produce a novel signal molecule, SAM-AMP, by conjugating ATP and SAM. SAM-AMP regulates a membrane effector of the CorA family to provide immunity. Here, we focus on NYN, a ribonuclease encoded within this system, probing its potential involvement in crRNA maturation. Structural modelling and in vitro ribonuclease assays reveal that NYN displays robust sequence-nonspecific, Mn2+-dependent ssRNA-cleavage activity. Our findings suggest a role for NYN in trimming crRNA intermediates into mature crRNAs, which is necessary for type III CRISPR antiviral defence. This study sheds light on the functional relevance of CRISPR-associated NYN proteins and highlights the complexity of CRISPR-mediated defence strategies in bacteria.

molecular biology↗

bioRxiv2023

Human pluripotent stem cell-derived hepatocyte-like cells for hepatitis D virus studies

Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study, we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co- infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg, HLCs effectively secrete infectious progeny virions. We also show that HLCs expressing HBsAg support extracellular spread of HDV, thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection, we have identified CD63 as a potential HDV/HBV co-entry factor, which was rate-limiting HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients, we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions. TeaserA human stem cell-derived hepatocyte culture model for hepatitis D virus studies

microbiology↗

bioRxiv2023

Antiviral Type III CRISPR signalling via conjugation of ATP and AdoMet

CRISPR systems are widespread in the prokaryotic world, providing adaptive immunity against mobile genetic elements (MGE) 1, 2. Type III CRISPR systems, with the signature gene cas10, use CRISPR RNA (crRNA) to detect non-self RNA, activating the enzymatic Cas10 subunit to defend the cell against MGE either directly, via the integral HD nuclease domain 3-5 or indirectly, via synthesis of cyclic oligonucleotide (cOA) second messengers to activate diverse ancillary effectors 6-9. A subset of type III CRISPR systems encode an uncharacterised CorA-family membrane protein and an associated NrN family phosphodiesterase predicted to function in antiviral defence. Here, we demonstrate that the CorA associated type III-B (Cmr) CRISPR system from Bacteroides fragilis provides immunity against MGE when expressed inE. coli. However, B. fragilis Cmr does not synthesise cOA species on activation, instead generating a previously undescribed sigalling molecule, SAM-AMP (3-adenylyl-AdoMet) by conjugating ATP to S-adenosyl methionine via a phosphodiester bond. Once synthesised, SAM-AMP binds to the CorA effector, presumably leading to cell death by disruption of the membrane integrity. SAM-AMP is degraded by CRISPR associated phosphodiesterases or a SAM-AMP lyase, providing an "off switch" analogous to cOA specific ring nucleases 10. SAM-AMP thus represents a new class of second messenger for antiviral signalling, which may function in different roles in diverse cellular contexts.

microbiology↗

bioRxiv2023

scMINER: a mutual information-based framework for identifying hidden drivers from single-cell omics data

The sparse nature of single-cell omics data makes it challenging to dissect the wiring and rewiring of the transcriptional and signaling drivers that regulate cellular states. Many of the drivers, referred to as "hidden drivers", are difficult to identify via conventional expression analysis due to low expression and inconsistency between RNA and protein activity caused by post-translational and other modifications. To address this issue, we developed scMINER, a mutual information (MI)-based computational framework for unsupervised clustering analysis and cell-type specific inference of intracellular networks, hidden drivers and network rewiring from single-cell RNA-seq data. We designed scMINER to capture nonlinear cell-cell and gene-gene relationships and infer driver activities. Systematic benchmarking showed that scMINER outperforms popular single-cell clustering algorithms, especially in distinguishing similar cell types. With respect to network inference, scMINER does not rely on the binding motifs which are available for a limited set of transcription factors, therefore scMINER can provide quantitative activity assessment for more than 6,000 transcription and signaling drivers from a scRNA-seq experiment. As demonstrations, we used scMINER to expose hidden transcription and signaling drivers and dissect their regulon rewiring in immune cell heterogeneity, lineage differentiation, and tissue specification. Overall, activity-based scMINER is a widely applicable, highly accurate, reproducible and scalable method for inferring cellular transcriptional and signaling networks in each cell state from scRNA-seq data. The scMINER software is publicly accessible via: https://github.com/jyyulab/scMINER.

systems biology↗

bioRxiv2022

Flavivirus prM interacts with MDA5 and MAVS to inhibit RLR antiviral signaling

Vector-borne flaviviruses, including tick-borne encephalitis virus (TBEV), Zika virus (ZIKA), West Nile virus (WNV), yellow fever virus (YFV), dengue virus (DENV), and Japanese encephalitis virus (JEV), pose a growing threat to public health worldwide, and have evolved complex mechanisms to overcome host antiviral innate immunity. However, the underlying mechanisms of flavivirus structural proteins to evade host immune response remain elusive. Here we show that TBEV structural proteins, including pre-membrane (prM), envelope, and capsid proteins, could inhibit type I interferon (IFN-I) production. Mechanically, TBEV prM interacted with both MDA5 and MAVS and interfered with the formation of MDA5-MAVS complex, thereby impeded the nuclear translocation and dimerization of IRF3 to inhibit RLR antiviral signaling. ZIKA and WNV prM was also demonstrated to interact with both MDA5 and MAVS, while dengue virus serotype 2 (DENV2) and YFV prM associated only with MDA5 or MAVS to suppress IFN-I production. In contrast, JEV prM could not suppress IFN-I production. Overexpression of TBEV and ZIKA prM significantly promoted the replication of Sendai virus. Our findings reveal the immune evasion mechanisms of flavivirus prM, which may contribute to understanding flavivirus pathogenicity, therapeutic intervention and vaccine development. Author SummaryAll flaviviruses must overcome the antiviral innate immunity to infect vertebrate host. The non-structural proteins of flaviviruses are mainly responsible for viral replication and host innate immune escape, and the structural proteins are involved in the virus assembly, which are potential targets for prevention and treatment of flaviviral infections. Whether flavivirus structural proteins participate in host innate immune escape remains to be determined. Here, we found that tick-borne encephalitis virus structural proteins precursor membrane (prM), capsid, and envelope proteins can antagonize type I interferon production, in which prM interacts with both MDA5 and MAVS to inhibit RLR antiviral signaling. Additionally, Zika virus and West Nile virus prMs also interact with both MDA5 and MAVS, while dengue virus serotype 2 and yellow fever virus prMs associate only with MDA5 or MAVS to suppress type I interferon production. In contrast, Japanese encephalitis virus prM cannot antagonize type I interferon production. Our findings reveal that flavivirus prM inhibits type I interferon production via interacting with MDA5 and/or MAVS in a species-dependent manner, which may contribute to understanding flavivirus pathogenicity, therapeutic intervention, and vaccine development.

microbiology↗

bioRxiv2022

The ITPRIPL1- CD3ϵ axis: a novel immune checkpoint controlling T cells activation

The immune system is critical to fighting infections and disease. The molecular recognition of harmful entities takes place when antigen-presenting cells (APC) harboring major histocompatibility complex (MHC) molecules bound to peptides derived from harmful antigens (ligand) dock on specific T cell receptor (TCR)-CD3 complex (receptor) at the surface of CD8+ T cells. The discovery of a general immune checkpoint mechanism to avoid the harmful impact of T cell hyperactivation provoked a paradigm shift. The clinical relevance of this mechanism is highlighted by the fact that PD-1 and PD-L1 inhibitors are very effective at boosting immune reactions. Still, immune evasion frequently happens. The observation that some PD-1/PD-L1 negative tumors have a poor immune response opens the door to identifying a novel immune checkpoint mechanism. Here, we discovered that ITPRIPL1, a gene with unknown function, impairs T cell activation. Surprisingly, we found that CD3{varepsilon} is the direct receptor of ITPRIPL1. This novel immune checkpoint was validated as a drug target using ITPRIPL1 KO mice and monoclonal antibodies. Thus, targeting the ITPRIPL1-CD3e axis, especially in PD-1 - PDL-1 negative patients, is a promising therapeutic strategy to reduce immune evasion.

immunology↗

bioRxiv2022

Coupling a Live Cell Directed Evolution Assay with Coevolutionary Landscapes to Engineer an Improved Fluorescent Rhodopsin Chloride Sensor

Our understanding of chloride in biology can be accelerated through the application of fluorescent protein-based sensors in living cells. Laboratory-guided evolution can be used to diversify and identify sensors with specific properties. Recently, we established that the fluorescent proton-pumping rhodopsin wtGR from Gloeobacter violaceus can be converted into a fluorescent sensor for chloride. To unlock this non-natural function, a single point mutation at the Schiff counterion position from D121V was introduced into wtGR fused with cyan fluorescent protein (CFP) resulting in GR1-CFP. Here, we have integrated coevolutionary analysis with directed evolution to understand how the rhodopsin sequence space can be explored and engineered to improve this starting point. We first show how evolutionary couplings are predictive of functional sites in the rhodopsin family and how a fitness metric based on sequence can be used to quantify known proton-pumping activities of GR-CFP variants. Then, we couple this ability to predict potential functional outcomes with a screening and selection assay in live Escherichia coli to reduce the mutational search space of five residues along the proton-pumping pathway in GR1-CFP. This iterative selection process results in GR2-CFP with four additional mutations, E132K, A84K, T125C, and V245I. Finally, bulk, and single fluorescence measurements in live E. coli reveal that GR2-CFP is a reversible, ratiometric fluorescent sensor for extracellular chloride with an improved dynamic range. We anticipate that our framework will be applicable to other systems, providing a more efficient methodology to engineer fluorescent-protein based sensors with desired properties. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=47 SRC="FIGDIR/small/474067v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1ddb801org.highwire.dtl.DTLVardef@15b4a9corg.highwire.dtl.DTLVardef@7f497corg.highwire.dtl.DTLVardef@b19c5b_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

GhCO and GhCRY1 accelerate floral meristem initiation in response to blue light to shorten cotton breeding

The shoot apical meristem (SAM) is a special category of tissue with pluripotency that forms new organs and individuals, especially floral individuals. However, little is known about the fate of cotton SAMs as a tunica corpus structure. Here, we demonstrate that cotton SAM fate decisions depend on light signals and circadian rhythms, and the genes GhFKF1, GhGI, GhCRY1 and GhCO were responsible for SAM fate decisions and highlighted via RNA sequencing (RNA-seq) analysis of different cotton cultivars, as confirmed by genetic analysis via the CRISPR-Cas9 system. In situ hybridization (ISH) analysis showed that the GhCO gene, induced by a relatively high blue light proportion, was highly upregulated during the initiation of floral meristems (FMs). Further blue light treatment analysis showed that the transition from vegetative to reproductive growth of SAM was promoted by a high proportion of blue light, coupled with high expression of the blue light-responsive genes GhCO and GhCRY1. Taken together, our study suggests that blue light signalling plays a key role in the fate decision of cotton SAM. These results provide a strategy to regulate the SAM differentiation of cotton by using the CRISPR-Cas9 system to change the ratio of red and blue light absorption to breed early-maturity cotton. One-sentence summaryThe SAM differentiation especially the initiation of floral meristem of upland cotton were mediated by genes GhCO and GhCRY1 which in response to blue light.

developmental biology↗

bioRxiv2021

A subset of Memory B-derived antibody repertoire from 3-dose vaccinees is ultrapotent against diverse and highly transmissible SARS-CoV-2 variants, including Omicron

Omicron, the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising unprecedented concerns about the effectiveness of antibody therapies and vaccines. We examined whether sera from individuals who received two or three doses of inactivated vaccine, could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2/60) and 95% (57/60) for 2- and 3-dose vaccinees, respectively. For three-dose recipients, the geometric mean neutralization antibody titer (GMT) of Omicron was 15, 16.5-fold lower than that of the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in 3-dose vaccinees, half of which recognize the receptor binding domain (RBD) and show that a subset of them (24/163) neutralize all SARS-CoV-2 variants of concern (VOCs), including Omicron, potently. Therapeutic treatments with representative broadly neutralizing mAbs individually or antibody cocktails were highly protective against SARS-CoV-2 Beta infection in mice. Atomic structures of the Omicron S in complex with three types of all five VOC-reactive antibodies defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to one major class of antibodies bound at the right shoulder of RBD through altering local conformation at the binding interface. Our results rationalize the use of 3-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are a rational target for a universal sarbecovirus vaccine. One sentence summaryA sub-set of antibodies derived from memory B cells of volunteers vaccinated with 3 doses of an inactivated SARS-CoV-2 vaccine work individually as well as synergistically to keep variants, including Omicron, at bay.

microbiology↗

bioRxiv2021

SAVED by a toxin: Structure and function of the CRISPR Lon protease.

CRISPR antiviral defense systems such as the well-known DNA-targeting Cas9- and the more complex RNA-targeting type III systems are widespread in bacteria and archea 1, 2. The type III systems can orchestrate a complex antiviral response that is initiated by the synthesis of cyclic oligoadenylates (cOAs) upon foreign RNA recognition 3-5. These second messenger molecules bind to the CARF (CRISPR associated Rossmann-fold) domains of dedicated effector proteins that are often DNAses, RNAses, or putative transcription factors 6. The activated effectors interfere with cellular pathways of the host, inducing cell death or a dormant state of the cell that is better suited to avoid propagation of the viral attack 7, 8. Among a large set of proteins that were predicted to be linked to the type III systems 9, 10, the CRISPR-Lon protein caught our attention. The protein was predicted to be an integral membrane protein containing a SAVED-instead of a CARF-domain as well as a Lon protease effector domain. Here, we report the crystal structure of CRISPR-Lon. The protein is a soluble monomer and indeed contains a SAVED domain that accommodates cA4. Further, we show that CRISPR-Lon forms a stable complex with the 34 kDa CRISPR-T protein. Upon activation by cA4, CRISPR-Lon specifically cleaves CRISRP-T, releasing CRISPR-T23, a 23 kDa fragment that is structurally very similar to MazF toxins and is likely a sequence specific nuclease. Our results describe the first cOA activated proteolytic enzyme and provide the first example of a SAVED domain connected to a type III CRISPR defense system. The use of a protease as a means to unleash a fast response against a threat has intriguing parallels to eukaryotic innate immunity.

biochemistry↗

bioRxiv2021

pChem: a modification-centric assessment tool for the performance of chemoproteomic probes

Chemoproteomics has emerged as a key technology to expand the functional space in complex proteomes for probing fundamental biology and for discovering new small molecule-based therapies. Here we report a modification-centric computational tool termed pChem to provide a streamlined pipeline for unbiased performance assessment of chemoproteomic probes. The pipeline starts with an experimental setting for isotopically coding probe-derived modifications (PDMs) that can be automatically recognized by pChem, with masses accurately calculated and sites precisely localized. Further, pChem exports on-demand reports by scoring the profiling efficiency, modification-homogeneity and proteome-wide residue selectivity of a tested probe. The performance and robustness of pChem were benchmarked by applying it to eighteen bioorthogonal probes. Of note, the analyses reveal that the formation of unexpected PDMs can be driven by endogenous reactive metabolites (e.g., bioactive aldehydes and glutathione). Together, pChem is a powerful and user-friendly tool that aims to facilitate the development of probes for the ever-growing field of chemoproteomics.

bioinformatics↗

bioRxiv2020

Exploring the microbiome-wide lysine acetylation, succinylation and propionylation in human gut microbiota

BackgroundLysine acylations are important post-translational modifications that are present in both eukaryotes and prokaryotes, regulating diverse cellular functions. Our knowledge of the microbiome lysine acylation remains limited due to the lack of efficient analytical and bioinformatics methods for complex microbial communities. ResultsWe show that serial enrichment using motif antibodies successfully captures peptides containing lysine acetylation, propionylation and succinylation from human gut microbiome samples. A new bioinformatic workflow consisting of unrestricted database search confidently identified >60,000 acetylated, and ~20,000 propionylated and succinylated gut microbial peptides. Characterization of these identified modification-specific metaproteomes, i.e. meta-PTMomes, demonstrates that lysine acylations are differentially distributed in microbial species with different metabolic capabilities. ConclusionThis study provides an analytical framework, consisting of a serial immunoaffinity enrichment and an open database search strategy, for the study of lysine acylations in microbiome, which enables functional microbiome studies at the post-translational level.

microbiology↗

bioRxiv2020

Single-cell analysis identifies TCF4 and ID3 as a molecular switch of mammary epithelial stem cell differentiation.

It is well known that the expansion of the mammary epithelium during the ovarian cycles in female mammals is supported by the transient increase in mammary epithelial stem cells (MaSCs). However, dissecting the molecular mechanisms that govern MaSC function and differentiation is poorly understood due to the lack of standardized methods for their identification and isolation. The development of robust single-cell mRNA sequencing () technologies and the computational methods to analyze them provides us with novel tools to approach the challenge of studying MaSCs in a completely unbiased way without. Here, we have performed the largest scRNA-seq analysis of individual mammary epithelial cells (~70,000 cells). Our study identified a distinct cell population presenting molecular features of MaSCs. Importantly, further purification and additional in-depth single-cell analysis of these cells revealed that they are not a fully homogenous entity. Instead, we identified three subpopulations representing early stages of lineage commitment. By tracking their molecular evolution through single-cell network analysis we found that one of these subpopulations represents bipotent MaSCs from which luminal and basal lineages diverge. Importantly, we also confirmed the presence of these cells in human mammary glands. Finally, through expression and network analysis studies, we have uncovered transcription factors that are activated early during lineage commitment. These data identified E2-2 (Tcf4) and ID3 as a potential molecular switch of mammary epithelial stem cell differentiation.

developmental biology↗

bioRxiv2020

SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damage and constitutes an antiviral target

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of the virus excessively damaging abilities remains unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is sufficient to cause acute respiratory distress syndrome (ARDS)-like damage in vitro and in vivo. Overexpression of 2-E protein induced rapid pyroptosis-like cell death in various susceptible cells and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damage in lung and spleen. Overexpressed 2-E protein formed cation channels in host cell membranes, eventually leading to membrane rupture. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent protective effects against the 2-E-induced damage both in vitro and in vivo. Importantly, their channel inhibition, cell protection and antiviral activities were positively correlated with each other, supporting 2-E is a promising drug target against SARS-CoV-2.

microbiology↗

bioRxiv2018

Open-pFind enables precise, comprehensive and rapid peptide identification in shotgun proteomics

Shotgun proteomics has grown rapidly in recent decades, but a large fraction of tandem mass spectrometry (MS/MS) data in shotgun proteomics are not successfully identified. We have developed a novel database search algorithm, Open-pFind, to efficiently identify peptides even in an ultra-large search space which takes into account unexpected modifications, amino acid mutations, semi- or non-specific digestion and co-eluting peptides. Tested on two metabolically labeled MS/MS datasets, Open-pFind reported 50.5-117.0% more peptide-spectrum matches (PSMs) than the seven other advanced algorithms. More importantly, the Open-pFind results were more credible judged by the verification experiments using stable isotopic labeling. Tested on four additional large-scale datasets, 70-85% of the spectra were confidently identified, and high-quality spectra were nearly completely interpreted by Open-pFind. Further, Open-pFind was over 40 times faster than the other three open search algorithms and 2-3 times faster than three restricted search algorithms. Re-analysis of an entire human proteome dataset consisting of [~]25 million spectra using Open-pFind identified a total of 14,064 proteins encoded by 12,723 genes by requiring at least two uniquely identified peptides. In this search results, Open-pFind also excelled in an independent test for false positives based on the presence or absence of olfactory receptors. Thus, a practical use of the open search strategy has been realized by Open-pFind for the truly global-scale proteomics experiments of today and in the future.

bioinformatics↗