Biology subjects

Huo, X.

Publications and source records attributed to Huo, X..

14 recordsLinked to original sources

bioRxiv2026

Stepwise reorganization of chromosome conformation and nuclear organization during stem cell differentiation

Nuclear organization is a fundamental feature of cell identity and cell fate determination. Compared with differentiated cells, pluripotent stem cells exhibit markedly distinct nuclear architecture at multiple levels, including chromosome folding, lamina association, histone modification landscapes and nuclear body organization. Although individual aspects of this reorganization have been characterized, how and in what order these features are remodeled as cells commit to a lineage remains poorly understood. Here, we temporally map nuclear reorganization across five stages of an in vitro differentiation system of human embryonic stem cells through definitive endoderm and hepatoblast intermediates into hepatocyte-like cells. By integrating Hi-C, RNA-seq, ATAC-seq, ChIP-seq, and CUT&RUN data, we establish a genome-wide framework linking structural, epigenetic, and transcriptional changes across differentiation. Combined with immunofluorescence imaging, chromosome painting, and liquid chromatin Hi-C (LC-Hi-C) we show that nuclear reorganization proceeds in a stepwise and temporally ordered manner through three major transitions. In the first transition, chromosomes condense into defined territories concurrent with transient anchoring of centromere-proximal regions to the nuclear periphery. In a second transition, active and inactive chromatin segregate, leading to stronger compartmentalization. This coincides with deposition and peripheralization of H3K9me2-marked chromatin and morphological changes in nuclear speckles, while chromatin conformation at speckle-associated regions stabilizes. In a third transition, after lineage-specific genes are activated, chromatin interactions globally stabilize, establishing a more stable nuclear architecture on top of these earlier large-scale structural rearrangements. Together, our results define a stepwise framework for nuclear reorganization during human embryonic stem cell differentiation and reveal that the transition from pluripotency to a differentiated state proceeds through coordinated, temporally ordered structural events.

genomics↗

bioRxiv2026

Gymnosperm FLS2 orthologues mediate conserved flg22 perception and divergent responses to flg22 variants

O_LIWhether gymnosperm FLAGELLIN-SENSING 2 (FLS2) orthologues are functional receptors and whether their flg22-recognition spectra have already diversified remain unclear, despite the central role of FLS2 in flagellin perception in angiosperms. C_LIO_LIHere, we identified two gymnosperm FLS2 orthologues, GbFLS2 from Ginkgo biloba and PtFLS2 from Pinus tabuliformis, and analysed their function using transient and stable expression in the Nicotiana benthamiana fls2 mutant, in planta cross-linking assays, AlphaFold3 modelling and structure-guided mutagenesis. C_LIO_LIBoth receptors restored flg22Pst-triggered reactive oxygen species production and MAPK activation, physically associated with flg22Pst, and required conserved residues for flg22Pst recognition. In stable transgenic plants, both receptors mediated flg22Pst-triggered PTI outputs and flg22Pst-induced resistance to Pseudomonas syringae. PtFLS2 additionally mediated responsiveness to flg22Rso and enhanced resistance to Ralstonia solanacearum, whereas GbFLS2 retained a typical flg22Pst-recognition profile. C_LIO_LIThese findings provide direct evidence that gymnosperm FLS2 orthologues can function as bona fide flagellin receptors in a heterologous angiosperm background, and further suggest that diversification in flg22 recognition had already emerged within gymnosperms. C_LI

plant biology↗

bioRxiv2025

Modeling Rotational Fluoroquinolone Therapy as a Novel Treatment for Ophthalmic MRSA Infections

Methicillin-resistant Staphylococcus aureus (MRSA) increasingly undermines the effectiveness of topical fluoroquinolone monotherapy in ophthalmic infections. We present a theoretical evaluation of rotational fluoroquinolone therapy, in which moxifloxacin and trovafloxacin are alternated to enhance bacterial suppression and mitigate resistance. Experimental time-kill data were fit using Bayesian parameter estimation via Markov Chain Monte Carlo (MCMC) to derive drug-specific growth and kill parameters, which were integrated into spatiotemporal pharmacodynamic models. These models incorporate radial anterior chamber geometry, intraocular diffusion, and aqueous humor pharmacokinetics governed by a circadian clearance function. Structural docking simulations of topoisomerase IV mutants reveal that common resistance mutations disrupt moxifloxacin binding while preserving trovafloxacin affinity, supporting a collateral-sensitivity mechanism. Under high-resistance conditions, rotational therapy improves bacterial clearance compared to monotherapy by increasing heterogeneity in antimicrobial exposure fronts and reducing overall bacterial burden. By alternating fluoroquinolones with complementary resistance profiles, rotational therapy leverages physiological clearance rhythms and spatial drug gradients to enhance anterior-segment efficacy, providing a quantitative foundation for its use as a rational alternative to monotherapy in ocular MRSA management.

microbiology↗

bioRxiv2025

Specific Pathogen Free Ten Gene-Edited Pig Donor for Xenotransplantation

Xenotransplantation has entered the clinical phase to fulfill the global organ shortage. However, recent clinical studies revealed that the xenograft from current gene-edited (GE) pigs still poses the risk of immune rejection, and biosafety concerns. In this study, we successfully constructed a large batch of 10- (GTKO/CMAHKO/ {beta}4GalNT2KO/hCD46/hCD55/hCD59/hTBM/hEPCR/hCD39/hCD47) GE cloned (GEC) donor pigs by utilizing gene editing and somatic cell cloning technology, and successfully obtained F1 generation. Phenotypic characterization of 10-GEC pigs showed the deletion of three xenoantigens along with expression of seven human transgenes in various tissues. Digital droplet polymerase chain reaction, and whole genome sequencing indicated 2 copies of hCD46/hCD55/hCD59/hTBM/hCD39 and 1 copy of hEPCR/hCD47 in pig genome without significant off-target and damage to the porcine own functional genes. The validation results showed that 10-GEC pigs effectively inhibited the attacks of human antibodies, complement, and macrophages on porcine endothelial cells and alleviated the coagulation abnormalities between pigs and humans. 10-GEC pigs were negative for all zoonotic pathogens (48) including cytomegalovirus, except streptococcal infections. Kidney, heart, and liver xenografts from these 10-GE pigs were transplanted to non-human primates (NHP), which started working normally without hyperacute rejection. Among them, the heart and liver transplant recipient died without resuscitation due to unexpected interruption of oxygen supply, while the 2 kidney transplant recipients survived for 23 and 16 days, respectively. Pathological analysis showed that 10-GE pig kidney xenografts showed mesenchymal congestion, and fibrosis, cellular hyperplasia, with minor antibody and complement deposition, and significantly reduced the infiltration of CD68+ macrophage. In summary, we successfully produced a group of specific pathogen free GEC donor pigs that effectively mitigated immune rejection upon multi-organ transplantation to NHP.

immunology↗

bioRxiv2024

Production of four-gene (GTKO/hCD55/hTBM/hCD39)-edited donor pigs and kidney xenotransplantation

BackgroundThe number of multigene-modified donor pigs for xenotransplantation is increasing with the advent of gene editing technologies. However, which gene combination is suitable for which organ transplantation remains unclear. MethodsIn this study, we utilized CRISPR/Cas9 gene editing technology, PiggyBac transposon system and somatic cell cloning to construct GTKO/hCD55/hTBM/hCD39 four-gene-edited cloned (GEC) pigs and performed kidney transplantation from pig to rhesus monkey to evaluate the effectiveness of these GEC pigs. ResultsFirst, 107 cell colonies were obtained through drug selection, of which 7 were 4-GE colonies. Two colonies were selected for somatic cell nuclear transfer, resulting in 7 fetuses, of which 4 were GGTA1 biallelic knockout. Both fetuses had higher expression of hCD55, hTBM and hCD39. Therefore, these two fetuses were selected for two consecutive rounds of cloning, resulting in a total of 97 live piglets. After phenotype identification, the GGTA1 gene of these pigs was inactivated, and hCD55, hTBM and hCD39 were expressed in cells and multiple tissues. Furthermore, the numbers of monkey IgM and IgG binding to the peripheral blood mononuclear cells (PBMCs) of the 4-GEC pigs were markedly reduced. Moreover, 4-GEC porcine PBMCs had greater survival rates than those from wild-type pigs through complement-mediated cytolysis assays. In pig-to-monkey kidney xenotransplantation, the kidney xenograft successfully survived for 11 days. All physiological and biochemical indicators were normal, and no hyperacute rejection or coagulation abnormalities were found after transplantation. ConclusionThese results indicate that the GTKO/hCD55/hTBM/hCD39 four-gene modification effectively alleviates immune rejection, and the pig kidney can functionally support the recipient monkeys life.

genetics↗

bioRxiv2024

Production and functional verification of 8-gene (GGTA1, CMAH, β4GalNT2, hCD46, hCD55, hCD59, hTBM, hCD39)-edited donor pigs for xenotransplantation

Gene-edited pig-to-human xenotransplantation continues to make breakthroughs and is expected to enter clinic to solve the global shortage of donor organs. However, which gene combination is suitable for which organ transplantation remains unclear. In this study, we utilized CRISPR/Cas9 gene editing technology, PiggyBac transposon system and somatic cell cloning to construct GTKO/CMAHKO/{beta}4GalNT2KO/hCD46/hCD55/hCD59/hCD39/hTBM 8 gene-edited cloned (GEC) donor pigs, and performed pig to non-human primate (NHP) transplantation to evaluate the effectiveness of these GEC pigs. The multiple vectors were co-transfected into fetal fibroblasts of Diannan miniature pig with O blood type, and 25 colonies were screened out, and one of them carried GGTA1, CMAH and {beta}4GalNT2 biallelic knockout and integration of hCD46, hCD55, hCD59, hTBM and hCD39 genes, which was used as a donor cell for cloning, and a 33-day-old viable fetus was obtained. The fetus was identified and confirmed for normal karyotype and the absence of three xenogeneic antigens -Gal, Neu5Gc and Sda, and expression of hCD46, hCD55, hCD59, hTBM and hCD39 genes, then the recloning was carried out and 28 cloned piglets were obtained by natural delivery. Molecular identification at DNA, mRNA and protein levels showed that 8 gene editing (GE) was successful in these GEC piglets. Moreover, antigen-antibody binding assay and complement-dependent cytotoxicity assay demonstrated that 8GE effectively reduced the immune incompatibility and kidney xenograft survived up to 15 and 17 days into two NHPs, respectively. During this period, the recipient serum antibodies IgA and IgM, complements C3 and C4, coagulation indicators PT, APTT, TT and FIB, as well as most electrolytes and liver function indicators remained relatively stable. The 24-hour urine output and serum creatinine remained normal at a period of post-transplantation. These results indicated that the 8GEC pigs effectively alleviated immune rejection and exerted life-supporting kidney function in the recipient.

genetics↗

bioRxiv2023

Engineering transposon-associated TnpB-1 ωRNA system for efficient gene editing and disease treatment in mouse

Transposon-associated ribonucleoprotein TnpB is known to be the ancestry endonuclease of diverse Cas12 effector proteins from type-V CRISPR system. Given its small size (409 aa), it is of interest to examine whether engineered TnpB could be used for efficient mammalian genome editing. Here, we showed that the gene editing activity of native TnpB in mouse embryos was already higher than previously identified small-sized Cas12f1. Further stepwise engineering of noncoding RNA ({omega}RNA or reRNA) component of TnpB significantly elevated the nuclease activity of TnpB. Notably, an optimized TnpB-{omega}RNA system could be efficiently delivered in vivo with single adeno-associated virus (AAV) and prevented the disease phenotype in a tyrosinaemia mouse model. Thus, the engineered miniature TnpB system represents a new addition to the current genome editing toolbox, with the unique feature of the smallest effector size that facilitate efficient AAV delivery for editing of cells and tissues.

molecular biology↗

bioRxiv2022

Meta-analysis of fecal metagenomes reveals global viral signatures and its diagnostic potential for colorectal cancer and adenoma

IntroductionGut microbiome plays an important role in maintaining human health. Although mounting evidence has revealed the critical function of the gut bacteriome in the progression of CRC, the contribution of gut viral community to CRC is rarely studied. ObjectivesThe present study aimed to reveal the gut virome signatures of colorectal adenoma patients and CRC patients and decipher the potential viral markers to build clinical predictive models for diagnosis. Methods1,282 available fecal metagenomes data from 9 published CRC studies were collected. A new virus database was constructed based on a reference-independent virome approach for further analysis. Viral markers were filtered by statistical methods and used to build machine learning models such as Random Forest and Least Absolute Shrinkage and Selection Operator (LASSO) to distinguish patients from controls. New fecal samples were collected to validate the generalization of predictive model. ResultsThe gut viral composition of CRC patients was drastically altered compared with healthy, as evidenced by changes in several Siphoviridae viruses and a reduction of Microviridae, whereas the virome variation in adenoma patients was relatively low. The viral markers contained the phages of Porphyromonas, Fusobacterium, Hungatella, and Ruminococcaceae. In 9 cohorts and independent validation cohorts, a random forest (RF) classifier and LASSO model got the optimal AUC 0.830 and 0.906, respectively. While the gut virome analysis of adenoma patients identified 88 differential viruses and achieved an optimal AUC of 0.772 for discriminating patients from controls. ConclusionOur findings demonstrate the distinctly different composition of gut virome between healthy controls and CRC patients, and highlight the potential of viral markers for clinical diagnosis.

microbiology↗

bioRxiv2022

The Insulin receptor regulates the persistence of mechanical nociceptive sensitization in flies and mice

Early phase diabetes is often accompanied by pain sensitization. In the fruit fly Drosophila, the insulin receptor (InR) regulates the persistence of injury-induced thermal nociceptive sensitization. Whether Drosophila InR also regulates the persistence of mechanical nociceptive sensitization remains unclear. Mice with a sensory neuron deletion of the gene encoding the Insulin receptor (Insr) show normal nociceptive baselines, however, it is not known whether deletion of Insr in nociceptive sensory neurons leads to persistent nociceptive hypersensitivity in an inflammatory pain paradigm. In this study, we used fly and mouse nociceptive sensitization models to address these questions. In flies, InR mutants and larvae with sensory neuron-specific expression of RNAi transgenes targeting InR exhibited persistent mechanical hypersensitivity, as previously observed for the thermal sensory modality. Mice with a specific deletion of the Insr gene in NaV1.8+ nociceptive sensory neurons showed normal nociceptive thermal and mechanical baselines similar to controls. In an inflammatory paradigm, however, these mutant mice showed persistent mechanical (but not thermal) hypersensitivity, particularly in female mice. Mice with the NaV1.8+ sensory neuron specific deletion of Insr did not show metabolic abnormalities that would be typical of a systemic defect in insulin signaling. Our results show that some aspects of the regulation of nociceptive hypersensitivity by the Insulin receptor are shared between flies and mice and that this regulation is likely independent of metabolic effects.

neuroscience↗

bioRxiv2020

Coordination of two enhancers drives expression of olfactory trace amine-associated receptors

Olfactory sensory neurons (OSNs) are functionally defined by their expression of a unique odorant receptor (OR). Mechanisms underlying singular OR expression are well studied, and involve a massive cross-chromosomal enhancer interaction network. Trace amine-associated receptors (TAARs) form a distinct family of olfactory receptors, and here we find that mechanisms regulating Taar gene choice display many unique features. The epigenetic signature of Taar genes in TAAR OSNs is different from that in OR OSNs. We further identify that two TAAR enhancers conserved across placental mammals are absolutely required for expression of the entire Taar gene repertoire. Deletion of either enhancer dramatically decreases the expression probabilities of different Taar genes, while deletion of both enhancers completely eliminates the TAAR OSN populations. In addition, both of the enhancers are sufficient to drive transgene expression in the partially overlapped TAAR OSNs. We also show that the TAAR enhancers operate in cis to regulate Taar gene expression. Our findings reveal a coordinated control of Taar gene choice in OSNs by two remote enhancers, and provide an excellent model to study molecular mechanisms underlying formation of an olfactory subsystem.

neuroscience↗

bioRxiv2020

G9a/GLP-Sensitivity of H3K9me2 Demarcates Two Types of Genomic Compartments

In the nucleus, chromatin is folded into hierarchical architecture that is tightly linked to various nuclear functions. However, the underlying molecular mechanisms that confer these architectures remain incompletely understood. Here, we investigated the functional roles of H3 lysine 9 dimethylation (H3K9me2), one of the abundant histone modifications, in three-dimensional (3D) genome organization. Unlike mouse embryonic stem cells (mESCs), inhibition of methyltransferases G9a and GLP in differentiated cells eliminated H3K9me2 predominantly at A-type (active) genomic compartments, and the level of residual H3K9me2 modification was strongly associated with genomic compartments in differentiated cells. Furthermore, chemical inhibition of G9a/GLP in mouse hepatocytes led to the decreased chromatin-nuclear lamina interactions mainly at G9a/GLP sensitive regions (GSRs), the increased degree of genomic compartmentalization, and the up-regulation of hundreds of genes that were associated with alterations of the 3D chromatin. Collectively, our data demonstrated essential roles of H3K9me2 in 3D genome organization.

genomics↗

bioRxiv2019

PI16 is a non-neuronal regulator of neuropathic pain

Chronic pain is a major clinical problem of which the mechanisms are incompletely understood. Here we describe the concept that PI16, a protein of unknown function mainly produced by fibroblasts, controls neuropathic pain. The spared nerve injury model of neuropathic pain increases PI16 protein levels in fibroblasts in DRG meninges and in the epi/perineurium of the sciatic nerve. We did not detect PI16 expression in neurons or glia in spinal cord, DRG and nerve. Mice deficient in PI16 are protected against neuropathic pain. In vitro, PI16 promotes trans-endothelial leukocyte migration. In vivo, PI16-/- mice show reduced endothelial barrier permeability, lower leukocyte infiltration and reduced activation of the endothelial barrier regulator MLCK and reduced phosphorylation of its substrate MLC2 in response to SNI. In summary, our findings support a model in which PI16 promotes neuropathic pain by mediating a cross talk between fibroblasts and the endothelial barrier leading to barrier opening, cellular influx and increased pain. Its key role in pain and its limited cellular and tissue distribution makes PI16 an attractive target for pain management.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=128 HEIGHT=200 SRC=\"FIGDIR/small/696542v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (22K):\norg.highwire.dtl.DTLVardef@172f530org.highwire.dtl.DTLVardef@3f62a8org.highwire.dtl.DTLVardef@16606ccorg.highwire.dtl.DTLVardef@1485156_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2018

Susceptibility of Spotted Doves (Streptopelia chinensis) to Experimental Infection with the SFTS Phlebovirus

BackgroundSevere fever with thrombocytopenia syndrome virus (SFTSV), an emerging human pathogen naturally transmitted by ticks, has spread widely during the last few years. Although SFTSV has been detected in wild birds, the natural reservoir and amplifying hosts for the virus have not been well-studied.\n\nMethodology/Principle FindingsHere we report an experimental infection of spotted doves (Streptopelia chinensis) with two strains of SFTSV, JS2010-14 (hereafter JS2010), a Chinese lineage strain and JS2014-16 (JS2014) from a Japanese lineage, which represent the main viral genotypes currently circulating in East Asia. We determined that spotted doves were susceptible to SFTSV and the severity of the viremia was dose-dependent. When challenged with 107 and 105 PFU, all doves developed viremia which peaked 3-5 days post-infection (dpi). A subset (25-62.5%) of the birds challenged at 103 PFU, developed viremia. Virulence of SFTSV in spotted doves appeared to be strain-dependent. Infection with the strain of JS2014 led to a death rate of 12.5% and higher viremia titers in experimentally inoculated birds. The doves inoculated with the JS2010 strain survived infection with relatively lower virus titers in the blood.\n\nConclusions/SignificanceOur results suggest that spotted doves, one of the most abundant bird species in China, could be a competent amplifying host of SFTSV, the strain of the Japanese lineage in particular, with higher viremia titers and play an important role in the transmission of SFTSV. Our observations shed light on the ecology of SFTSV which could benefit the implementation of future surveillance and control programs.\n\nAuthor SummarySevere fever with thrombocytopenia syndrome virus (SFTSV), an emerging human pathogen naturally transmitted by ticks. Our recent study have showed that some species of migratory birds, such as swan geese and spotted doves, could be parasitized by H. longicornis, and antibodies against the virus could also be determined in these birds, which showed that migratory birds could be infected by SFTSV naturally. Other studies have reported that migratory bird routes and the distribution of H. longicornis in East Asia overlap with the geographic distribution of SFTSV. Migratory birds are known to be carriers and transmitters of infectious agents, like the causative agents of influenza, West Nile encephalitis, and Lyme disease. Wild birds often travel long distances carrying various parasites, including ticks, which may be infected with viruses and bacteria. It is therefore reasonable to hypothesize that migratory birds may have played an important role in dispersing H. longicornis-borne SFTSV in both scenarios, either the birds are infected directly with the virus or the birds are carriers of parasitic ticks that are infected with the virus. Here we report an experimental infection of spotted doves (Streptopelia chinensis) with two strains of SFTSV, JS2010-14 (hereafter JS2010), a Chinese lineage strain and JS2014-16 (JS2014) from a Japanese lineage, which represent the main viral genotypes currently circulating in East Asia. We determined that spotted doves were susceptible to SFTSV and the severity of the viremia was dose-dependent.\n\nInterestingly, virulence of SFTSV in spotted doves appeared to be strain-dependent. Infection with the strain of JS2014 led to a death rate of 12.5% and higher viremia titers in experimentally inoculated birds. The doves inoculated with the JS2010 strain survived infection with relatively lower virus titers in the blood. These findings provide novel insights for understanding the rapid spread of the virus in a short time span, especially the SFTSV strains from the Japanese lineage (genotype E), which presented cross ocean transmission.

microbiology↗

bioRxiv2017

Dr.seq2: A Quality Control And Analysis Pipeline For Parallel Single Cell Transcriptome And Epigenome Data

An increasing number of single cell transcriptome and epigenome technologies, including single cell ATAC-seq (scATAC-seq), have been recently developed as powerful tools to analyze the features of many individual cells simultaneously. However, the methods and software were designed for one certain data type and only for single cell transcriptome data. A systematic approach for epigenome data and multiple types of transcriptome data is needed to control data quality and to perform cell-to-cell heterogeneity analysis on these ultra-high-dimensional transcriptome and epigenome datasets. Here we developed Dr.seq2, a Quality Control (QC) and analysis pipeline for multiple types of single cell transcriptome and epigenome data, including scATAC-seq and Drop-ChIP data. Application of this pipeline provides four groups of QC measurements and different analyses, including cell heterogeneity analysis. Dr.seq2 produced reliable results on published single cell transcriptome and epigenome datasets. Overall, Dr.seq2 is a systematic and comprehensive QC and analysis pipeline designed for parallel single cell transcriptome and epigenome data. Dr.seq2 is freely available at: http://www.tongji.edu.cn/~zhanglab/drseq2/ and https://github.com/ChengchenZhao/DrSeq2.

bioinformatics↗