Biology subjects

Chen, X.

Publications and source records attributed to Chen, X..

At least 685 records · Page 38Linked to original sources

bioRxiv2024

Secondary microglia formation center in the human fetal brain

Yolk sac-derived microglia migrate and populate the brain during development, constituting 10-15% of the total brain cells. The human brain is the largest and most complex brain with the highest cognitive capacity among all species. Therefore, the limitations of rodent brain studies in interpreting the human brain are evident. By co-immunostaining microglia in 50 {micro}m fetal brain sections from 7.5 to 16 gestational weeks (gw) and combining high-resolution scanning, we identified a highly proliferative microglia aggregate (0.108-2.129 mm2) that expanded in Downs Syndrome fetal brain (4.168 mm2) and was located near the ganglion eminence, in which Ki67+ microglia accounted for 23.4% of total microglia compared to 6.3% in other brain regions. The microglia in the aggregates lack phagocytic bulbs, membrane ruffles, and long/branching processes compared to microglia in other brain regions. Introducing human microglia into cortical organoids, but not macrophages, replicated proliferative microglial aggregates on the brain organoid surface and sufficiently penetrated deeper regions of the cortical organoids. Penetrating microglia display phagocytic capacity, enhance immunity, and accelerate the maturation of brain organoids. The large proliferative microglial aggregate may be a unique secondary microglial formation center in the human fetal brain to compensate for the enormous microglial demands during brain expansion.

neuroscience↗

bioRxiv2024

LRRK2 G2019S mutation suppresses differentiation of Th9 and Treg cells via JAK/STAT3

The Leucine-rich repeat kinase-2 (LRRK2) G2019S mutation, resulting in aberrantly enhanced kinase activity, is one of the well-recognized genetic risk factors in Parkinsons Disease (PD). Increased LRRK2 activity was also observed in immune cells from PD patients. Emerging results have also unveiled an upsurge in -synuclein (-syn)-specific CD4+ T cell responses in PD patients. Given that LRRK2 mutations in PD are germline mutations, there are unmet meets to explore whether LRRK2 G2019S mutation contributes to the pathogenesis of PD via altering CD4+ T-cell functions. To fill this knowledge gap, we generated a new T cell receptor (TCR) transgenic mouse strain bearing LRRK2 G2019S knock-in mutation, OT-II/LRRK2 (Refer to Mut). As CD4+ T cells from OT-II mice specifically recognize ovalbumin, this new strain enables us to explore the impact of LRRK2 G2019S mutation on T-cell functions in an antigen-specific manner. We found that the abundance and proliferation of major immune subsets in spleen tissue from Mut mice are comparable to wild-type (OT-II, Refer to WT) control. However, when we characterized T cell differentiation in these two strains, T cells derived from Mut mice displayed increased Th2 differentiation (IL-4) and decreased Th9 (IL-9) and Treg (Foxp3+ %) differentiation. LRRK2 G2019S mutation significantly altered the expression levels of master transcription factors (TFs) for T cell differentiation. Specifically, Mut T cells displayed an increase in mRNA expression of Gata3 (TF for Th2), a decrease in expression of Irf4 and Foxp3 (TFs for Th9 and Treg, respectively). Mechanistically, LRRK2 mutation decreased IL-9 production and Treg cell population through the JAK/STAT3 signaling. In conclusion, LRRK2 plays a critical role in regulating T cell differentiation, warranting further studies to evaluate the impacts of altered T cell differentiation led by LRRK2 mutation in dopaminergic neuron damages.

immunology↗

bioRxiv2024

Canonical AREs are tumor suppressive regulatory elements in the prostate

The androgen receptor (AR) is the central determinant of prostate tissue identity and differentiation, controlling normal, growth-suppressive prostate-specific gene expression1. It is also a key driver of prostate tumorigenesis, becoming "hijacked" to drive oncogenic transcription2-5. However, the regulatory elements determining the execution of the growth suppressive AR transcriptional program, and whether this can be reactivated in prostate cancer (PCa) cells remains unclear. Canonical androgen response element (ARE) motifs are the classic DNA binding element for AR6. Here, we used a genome-wide strategy to modulate regulatory elements containing AREs to define distinct AR transcriptional programs. We find that activation of these AREs is specifically associated with differentiation and growth suppressive transcription, and this can be reactivated to cause death in AR+ PCa cells. In contrast, repression of AREs is well tolerated by PCa cells, but deleterious to normal prostate cells. Finally, gene expression signatures driven by ARE activity are associated with improved prognosis and luminal phenotypes in human PCa patients. This study demonstrates that canonical AREs are responsible for a normal, growth-suppressive, lineage-specific transcriptional program, that this can be reengaged in PCa cells for potential therapeutic benefit, and genes controlled by this mechanism are clinically relevant in human PCa patients.

cancer biology↗

bioRxiv2024

Stereotyped cell lineage trees support robust development

Multicellular organisms must have robust development to ensure physiological stability in the face of environmental changes or perturbations. While various mechanisms contributing to developmental robustness have been identified at the subcellular level, those at the intercellular and tissue level remain largely unknown. Our study explores this question using an in vitro directed differentiation model of human embryonic stem cells (hESCs) into lung progenitor cells. Integrated analysis of single-cell transcriptomes and high-density cell lineage trees (CLTs) of the same colonies allowed a fine-resolution recapitulation of known cell types, as well as their differentiation hierarchies and developmental trajectories. Most importantly, we observed stable cell type compositions among many sub-CLTs across biological replicates. Systematic comparison among CLTs by a novel computational framework for CLT alignment suggests that stereotypical development extends beyond stable cell type composition to a degree of significant resemblance in sub-CLT topology. The existence of such sub-CLTs resembling each other not only deepens our understanding of developmental robustness by demonstrating the existence of a stereotyped program, but also suggests a novel perspective for the function of specific cell types in the context of stereotyped sub-CLTs.

developmental biology↗

bioRxiv2024

Chlorpromazine inhibits EAG1 channels by altering the coupling between the PAS, CNBH and pore domains

EAG1 depolarization-activated potassium selective channels are important targets for treatment of cancer and neurological disorders. EAG1 channels are formed by a tetrameric subunit assembly with each subunit containing an N-terminal Per-Arnt-Sim (PAS) domain and C-terminal cyclic nucleotide-binding homology (CNBH) domain. The PAS and CNBH domains from adjacent subunits interact and form an intracellular tetrameric ring that regulates the EAG1 channel gating, including the movement of the voltage sensor domain (VSD) from closed to open states. Small molecule ligands can inhibit EAG1 channels by binding to their PAS domains. However, the allosteric pathways of this inhibition are not known. Here we show that chlorpromazine, a PAS domain small molecule binder, alters interactions between the PAS and CNBH domains and decreases the coupling between the intracellular tetrameric ring and the pore of the channel, while having little effect on the coupling between the PAS and VSD domains. In addition, chlorpromazine binding to the PAS domain did not alter Cole-Moore shift characteristic of EAG1 channels, further indicating that chlorpromazine has no effect on VSD movement from the deep closed to opened states. Our study provides a framework for understanding global pathways of EAG1 channel regulation by small molecule PAS domain binders.

biophysics↗

bioRxiv2024

Ilamycins are dual inhibitors of ClpX and ClpC1 involved in proteostasis in Mycobacteria

The mycobacterial caseinolytic protease (Clp) system has been recognized as a promising therapeutic target. In this study, we identified two novel ilamycin analogs, ilamycin E (ILE) and ilamycin F (ILF), both targeting the ClpC1 component of the ClpC1P1P2 proteasome. ILE potently disrupts ClpC1P1P2-mediated proteolysis, leading to delayed bactericidal activity, while ILF also binds ClpC1, albeit with lower affinity. Notably, we discovered a unique mutation in clpX and a novel insertion in clpC1, both conferring resistance to ILE and ILF in mycobacterium, validated by gene editing. Furthermore, ILE can also inhibit the proteolytic activity of ClpXP1P2 in a manner dependent on the substrates tag sequence and adaptor. This first demonstration of clpX- and clpC1-mediated ilamycin resistance underscores the potential of ilamycins to target multiple components of the Clp protease system, offering a novel dual-target strategy for combating mycobacterial infections.

microbiology↗

bioRxiv2024

Arginine Di-methylation of RIPK3 Safeguards Necroptosis for Intestinal Homeostasis

The necroptosis mediated by RIPK3 is stringently regulated for intestinal homeostasis. Here we found that mice lacking Prmt5 (Protein arginase methyltransferase 5) in intestinal epithelial cells (IECs) caused premature death with IECs necroptosis, villus atrophy and loss of Paneth cells. This pathology can be partially rescued by antibiotic treatment, germ-free breeding condition and pharmaceutical inhibition of RIPK1 and RIPK3, but aggravated for embryonic lethality by Caspase-8 deficiency, which demonstrating the importance of commensal bacteria and necroptosis for the Prmt5-IEC deficiency. Intriguingly, tumor-necrosis factor (TNF) receptor 1(Tnfr1) deficiency could not completely rescue the pathology, and mice deficit in Z- DNA binding protein 1(ZBP1) exhibited shorter lifespan compared with Prmt5 null mice, suggesting Prmt5 loss might trigger TNFR-RIPK1-depenfent and ZBP1- dependent necroptosis. Mechanically, we identified the 479-arginine residue of RIPK3 di-methylated by PRMT5 was an endogenous checkpoint for necroptosis. Furthermore, RIPK3-R479K mutation had higher affinity with both RIPK1 and ZBP1 by immunoprecipitation and STORM (Stochastic Optical Reconstruction Microscopy) analysis, which might explain the endogenous necroptosis triggered by mutated RIPK3 even without upstream stimuli. Moreover, the peptide of RIPK3-SDMA (Symmetric dimethylarginine of 479) could rescue lethality of Prmt5 lacking mice through necrosome formation inhibition, which demonstrating the great potential for necroptosis-related disease treatment through RIPK3 dimethylation targeting.

cell biology↗

bioRxiv2024

LXA4 promotes the browning of white adipose through miR-133a-3p/SIRT1 pathway

Lipoxin A4 (LXA4) promotes the browning of white adipose and energy consumption. The specific mechanism of which involved in white adipose browning is less clear. A high-fat diet (HFD) mouse model was constructed. Different groups of mice were treated with LXA4 accordingly. The body weight of mouse, subcutaneous and visceral fat, and food intake were recorded. The effect of LXA4 was examined by observing changes in pathology, serum insulin and lipid accumulation indices. The effects of LXA4/miR-133a-3p/Sirtuin1 on lipid droplet formation, fat browning-related genes, and the insulin receptor-AKT pathway in cells were examined after induction of adipocyte differentiation in 3T3-L1 precursors. At the cellular level, LXA4 promoted lipid droplet formation, expressions of fat browning genes and activation of the insulin receptor-AKT pathway in differentiated 3T3-L1 cells. MiR-133a-3p agomir partially offset the effects of LXA4. SIRT1 was a downstream target gene of miR-133a-3p, participating in the promotive effects of LXA4 on fat browning. LXA4 promotes white adipose browning and relieves insulin resistance through miR-133a-3p/SIRT1 pathway.

biochemistry↗

bioRxiv2024

The Drosophila histone methyl-transferase SET1 coordinates multiple signaling pathways in regulating male germline stem cell maintenance and differentiation

Many cell types come from tissue-specific adult stem cells that maintain the balance between proliferation and differentiation. Here, we study how the H3K4me3 methyltransferase, Set1, regulates early-stage male germ cell proliferation and differentiation in Drosophila. Early-stage germline-specific knockdown of set1 results in a temporally progressed defects, arising as germ cell loss and developing to overpopulated early-stage germ cells. These germline defects also impact the niche architecture and cyst stem cell lineage in a non-cell-autonomous manner. Additionally, wild-type Set1, but not the catalytically inactive Set1, could rescue the set1 knockdown phenotypes, highlighting the functional importance of the methyl-transferase activity of the Set1 enzyme. Further, RNA-seq experiments reveal key signaling pathway components, such as the JAK-STAT pathway gene stat92E and the BMP pathway gene mad, that are upregulated upon set1 knockdown. Genetic interaction assays support the functional relationships between set1 and JAK-STAT or BMP pathways, as mutations of both the stat92E and mad genes suppress the set1 knockdown phenotypes. These findings enhance our understanding of the balance between proliferation and differentiation in an adult stem cell lineage. The germ cell loss followed by over-proliferation phenotypes when inhibiting a histone methyl-transferase raise concerns about using their inhibitors in cancer therapy.

developmental biology↗

bioRxiv2024

Accessible chromatin maps of inflammatory bowel disease intestine nominate cell-type mediators of genetic disease risk

Inflammatory Bowel Disease (IBD) is a chronic autoinflammatory disorder with rising incidence in pediatrics. TNFa inhibition (TNFi) is the first-line biologic therapy in children, but many do not achieve mucosal healing. Identifying which patients will benefit from TNFi and the underlying nonresponse mechanisms is critical. We built a novel resource: whole genome sequencing linked to multiome-seq (single-nuclei transcriptome and chromatin accessibility) of intestinal biopsies from a cohort of children with IBD, whose TNFi response was defined by mucosal healing. Our study uncovers links between IBD genetic risk and TNFi response. First, classifiers integrating genetic data with clinical variables identified the IBD polygenic risk score as a top predictor of TNFi response. Second, multiome-seq analysis implicated IBD risk variants in persistent cytokine signaling in monocytes, macrophage and fibroblasts of nonresponders. These data reveal genetic mechanisms of treatment response in pediatric IBD and suggest alternative therapeutic approaches for TNFi nonresponders.

genomics↗

bioRxiv2024

Direct, bisulfite-free 5mC and 5hmC sequencing at single-cell resolution

We report the first direct sequencing methodologies for quantitative detection of 5mC and 5hmC at single-base resolution and single-cell level, termed scTAPS (for 5mC + 5hmC) and scCAPS+ (for 5hmC specifically). With [~]90% mapping efficiency, our methods accurately benchmark 5mC and 5hmC profiles in CD8+ T and mES cells, respectively. Notably, scCAPS+ revealed a global increase in 5hmC within the hippocampus of aging mice, both in neurons and in non-neurons.

genomics↗

bioRxiv2024

Fourfold increase in photocurrent generation of Synechocystis sp. PCC 6803 by exopolysaccharide deprivation

Photosynthetic microorganisms, including algae and cyanobacteria, export electrons in a light-stimulated phenomenon called exoelectrogenesis. However, the route(s) by which electrons reach an external electrode from the cell remain(s) unclear. For the model cyanobacterium Synechocystis sp. PCC 6803, it has been established that electron transfer does not depend on direct extracellular electron transfer by type IV pili. However, the role of the exopolysaccharide matrix in which cells are embedded has not been investigated. We show that a Synechocystis mutant with substantially reduced exopolysaccharide production has a four-fold greater photocurrent than wild-type cells. This increase is due in part to increased adhesion of exopolysaccharide-deficient cells to electrodes. Stirred system experiments reveal that a substantial portion of the photocurrent depends on an endogenous diffusible electron mediator, supporting indirect extracellular electron transfer as the bioelectrochemical mechanism of exoelectrogenesis. These findings will be important in harnessing exoelectrogenesis for sustainable electricity generation in biophotovoltaic devices.

microbiology↗

bioRxiv2024

Broad de-regulated U2AF1 splicing is prognostic and augments leukemic transformation via protein arginine methyltransferase activation

The role of splicing dysregulation in cancer is underscored by splicing factor mutations; however, its impact in the absence of such rare mutations is poorly understood. To reveal complex patient subtypes and putative regulators of pathogenic splicing in Acute Myeloid Leukemia (AML), we developed a new approach called OncoSplice. Among diverse new subtypes, OncoSplice identified a biphasic poor prognosis signature that partially phenocopies U2AF1-mutant splicing, impacting thousands of genes in over 40% of adult and pediatric AML cases. U2AF1-like splicing co-opted a healthy circadian splicing program, was stable over time and induced a leukemia stem cell (LSC) program. Pharmacological inhibition of the implicated U2AF1-like splicing regulator, PRMT5, rescued leukemia mis-splicing and inhibited leukemic cell growth. Genetic deletion of IRAK4, a common target of U2AF1-like and PRMT5 treated cells, blocked leukemia development in xenograft models and induced differentiation. These analyses reveal a new prognostic alternative-splicing mechanism in malignancy, independent of splicing-factor mutations. Statement of significanceUsing a new in silico strategy we reveal counteracting determinants of patient survival in Acute Myeloid Leukemia that co-opt well-defined mutation-dependent splicing programs. Broad poor-prognosis splicing and leukemia stem cell survival could be rescued through pharmacological inhibition (PRMT5) or target deletion (IRAK4), opening the door for new precision therapies. Competing InterestsConflict-of-interest disclosure: DTS. serves on the scientific advisory board at Kurome Therapeutics; is a consultant for and/or received funding from Kurome Therapeutics, Captor Therapeutics, Treeline Biosciences, and Tolero Therapeutics; and has equity in Kurome Therapeutics. AV has received research funding from GlaxoSmithKline, BMS, Jannsen, Incyte, MedPacto, Celgene, Novartis, Curis, Prelude and Eli Lilly and Company, has received compensation as a scientific advisor to Novartis, Stelexis Therapeutics, Acceleron Pharma, and Celgene, and has equity ownership in Throws Exception and Stelexis Therapeutics.

cancer biology↗

bioRxiv2024

Enhancing Plant Photosynthesis using Carbon Dots as Light Converter and Photosensitizer

Improving photosynthetic efficiency is pivotal for CO2-based biomanufacturing and agriculture purposes. Despite the progress on photosynthetic biohybrids integrating biocatalysts with synthetic materials, nanomaterials with improved optical and photoelectrochemical properties are still needed to increase the energy-conversion efficiency. Here, we present a novel approach using carbon dots (CDs) as both intracellular photosensitizers and light converters for enhancing solar energy utilization in photosynthetic organisms. The CDs were produced from cyanobacterial biomass and used to convert a broad spectrum of solar irradiation to red light. We demonstrated that the nanosized CDs were incorporated into cyanobacterial cells and transferred light-excited electrons into the photosynthetic electron transfer chain. The biohybrids consisting of the CDs and Synechococcus elongatus exhibited increased growth rates, enhanced activities of both photosystems, and accelerated linear electron transport, compared with the cyanobacterial cells only. The supplementation of the CDs increased CO2-fixation rate and CO2-to-glycerol production by 2.4-fold and 2.2-fold, respectively. Furthermore, the CDs were shown to enhance photosynthesis and promote growth of Arabidopsis thaliana. The fresh weight of plant was increased 1.8-fold by CDs addition. These results reveal that simultaneous photosensitization and spectral modification could substantially improve the efficiency of natural photosynthesis. This study presents CDs as an attractive nanomaterial with great application potential in agriculture and solar-powered biomanufacturing.

synthetic biology↗

bioRxiv2024

Large-scale multi-omics analyses identified root-microbiome associations underlying plant nitrogen nutrition

The microbiome determines the performance and fitness of the host plant. Nevertheless, the causal interaction between host genetic variation, gene regulation and the impact of the microbiome on the host phenotype remain unknown. Here we generated 1,341 paired root transcriptome, rhizosphere microbiome and root ionome samples and performed a multi-omics analyses of the host-microbe association at the root-soil interface using 175 rapeseeds (Brassica napus L.) resequenced ecotypes at two field environments. We observed the highest statistically explained variance for root nitrogen uptake among natural ionomic variation by overall transcriptome-wide gene expression and microbial abundance variation. Moreover, we identified significant genome-wide associations for 203 highly heritable amplicon sequence variants (ASVs) at multiple genetic loci regulated by eQTL hotspots associated with nitrogen metabolism components. These associations involved a central bacterial genus (Sphingopyxis), which plays a dominant role on gene regulatory effect on its variation regulated by eQTL hotspots. In addition, we performed high-throughput bacterial cultivation from rapeseed roots and subjected Sphingopyxis to whole genome sequencing. Finally, targeted metabolite profiling and confocal imaging assays demonstrated a host-microbiome regulatory effect on Sphingopyxis established by lateral root development and plant nitrogen nutrition. In summary, our integrative approach reveals the genetic basis of host-microbiome trait associations in the transcriptional, nutritional and environmental domains and suggests that the microbiome might have causal effects on root development with implications towards the breeding of nutrient-efficient crops.

plant biology↗

bioRxiv2024

TMEM63B functions as a mammalian thirst receptor

Thirst drives animals to reinstate water homeostasis by fluid intake. An increase of blood osmolality is thought to induce thirst by activating a thirst receptor expressed in the subfornical organ (SFO), but the molecular identity of this receptor remains elusive. Here, we provide behavioral and functional evidence to show that TMEM63B functions as a mammalian thirst receptor in the SFO and mediates osmotic and dehydrated thirst. First, we showed that TMEM63B is expressed in SFO excitatory neurons and required for the neuronal responses to hypertonic stimulation. Heterologously expressed TMEM63B is activated by hypertonic stimuli and point mutations can alter the reversal potential of the channel. More importantly, purified TMEM63B in liposomes establishes osmolarity-gated currents. Finally, Tmem63b knockout mice have profound deficits in thirst, and deleting TMEM63B within the SFO neurons recapitulated this phenotype. Taken together, these results provide a molecular basis for thirst 82and demonstrate TMEM63B is the long-sought mammalian thirst receptor.

neuroscience↗

bioRxiv2024

Conserved Pseudouridines in Helix 69 of the Ribosome are Important for Ribosome Dynamics in Translation

The widespread distribution of pseudouridine ({Psi}), an isomer of the canonical uridine base, in RNA indicates its functional importance to the cell. In eukaryotes, it is estimated that around 2% of ribosomal RNA nucleotides are pseudouridines, most of which are located in functional regions of the ribosome. Defects in RNA pseudouridylation induce a range of detrimental effects from compromised cellular protein biosynthesis to disease phenotypes in humans. However, genome-wide changes to mRNA translation profiles by ribosomes lacking specific conserved pseudouridines have not been extensively studied. Here, using a new genomic method called 5P-Seq and in vitro biochemistry, we investigated changes in ribosome dynamics and cellular translation profiles upon loss of {Psi}2258 and {Psi}2260 in helix 69, the two most conserved pseudouridines in the ribosomes. We found that inhibiting the formation of these two pseudouridines challenges ribosomes to maintain the correct open reading frame and causes generally faster ribosome dynamics in translation. Furthermore, mutant ribosomes are more prone to pause while translating a subset of GC-rich codons, especially rare codons such as Arg (CGA) and Arg (CGG). Our data suggest the presence of {Psi}2258 and {Psi}2260 contributes to the dynamics of the H69 RNA stem-loop, and helps to maintain functional interactions with the tRNAs as they move within the ribosome. The optimality of this ribosome-tRNA interaction is likely to be more critical for those limited tRNAs that decode rare codons. Consistent with the changes in ribosome dynamics, our data also show that IRES-mediated translation is compromised in the mutant ribosome. These results explain the importance of {Psi}2258 and {Psi}2260 in H69 to maintain cellular fitness. The strong conservation of {Psi}2258 and {Psi}2260 in the ribosomes from bacteria to humans indicates their functional significance in modulating ribosome functions. We anticipate that the identified functions of these covalent modifications will be conserved in other species.

biochemistry↗

bioRxiv2024

Comprehensive genome annotation of the model ciliate Tetrahymena thermophila by in-depth epigenetic and transcriptomic profiling

The ciliate Tetrahymena thermophila is a well-established unicellular model eukaryote, contributing significantly to foundational biological discoveries. Despite its acknowledged importance, current Tetrahymena biology studies face challenges due to gene annotation inaccuracy, particularly the notable absence of untranslated regions (UTRs). To comprehensively annotate the Tetrahymena macronuclear genome, we collected extensive transcriptomic data spanning various cell stages. To ascertain transcript orientation and transcription start/end sites, we incorporated data of epigenetic marks displaying enrichment towards the 5 end of gene bodies, including H3 lysine 4 tri-methylation (H3K4me3), H2A.Z, nucleosomes, and N6-methyldeoxyadenine (6mA). Additionally, we integrated Nanopore direct sequencing (DRS), strand-specific RNA-seq, and ATAC-seq data. Using a newly-developed bioinformatic pipeline, coupled with manual curation and experimental validation, our work yielded substantial improvements to the current gene models, including the addition of 2,481 new genes, updates to 6,257 existing genes, and the incorporation of 5,917 alternatively spliced isoforms. Furthermore, novel UTR information was annotated for 26,223 high-confidence genes. Intriguingly, 16% of protein-coding genes were identified to have natural antisense transcripts (NATs) characterized by high diversity in alternative splicing, thus offering insights into understanding transcriptional regulation. Our work will enhance the utility of Tetrahymena as a robust genetic toolkit for advancing biological research.

bioinformatics↗