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Chen, W.

Publications and source records attributed to Chen, W..

At least 271 records · Page 15Linked to original sources
bioRxiv2023

Multimodal single cell-resolved spatial proteomics reveals pancreatic tumor heterogeneity

Despite the advances in antibody-guided cell typing and mass spectrometry-based proteomics, their integration is hindered by challenges for processing rare cells in the heterogeneous tissue context. Here, we introduce Spatial and Cell-type Proteomics (SCPro), which combines multiplexed imaging and flow cytometry with ion exchange-based protein aggregation capture technology to characterize spatial proteome heterogeneity with single cell resolution. The SCPro was employed to explore the pancreatic tumor microenvironment and revealed the spatial alternations of over 5,000 proteins by automatically dissecting up to 100 single cells guided by multi-color imaging of centimeter-scale formalin-fixed, paraffin-embedded tissue slide. To enhance cell-type resolution, we characterized the proteome of 14 different cell types by sorting up to 1,000 cells from the same tumor, which allows us to deconvolute the spatial distribution of immune cell subtypes and leads to the discovery of a novel subtype of regulatory T cells. Together, the SCPro provides a multimodal spatial proteomics approach for profiling tissue proteome heterogeneity.

systems biology↗
bioRxiv2023

Robust and Accurate Doublet Detection of Single-Cell Sequencing Data via Maximizing Area Under Precision-Recall Curve

Single-cell sequencing has revolutionized our understanding of cellular heterogeneity by offering detailed profiles of individual cells within diverse specimens. However, due to the limitations of sequencing technology, two or more cells may be captured in the same droplet and share the same barcode. These incidents, termed doublets or multiplets, can lead to artifacts in single-cell data analysis. While explicit experimental design can mitigate these issues with the help of auxiliary cell markers, computationally annotating doublets has a broad impact on analyzing the existing public single-cell data and reduces potential experimental costs. Considering that doublets form only a minor fraction of the total dataset, we argue that current doublet detection methods, primarily focused on optimizing classification accuracy, might be inefficient in performing well on the inherently imbalanced data in the area under the precision-recall curve (AUPRC) metric. To address this, we introduce RADO (Robust and Accurate DOublet detection) - an algorithm designed to annotate doublets by maximizing the AUPRC, effectively tackling the imbalance challenge. Benchmarked on 18 public datasets, RADO outperforms other methods in terms of doublet score and achieves similar performance to the current best methods in doublet calling. Furthermore, beyond its application in single-cell RNA-seq data, we demonstrate RADOs adaptability to single-cell assays for transposase-accessible chromatin sequencing (scATAC-seq) data, where it outperforms other scATAC-seq doublet detection methods. RADOs open-source implementation is available at: https://github.com/poseidonchan/RADO.

bioinformatics↗
bioRxiv2023

Molecular mechanism of NAD+ binding to the Nudix homology domains of DBC1

DBC1 (deleted in breast cancer 1) is a human nuclear protein that modulates the activities of various proteins. NAD+ (oxidized form of nicotinamide adenine dinucleotide) is thought to potentially bind to the Nudix homology domains (NHDs) of DBC1, thereby regulating DBC1-PARP1 [poly (adenosine diphosphate-ribose) polymerase] interactions, the modulation of which may restore DNA repair to protect against cancer, radiation, and aging. Therefore, our study comprehensively employed methods including NMR (Nuclear Magnetic Resonance), ITC (isothermal titration calorimetry), genetic mutation, and computer biology to thoroughly investigate the molecular mechanism of the binding interaction between NAD+ and its precursor NMN with the NHD domain of DBC1 (DBC1354-396). The results from NMR and ITC indicate that NAD+ likely interacts with DBC1354-396 through hydrogen bonding, with a binding affinity nearly twice that of NMN. The key binding sites are primarily E363 and D372. Molecular Docking further revealed the importance of conventional hydrogen bonds and carbon-hydrogen bonds in the binding process. These findings may lead to a better understanding of how NAD+ regulates the physiological functions of DBC1, thereby offering guiding principles for the development of targeted therapies and drug research focused on tumor diseases associated with DBC1.

biochemistry↗
bioRxiv2023

MatK impacts Differential Chloroplast Translation of Ribosomal and Photosynthetic genes by limiting spliced tRNA-K(UUU) abundance

The protein levels of chloroplast photosynthetic genes and genes related to the chloroplast genetic apparatus vary to adapt to different conditions. However, the underlying mechanisms governing these variations remain unclear. The chloroplast intron Maturase K is encoded within the trnK intron and has been suggested to be required for splicing several group IIA introns, including the trnK intron. In this study, we employed RNA immunoprecipitation followed by high-throughput sequencing (RIP-Seq) to identify MatKs preference for binding to group IIA intron domains I and VI within target transcripts. Importantly, these domains are crucial for branch point selection, and we discovered alternative branch points in three MatK target introns, the first observed instances of alternative splicing in chloroplasts. The alternative trnK lariat structure showed increased accumulation during heat acclimation. The cognate codon of tRNA-K(UUU) is highly enriched in mRNAs encoding ribosomal proteins and ribosome profiling in a trnK-matK over-expressor exhibited elevated levels of the spliced tRNA-K(UUU). Our analysis revealed a significant up-shift in the translation of ribosomal proteins compared to photosynthetic genes. Our findings suggest the existence of a novel regulatory mechanism linked to the abundance of tRNA-K(UUU), enabling the differential expression of functional chloroplast gene groups.

plant biology↗
bioRxiv2023

Distinct eLPB ChAT projections for methamphetamine anxiety and relapse

Choline acetyltransferase-positive neurons in the external lateral parabrachial nucleus (eLPBChAT) send projections to PKC{delta}-positive (PKC{delta}+) neurons in lateral portion of central nucleus of amygdala (lCeAPKC{delta}) and oval portion of bed nucleus of the stria terminalis (ovBNSTPKC{delta}), forming eLPBChAT-lCeAPKC{delta} and eLPBChAT-ovBNSTPKC{delta} pathways. At least in part, the eLPBChAT neurons positively innervate lCeAPKC{delta} and ovBNSTPKC{delta} through regulating synaptic elements of presynaptic acetylcholine (Ach) release and postsynaptic nicotinic acetylcholine receptors (nAChRs). Methamphetamine (METH) withdrawal anxiety and METH-primed reinstatement of conditioned place preference (CPP) recruit eLPBChAT-lCeAPKC{delta} pathway and eLPBChAT-ovBNSTPKC{delta} pathway in male METH-exposed mice, respectively.

neuroscience↗
bioRxiv2023

Mechanism and cellular function of direct membrane binding by the ESCRT and ERES-associated Ca2+-sensor ALG-2

Apoptosis Linked Gene-2 (ALG-2) is a multifunctional intracellular Ca2+ sensor and the archetypal member of the penta-EF hand protein family. ALG-2 functions in the repair of damage to both the plasma and lysosome membranes and in COPII-dependent budding at endoplasmic reticulum exit sites (ERES). In the presence of Ca2+, ALG-2 binds to ESCRT-I and ALIX in membrane repair and to SEC31A at ERES. ALG-2 also binds directly to acidic membranes in the presence of Ca2+ by a combination of electrostatic and hydrophobic interactions. By combining GUV-based experiments and molecular dynamics simulations, we show that charge-reversed mutants of ALG-2 at these locations disrupt membrane recruitment. ALG-2 membrane binding mutants have reduced or abrogated ERES localization in response to Thapsigargin-induced Ca2+ release but still localize to lysosomes following lysosomal Ca2+ release. In vitro reconstitution shows that the ALG-2 membrane-binding defect can be rescued by binding to ESCRT-I. These data thus reveal the nature of direct Ca2+-dependent membrane binding and its interplay with Ca2+-dependent protein binding in the cellular functions of ALG-2.

biophysics↗
bioRxiv2023

A molecularly distinct cell type in the dmPAG regulates intermale aggression behaviors in mice

BackgroundThe periaqueductal gray (PAG) is a central hub for regulation of aggression, while little is known on the circuitry and molecular mechanisms that govern this regulation. We investigate the role of a distinct cell type, Tachykinin 2-expressing (Tac2+) neurons, located in the dorsomedial PAG (dmPAG), in modulating aggression in mice. MethodsWe combined activity mapping, in vivo Ca2+ recording, chemogenetic and pharmacological manipulation, and a viral-based translating ribosome affinity purification (TRAP) profiling using a mouse resident-intruder model. ResultsWe reveal that the dmPAGTac2 neurons were selectively activated during fighting behaviors. Activation of the dmPAGTac2 neurons evoked, while inhibition or genetic ablation of the dmPAGTac2 neurons suppressed fighting behaviors. TRAP profiling of dmPAGTac2 neurons revealed that fighting behaviors specifically induced enrichment of serotonin-associated transcripts to the dmPAGTac2 neurons. Last, we validated these findings by selectively delivering pharmacological agent into the dmPAG and reversed the behavioral outcomes induced by chemogenetic manipulation. ConclusionsWe identify that the dmPAGTac2 neuron can regulate mouse aggressive behavior and thus suggest a distinct molecular target for the treatment of exacerbated aggressive behaviors in populations that exhibit high-level of violence. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/562724v3_ufig1.gif" ALT="Figure 1"> View larger version (75K): org.highwire.dtl.DTLVardef@afc876org.highwire.dtl.DTLVardef@91e355org.highwire.dtl.DTLVardef@1e41e43org.highwire.dtl.DTLVardef@17ef3f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗
bioRxiv2023

GRAMD2+ alveolar type I cell plasticity facilitates cell state transitions in organoid culture

Alveolar epithelial regeneration is critical for normal lung function and becomes dysregulated in disease. While alveolar type 2 (AT2) and club cells are known distal lung epithelial progenitors, determining if alveolar epithelial type 1 (AT1) cells also contribute to alveolar regeneration has been hampered by lack of highly specific mouse models labeling AT1 cells. To address this, the Gramd2CreERT2 transgenic strain was generated and crossed to RosamTmG mice. Extensive cellular characterization, including distal lung immunofluorescence and cytospin staining, confirmed that GRAMD2+ AT1 cells are highly enriched for green fluorescent protein (GFP). Interestingly, Gramd2CreERT2 GFP+ cells were able to form organoids in organoid co-culture with Mlg fibroblasts. Temporal scRNAseq revealed that Gramd2+ AT1 cells transition through numerous intermediate lung epithelial cell states including basal, secretory and AT2 cell in organoids while acquiring proliferative capacity. Our results indicate that Gramd2+ AT1 cells are highly plastic suggesting they may contribute to alveolar regeneration.

cell biology↗
bioRxiv2023

Myeloperoxidase activity predicts atherosclerotic plaque disruption and atherothrombosis

BackgroundUnstable atherosclerotic plaque is characterized by increased myeloperoxidase (MPO) activity. As unstable plaque is vulnerable to disruption and ensuing thrombosis, we examined whether plaque MPO activity predicts atherothrombosis in a pre-clinical model and correlates with ruptured human atheroma. MethodsTo assess if plaque MPO activity predicts atherothrombosis, rabbits were subjected to aortic endothelial denudation, cholesterol feeding, in vivo magnetic resonance imaging (MRI) of MPO activity using MPO-Gd (gadolinium), followed by pharmacological triggering of atherothrombosis, histology, and MPO activity determined by liquid chromatography tandem mass spectrometry (LC-MSMS) by quantifying the MPO-specific product of hydroethidine, 2-chloroethidium. To correlate plaque MPO activity to ruptured human atheroma, ex vivo determination of MPO activity by MPO-Gd retention in carotid endarterectomy (CEA) specimens was correlated with in vivo MRI plaque phenotyping in patients, histology, and MPO activity determined by LC-MSMS. ResultsIn rabbits, pre-trigger in vivo MPO activity, validated by LC-MSMS and histology, was higher in thrombosis-prone than thrombosis-resistant plaques and lesion-free segments (R1 relaxation rate = 2.2 {+/-} 0.4 versus 1.6 {+/-} 0.2 and 1.5 {+/-} 0.2 s-1, respectively, p<0.0001), and it predicted atherothrombosis. In CEA specimens, MPO-Gd retention was greater in histologic and MRI-graded American Heart Association (AHA) type VI than types III, IV and V plaques ({Delta}R1 relaxation rate from baseline = 48 {+/-} 6 versus 16 {+/-} 7, 17 {+/-} 8 and 23 {+/-} 8%, respectively, p<0.0001). This association was confirmed by comparing AHA grade to MPO activity determined by LC-MSMS (277 {+/-} 338 versus 7 {+/-} 6, 11 {+/-} 12 and 42 {+/-} 39 pmol 2-chloroethidium/mg protein for type VI versus type III-V plaques, respectively, p=0.0008). ConclusionsMPO activity is elevated in thrombosis-prone rabbit and ruptured human atheroma. Non-invasive molecular imaging of MPO activity predicts atherothrombosis, highlighting the potential of arterial MPO activity to detect vulnerable, destabilized atherosclerosis.

biochemistry↗
bioRxiv2023

Recognition of the Nucleotide-Dependent Feature Facilitates the Microtubule End-Binding of MCAK

The growing plus-end is a key regulatory site for microtubule dynamics. MCAK (mitotic centromere-associated kinesin), a microtubule depolymerizing kinesin, is an end-binding regulator of catastrophe frequency. It is intriguing how MCAK specifically binds to growing microtubule ends. Here, we measure the end-binding kinetics of MCAK using single-molecule imaging, and reveal that MCAK binds to the entire GTP cap, including the region where EB1 preferentially binds and the distalmost tip. Further analysis shows that MCAK strongly binds to GTP{gamma}S microtubules, and this binding preference is dependent on the nucleotide state of MCAK. This finding suggests that MCAK recognizes the nucleotide-dependent feature of microtubules. Moreover, we show that although MCAK and XMAP215 partly share binding regions at the distalmost ends, their regulatory effects can be additive, highlighting a simple rule of how the end-binding regulators coordinate their activities. Overall, our findings provide new insights into how MCAK regulates microtubule end dynamics.

biochemistry↗
bioRxiv2023

Harnessing the endogenous Type I-C CRISPR-Cas system for genome editing in Bifidobacterium breve

Bifidobacterium breve, one of the main bifidobacterial species colonizing the human gastrointestinal tract in early life, has received extensive attention for its purported beneficial effects on human health. However, exploration of the mode of action of such beneficial effects exerted by B. breve is cumbersome due to the lack of effective genetic tools, which limits its synthetic biology application. Given the widespread presence of endogenous CRISPR-Cas systems in B. breve, the current study developed an endogenous CRISPR-based gene editing toolkit for genetic manipulation of B. breve. Deletion of the gene coding uracil phosphoribosyl-transferase (upp) was achieved in two different B. breve strains using this system. In addition, translational termination of uracil phosphoribosyl-transferase was successfully achieved in B. breve FJSWX38M7 by single-base substitution of the upp gene and insertion of three stop codons. The gene encoding linoleic acid isomerase (bbi) in B. breve, being a characteristic trait, was deleted after plasmid curing, which rendered it unable to convert linoleic acid into conjugated linoleic acid, demonstrating the feasibility of successive editing. This study expanded the gene manipulation toolkit of B. breve and provides a reference for functional genome editing and analysis using an endogenous CRISPR-Cas system in Bifidobacterium. ImportanceThe lack of effective genetic tools for Bifidobacterium breve is an obstacle to studying the molecular mechanisms of its health-promoting effects, hindering the development of next-generation probiotics. Here, we introduce a gene editing method based on the endogenous CRISPR-Cas system, which can achieve gene deletion, single base substitution, gene insertion and continuous gene editing in B. breve. This study will promote the excavation of functional genes and elucidation of molecular mechanisms of B. breve.

molecular biology↗
bioRxiv2023

Gut microbial genomes with paired isolates from China signify probiotic and cardiometabolic effects

The gut microbiome displays significant genetic differences between populations while systematic characterization of the genomic landscape of the gut microbiome in Asia populations remains limited. Here, we present the Chinese gut microbial reference (CGMR) set, comprising 101,060 high quality metagenomic assembled genomes (MAGs) of 3,707 non-redundant species paired with 1,376 live isolates from a national wide collection of 3,234 fecal samples across China. This improved reference set contains 987 novel species compared with existing resources worldwide. By associating MAGs with geographic and phenotypic characteristics, we observed regional-specific coexisting MAGs and MAGs with probiotic and cardiometabolic functionalities. We further conducted mice experiments to confirm the probiotic effects of two Faecalibacterium intestinalis isolates in alleviating constipation, the cardiometabolic influences of three Bacteroides fragilis_A isolates in obesity, and the functional potential of isolates from the two new species belonging to the genera Parabacteroides and Lactobacillus in host lipids metabolism. Our study not only expands the current microbial genomes with paired isolates but also demonstrates their probiotic and cardiometabolic effects on hosts, contributing to the mechanistic understanding of host-microbe interactions and the translation of microbiome-based personalized therapies.

microbiology↗
bioRxiv2023

Computational design of sequence-specific DNA-binding proteins

Sequence-specific DNA-binding proteins (DBPs) play critical roles in biology and biotechnology, and there has been considerable interest in the engineering of DBPs with new or altered specificities for genome editing and other applications. While there has been some success in reprogramming naturally occurring DBPs using selection methods, the computational design of new DBPs that recognize arbitrary target sites remains an outstanding challenge. We describe a computational method for the design of small DBPs that recognize specific target sequences through interactions with bases in the major groove, and employ this method in conjunction with experimental screening to generate binders for 5 distinct DNA targets. These binders exhibit specificity closely matching the computational models for the target DNA sequences at as many as 6 base positions and affinities as low as 30-100 nM. The crystal structure of a designed DBP-target site complex is in close agreement with the design model, highlighting the accuracy of the design method. The designed DBPs function in both Escherichia coli and mammalian cells to repress and activate transcription of neighboring genes. Our method is a substantial step towards a general route to small and hence readily deliverable sequence-specific DBPs for gene regulation and editing.

synthetic biology↗
bioRxiv2023

GametesOmics: A Comprehensive Multi-Omics Database for Exploring the Gametogenesis in Humans and Mice

Gametogenesis plays an important role in the reproduction and evolution of species. The transcriptomic and epigenetic alterations in this process can influence the reproductive capacity, fertilization, and embryonic development. The rapidly increasing single-cell studies have provided valuable multi-omics resources. However, data from different layers and sequencing platforms have not been uniformed and integrated, which greatly limits their use for exploring the molecular mechanisms that underlie oogenesis and spermatogenesis. Here, we developed GametesOmics, a comprehensive database that integrated the data of gene expression, DNA methylation, and chromatin accessibility during oogenesis and spermatogenesis in humans and mice. GametesOmics provides a user-friendly website and various tools, including Search and Advanced Search for querying the expression and epigenetic modification of each gene; Analysis Tools with Differentially Expressed Gene (DEG) analysis for identifying DEGs, Correlation analysis for demonstrating the genetic and epigenetic changes, Visualization for displaying single-cell cluster and screening marker genes as well as master transcription factors (TFs), and MethylView for studying the genomic distribution of epigenetic modifications. GametesOmics also provides Genome Browser and Orthologs for tracking and comparing gene expression, DNA methylations, as well as chromatin accessibilities between humans and mice. GametesOmics offers a comprehensive resource for biologists and clinicians to decipher the cell fate transition in germ cell development, and can be accessed at http://gametesomics.cn/.

bioinformatics↗
bioRxiv2023

A Stapled Peptide Inhibitor of METTL3-METTL14 for Cancer Therapy

METTL3, a primary methyltransferase catalyzing RNA N6-methyladenosine (m6A) modification, has been identified as an oncogene in several cancer types and thus nominated as a potentially effective target for therapeutic inhibition, although current options using this strategy are limited. In this study, we targeted protein-protein interactions at the METTL3-METTL14 binding interface to inhibit complex formation and subsequent catalysis of RNA m6A modification. Among candidate peptides, RM3 exhibited the highest anti-cancer potency, inhibiting METTL3 activity while also facilitating its proteasomal degradation. We then designed a stapled peptide inhibitor (RSM3) with enhanced peptide stability and formation of the -helical secondary structure required for METTL3 interaction. Functional and transcriptomic analysis in vivo indicated that RSM3 induced upregulation of programmed cell death-related genes while inhibiting cancer-promoting signals. Furthermore, tumor growth was significantly suppressed while apoptosis was enhanced upon RSM3 treatment, accompanied by in-creased METTL3 degradation, and reduced global RNA methylation levels in two in vivo tumor models. This peptide inhibitor thus exploits a mechanism distinct from other competitive-binding small molecules to inhibit oncogenic METTL3 activity. Our findings collectively highlight the potential of targeting METTL3 in cancer therapies through peptide-based inhibition of complex formation and proteolytic degradation.

biochemistry↗
bioRxiv2023

The A53T mutation in α-synuclein enhances pro-inflammatory activation in human microglia

Parkinsons disease (PD) is characterized by the aggregation of -synuclein into Lewy bodies and Lewy neurites in the brain. Microglia-driven neuroinflammation may contribute to neuronal death in PD, however the exact role of microglia remains unclear and has been understudied. The A53T mutation in the gene coding for -synuclein has been linked to early-onset PD, and exposure to A53T-mutant human -synuclein increases the potential for inflammation of murine microglia. To date, its effect has not been studied in human microglia. Here, we used 2-dimensional cultures of human iPSC-derived microglia and transplantation of these cells into the mouse brain to assess the effects of the A53T mutation on human microglia. We found that A53T-mutant human microglia had an intrinsically increased propensity towards pro-inflammatory activation upon inflammatory stimulus. Additionally, A53T mutant microglia showed a strong decrease in catalase expression in non-inflammatory conditions, and increased oxidative stress. Our results indicate that A53T mutant human microglia display cell-autonomous phenotypes that may worsen neuronal damage in early-onset PD.

neuroscience↗
bioRxiv2023

Population genomic analyses reveal extensive genomic regions within selective sweeps associated with adaptation and demographic history of a wheat fungal pathogen

Plant pathogens can quickly evolve in response to the changing host and environment, resulting in destructive epidemics. The fungus Puccinia striiformis f. sp. tritici (Pst), causing wheat stripe rust disease worldwide, is one such pathogen. In the arms race between pathogens and hosts, changes in host populations exerted continuous selective forces on the dynamics of pathogens. However, the footprints of these selection forces and the demography of Pst remain poorly explored, limited our understanding the evolutionary processes that shape the spread and evolution of pathogens. In this study, we revealed several features of worldwide Pst populations through population genomic analyses. There might be limited gene flow between Pst populations from China and other countries. A slower rate of linkage disequilibrium decay was detected comparing to rust fungi with known sexual reproduction. Furthermore, we detected extensive hard and soft sweeps associated with Pst adaptation. Genes within the selective sweeps were enriched in secreted proteins and effectors and showed functions related to pathogenicity or virulence, temperature tolerance, and fungicide resistance implying that Chinese Pst populations suffered positive selection pressures from host and abiotic factors. Moreover, demographic history indicated Pst populations experienced strong bottlenecks at the beginning of the wheat domestication around 10,000 years ago and during modern agriculture around 100 years ago, suggesting that the crop domestication and breeding programs could continuously contribute to the decline of pathogen effective population sizes. Our results provided insights into the evolution of the Pst genome and highlighted the role of modern agriculture on pathogen demography. Author SummaryWheat stripe rust, as a pathogen of wheat, is capable of inducing significant yield reduction on a global scale. Traditional epidemiological investigations have elucidated the causative agent behind this disease, the wheat stripe rust pathogen, as an organism exhibiting swift adaptive evolution in response to its environment. However, the precise mechanisms underpinning its adaptive strategies and the ensuing alterations in population dynamics remain relatively unexplored. In our research, we have undertaken an analysis encompassing wheat stripe rust samples throughout the world. Employing population genomics methods, we have sought to discern the genomic footprints corresponding to the adaptive process within the pathogen. These genomic footprints have illuminated the operation of positive selection pressures caused by both the host plant and the ecological context. Concurrently, it have provided insights into the population size that the wheat stripe rust pathogen has undergone over millennia. Notably, its change in population size revealed the demographic history associated with wheat domestication.

microbiology↗