Biology subjects

Wen, L.

Publications and source records attributed to Wen, L..

31 records · Page 2Linked to original sources

bioRxiv2023

Identification of a carbohydrate-recognition motif of purinergic receptors

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

molecular biology↗

bioRxiv2023

GenBank genomics highlight the genomic features, genetic diversity and regulation of morphological, metabolic and disease-resistance traits in Nicotiana tabacum

Nicotiana tabacum is a model organism in plant molecular and pathogenic research and has significant potential in the production of biofuels and active pharmaceutical compounds in synthetic biology. Because of the large allotetraploid genome of tobacco, its genomic features, genetic diversity and genetic regulation of many complex traits remain unknown. In this study, we present a nearly complete chromosome-scale assembly of N. tabacum and provide evidence that homoeologous exchange between subgenomes and epigenetic remodelling are likely mechanisms of genome stabilization and subgenome coordination following polyploidization. By leveraging GenBank-scale sequencing and phenotyping data from 5196 lines, geography at the continent scale, rather than types assigned on the basis of curing crop practices, was found to be the most important correlate of genetic structure. Using 178 marker{square}trait associations detected in genome-wide association analysis, a reference genotype-to-phenotype map was built for 39 morphological, developmental, and disease-resistance traits. A novel gene, auxin response factor 9 (Arf9), associated with wider leaves after being knocked out, was fine-mapped to a single nucleotide polymorphism (SNP). This point mutation alters the translated amino acid from Ala203 to Pro203, likely preventing homodimer formation during DNA binding. Our analysis also revealed signatures of positive and polygenic selection for multiple traits during the process of selective breeding. Overall, this study demonstrated the power of leveraging GenBank genomics to gain insights into the genomic features, genetic diversity, and regulation of complex traits in N. tabacum, laying a foundation for future research on plant functional genomics, crop breeding, and the production of biopharmaceuticals and biofuels.

genetics↗

bioRxiv2022

Nuclear speckle-localized RNAs exhibit preferential positioning and orientation

RNA molecules often play critical roles in assisting the formation of membraneless organelles in eukaryotic cells. Yet, little is known about the organization of RNAs within membraneless organelles. Here, using super-resolution imaging and nuclear speckles as a model system, we demonstrate that different sequence domains of RNA transcripts exhibit differential spatial distributions within speckles. Specifically, we image transcripts containing a region enriched in SR protein binding motifs and another region enriched in hnRNP binding motifs. We show that these transcripts localize to the outer shell of speckles, with the SR motif-rich region localized closer to the speckle center relative to the hnRNP motif-rich region. Further, we identify that this intra-speckle RNA organization is driven by the strength of RNA-protein interactions inside and outside speckles. Our results hint at novel functional roles of nuclear speckles and likely other membraneless organelles in organizing RNA substrates for biochemical reactions.

biophysics↗

bioRxiv2022

IRE-1α is a key switch of pyroptosis and necroptosis in mice by dominating Gasdermin D

Necroptosis and pyroptosis are lytic and inflammatory types of programmed cell death that require the membrane destruction predominantly driven by the mixed lineage kinase domain-like (MLKL) and gasdermin D (GSDMD) proteins, respectively. However, the crosstalk between them remains largely unknown. Here, our research discloses that endoplasmic reticulumn transmembrane protein inositol-requiring enzyme-1 (IRE-1) is a potential modulator of both necroptosis and pyroptosis, paricularly in liver pathology. Interestingly, enhanced expression of IRE-1 triggers hepatic pyroptosis, while defective IRE-1 level activates hepatic necroptosis, and both processes are closed related to the activity of GSDMD in mice. To elucidate unknown crosstalk, by using pharmacological and genetic methods, we first demonstrated that IRE-1 suppresses necroptosis by promoting the expression of GSDMD and cleaves caspase-8 and by inhibiting the expression of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3 and MLKL. Unexpectedly, excess IRE-1 initiates pyroptosis by increasing GSDMD and NLRP3 levels. Our work clearly provides insight into the modulation of IRE-1 to dominate necroptosis and pyroptosis and suggests that IRE-1 may be a promising therapeutic target for drug discovery in both types of tissue injuries.

cell biology↗

bioRxiv2021

Tomato SlBES1.8 influences leaf morphogenesis by mediating gibberellin metabolism and signaling

Leaf morphogenetic activity determines its shape diversity. However, our knowledge to the regulatory mechanism in maintaining leaf morphogenetic capacity is still limited. In tomato, gibberellin (GA) negatively regulates leaf complexity by shortening the morphogenetic window. We here reported a tomato BRI1-EMS-SUPPRESSOR 1 (BES1) transcription factor, SlBES1.8, that promoted the simplification of leaf pattern in a similar manner as GA functions. Enhanced level of SlBES1.8 dramatically decreased the sensibility of tomato to GA whereas increased the sensibility to the GA biosynthesis inhibitor, PAC. In line with the phenotypic observation, the endogenous bioactive GA contents were increased in OE-SlBES1.8 lines, which certainly promoted the degradation of the GA signaling negative regulator, SlDELLA. Moreover, transcriptomic analysis uncovered a set of overlapping genomic targets of SlBES1.8 and GA, and most of them were regulated in the same way. Expression studies showed the repression of SlBES1.8 to the transcriptions of two GA deactivated genes, SlGA2ox2 and SlGA2ox6, and one GA receptor, SlGID1b-1. Further experiments confirmed the direct regulation of SlBES1.8 to their promoters. On the other hand, SlDELLA physically interacted with SlBES1.8 and further inhibited its transcriptional regulation activity by abolishing SlBES1.8-DNA binding. Conclusively, by mediating GA deactivation and signaling, SlBES1.8 greatly influenced tomato leaf morphogenesis. HighlightThe BRI1-EMS-SUPPRESSOR 1 (BES1) family protein SlBES1.8 promotes leaf simplification by inhibiting gibberellin deactivation and physically interacting with DELLA protein in tomato.

plant biology↗

bioRxiv2021

Carbonyl Post-Translational Modification Associated with Early Onset Type 1 Diabetes Autoimmunity

Inflammation and oxidative stress in pancreatic islets amplify the appearance of various post-translational modifications (PTMs) to self-proteins. Herein, we identified a select group of carbonylated islet proteins arising before the onset of hyperglycemia in non-obese diabetic (NOD) mice. Of particular interest, we identified carbonyl modification of the prolyl-4-hydroxylase beta subunit (P4Hb) that is responsible for proinsulin folding and trafficking as an autoantigen in both human and murine type 1 diabetes. We found the carbonylated-P4Hb is amplified in stressed islets coincident with decreased glucose-stimulated insulin secretion and altered proinsulin to insulin ratios. Moreover, circulating autoantibodies against P4Hb were detected in prediabetic NOD mice and in early human type 1 diabetes prior to the onset of anti-insulin autoimmunity. Our studies provide mechanistic insight into the pathways of proinsulin metabolism and those creating autoantigenic forms of insulin in type 1 diabetes.

immunology↗

bioRxiv2020

CD45 pre-exclusion from the tips of microvilli establishes a phosphatase-free zone for early TCR triggering

The tyrosine phosphatase CD45 is a major gatekeeper for restraining T cell activation. Its exclusion from the immunological synapse (IS) is crucial for TCR signal transduction. Here, we used expansion super-resolution microscopy to reveal that CD45 is pre-excluded from the tips of microvilli on primary T cells prior to antigen encounter. This pre-exclusion was diminished by depleting cholesterol or by engineering the transmembrane domain of CD45 to increase its membrane integration length, but was independent of the CD45 extracellular domain. We further show that brief microvilli-mediated contacts can induce Ca2+ influx in mouse antigen-specific T cells engaged by antigen-pulsed APCs. We propose that the absence of CD45 phosphatase activity at the tips of microvilli enables or facilitates TCR triggering from brief T cell-APC contacts before formation of a stable IS, and that these microvilli-mediated contacts represent the earliest step in the initiation of a T cell adaptive immune response. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/109074v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@24d116org.highwire.dtl.DTLVardef@11b029borg.highwire.dtl.DTLVardef@4817e9org.highwire.dtl.DTLVardef@119ec32_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2020

DNA Encoded Glycan Libraries as a next-generation tool for the study of glycan-protein interactions

Interactions between glycans and glycan-binding proteins (GBPs) mediate diverse cellular functions, and therefore are of diagnostic and therapeutic significance. Current leading strategies for studying glycan-GBP interactions require specialized knowledge and instrumentation. In this study, we report a strategy for studying glycan-GBP interactions that uses PCR, qPCR and next-generation sequencing (NGS) technologies that are more routinely accessible. Our headpiece conjugation-code ligation (HCCL) strategy couples glycans with unique DNA codes that specify glycan sugar moieties and glycosidic linkages when sequenced. We demonstrate the technology by synthesizing a DNA encoded glycan library of 50 biologically relevant glycans (DEGL-50) and probing interactions against 25 target proteins including lectins and antibodies. Data show glycan-GPB interactions in solution that are consistent with lower content, lower throughput ELISA assays. Data further demonstrate how monovalent and multivalent headpieces can be used to increase glycan-GPB interactions and enrich signals while using smaller sample sizes. The flexibility of our modular HCCL strategy has potential for producing large glycan libraries, facilitating high content-high throughput glycan binding studies, and increasing access to lower cost glyco-analyses. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/017012v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@fb1c1dorg.highwire.dtl.DTLVardef@1f2c3ceorg.highwire.dtl.DTLVardef@11546e7org.highwire.dtl.DTLVardef@1e21a83_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2020

Dissecting endothelial to haematopoietic stem cell transition by single-cell transcriptomic and functional analyses

Haematopoietic stem cells (HSCs) in adults are believed to be born from hemogenic endothelial cells (HECs) in mid-gestational mouse embryos. Due to rare and transient nature, the HSC-competent ECs have never been stringently identified and accurately captured, let alone their genuine vasculature precursors. Here, we firstly used high-precision single-cell transcriptomics to unbiasedly examine relevant EC populations at continuous developmental stages and transcriptomically identified putative HSC-primed HECs. Combining computational prediction and in vivo functional validation, we precisely captured HSC-competent HECs by newly constructed Neurl3-EGFP reporter mouse model, and realized enrichment further by surface marker combination. Surprisingly, endothelial-haematopoietic bi-potential was rarely but reliably witnessed in culture of single HECs. Noteworthy, primitive vascular ECs experienced two-step fate choices to become HSC-primed HECs, resolving several previously observed contradictions. Taken together, comprehensive understanding of endothelial evolutions and molecular programs underlying HSC-primed HEC specification in vivo will facilitate future investigations directing HSC production in vitro.

developmental biology↗

bioRxiv2019

Enhancing single-cell cellular state inference by incorporating molecular network features

In biological systems, genes function in conjunction rather than in isolation. However, traditional single-cell RNA-seq (scRNA-seq) analyses heavily rely on the transcriptional similarity of individual genes, ignoring the inherent gene-gene interactions. Here, we present SCORE, a network-based method, which incorporates the validated molecular network features to infer cellular states. Using real scRNA-seq datasets, SCORE outperforms existing methods in accuracy, robustness, scalability, data integration and removal of batch effect. When applying SCORE to a newly generated human ileal scRNA-seq dataset, we identified several novel stem/progenitor clusters, including a Cripto-1+ cluster. Moreover, two distinct groups of goblet cells were identified and only one of them tended to secrete mucus. Besides, we found that the recently identified BEST4+OTOP2+ microfold cells also highly expressed CFTR, which is different from their colonic counterparts. In summary, SCORE enhances cellular state inference by simulating the dynamic changes of molecular networks, providing more biological insights beyond statistical interpretations.

bioinformatics↗

bioRxiv2018

Robust Antibacterial Activity of Tungsten Oxide (WO3-X) Nanodots

Antibacterial agents are an important tool in the prevention of bacterial infections. Inorganic materials are attractive due to their high stability under a variety of conditions compared to organic antibacterial agents. Herein tungsten oxide nanodots (WO3-X), synthesized by a simple one-pot synthetic approach, was found to exhibit efficient antibacterial capabilities. The analyses with colony-forming units (CFU) showed excellent antibacterial activity of WO3-X against both gram-negative E. coli (Escherichia coli) and gram-positive S. aureus (Staphylococcus aureus) strains. The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images revealed clear damage to the bacterial cell membranes, which was further confirmed by molecular dynamics simulations. Additionally, exposure to simulated sunlight was found to further increase germicidal activity of WO3-X nanodots - a 30-minute exposure to sunlight (combining 50 g/mL WO3-X nanodots) showed a 70% decrease in E. coli viability compared to without exposure. Electron spin resonance spectroscopy (ESR) was used to elucidate the underlying mechanism of this photocatalytic activity through the generation of hydroxyl radical species. Cell counting kit-8 (CCK-8) and the live/dead assay were further employed to evaluate the cytotoxicity of WO3-X nanodots on eukaryotic cells, which demonstrated their general biocompatibility. In all, our results suggest WO3-X nanodots have considerable potential in antibacterial applications, while also being biocompatible at large.

pharmacology and toxicology↗

bioRxiv2018

Loss of SDHB reprograms energy metabolisms and inhibits high fat diet induced metabolic syndromes

Mitochondrial respiratory complex II utilizes succinate, key substrate of the Krebs cycle, for oxidative phosphorylation, which is essential for glucose metabolism. Mutations of complex II cause cancers and mitochondrial diseases, raising a critical question of the (patho-)physiological functions. To address the fundamental role of complex II in systemic energy metabolism, we specifically knockout SDHB in mice liver, a key complex II subunit that tethers the catalytic SDHA subunit and transfers the electrons to ubiquinone, and found that SHDB deficiency abolishes the assembly of complex II without affecting other respiration complexes while largely retaining SDHA stability. SHDB ablation reprograms energy metabolism and hyperactivates the glycolysis, Krebs cycle and {beta}-oxidation pathways, leading to catastrophic energy deficit and early death. Strikingly, sucrose supplementation or high fat diet resumes both glucose and lipid metabolism and prevent early death. Also, SDHB deficient mice are completely resistant to high fat diet induced obesity. Our findings reveal that the unanticipated role of complex II orchestrating both lipid and glucose metabolisms, and suggest that SDHB is an ideal therapeutic target for combating obesity.

molecular biology↗

bioRxiv2017

Integration of A New Sensory Skill with Vision After Less Than 3 Hours of Training

Humans are effective at dealing with noisy, probabilistic information in familiar settings. One hallmark of this is Bayesian Cue Combination: combining multiple noisy estimates to increase precision beyond the best single estimate, taking into account their reliabilities. Here we show that adults also combine a novel audio cue to distance, akin to human echolocation, with a visual cue. Following two hours of training, subjects were more precise given both cues together versus the best single cue. This persisted when we changed the novel cues auditory frequency. Reliability changes also led to a re-weighting of cues without feedback, showing that they learned something more flexible than a rote decision rule for specific stimuli. The main findings replicated with a vibrotactile cue. These results show that the mature sensory apparatus can learn to flexibly integrate new sensory skills. The findings are unexpected considering previous empirical results and current models of multisensory learning.

animal behavior and cognition↗