Biology subjects

Yin, Y.

Publications and source records attributed to Yin, Y..

At least 109 records · Page 6Linked to original sources

bioRxiv2024

Restoring O-glycosylation and expression of MUC2 limits progression of colorectal cancer

This study investigated the molecular mechanisms underlying the regulation of MUC2 expression and O-glycosylation modification in colorectal cancer. In addition, the potential of rosiglitazone to inhibit colorectal cancer by improving MUC2 glycosylation to protect intestinal barrier function was explored. In vitro, lectin staining combined with Co-IP assay was used to detect glycosyltransferases regulating MUC2 O-glycosylation. ChIP and Luciferase experiments were used to verify the transcription factors regulating MUC2 expression level. Samples from CRC patients were used to detect differences in multimolecular expression. The AOM/DSS mouse model was used to validate the effect of rosiglitazone on inhibiting colorectal cancer progression. Our results showed that B3GNT6 acts as a glycosyltransferase to enhance the O-glycosylation level of MUC2 and maintain protein stability to resist degradation by StcE secreting from pathogenic bacteria. Furthermore, KLF4 directly promotes the transcription of B3GNT6 and MUC2, which are regulated by PPARg. Rosiglitazone activated PPARg-KLF4-B3GNT6 axis which increased the expression level and glycosylation of MUC2 and further improved the intestinal mucosal barrier function to delay the development of colorectal cancer in mice. These data suggest that O-glycosylation and expression of MUC2 is key to the maintenance of functional intestinal mucosa and rosiglitazone is a potential colorectal cancer therapeutic agent.

cancer biology↗

bioRxiv2024

Tmc7 deficiency causes acrosome biogenesis defects and male infertility in mice

Transmembrane channel-like (TMC) proteins are a highly conserved ion channel family consisting of eight members (TMC1-TMC8) in mammals. TMC1/2 are components of the mechanotransduction channel in hair cells, and mutations of TMC1/2 cause deafness in humans and mice. However, the physiological roles of other TMC proteins remain largely unknown. Here, we show that Tmc7 is specifically expressed in the testis and that it is required for acrosome biogenesis during spermatogenesis. Tmc7-/- mice exhibited abnormal sperm head, disorganized mitochondrial sheaths, and reduced number of elongating spermatids, similar to human oligo-astheno-teratozoospermia. We further demonstrate that TMC7 is colocalized with GM130 at the cis-Golgi region in round spermatids. TMC7 deficiency leads to aberrant Golgi morphology and impaired fusion of Golgi-derived vesicles to the developing acrosome. Moreover, upon loss of TMC7 intracellular ion homeostasis is impaired and ROS levels are increased, which in turn causes Golgi and endoplasmic reticulum (ER) stress. Taken together, these results suggest that TMC7 is required to maintain pH and ion homeostasis, which is needed for acrosome biogenesis. Our findings unveil a novel role for TMC7 in acrosome biogenesis during spermiogenesis.

developmental biology↗

bioRxiv2024

Carbohydrate-active enzyme annotation in microbiomes using dbCAN

CAZymes or carbohydrate-active enzymes are critically important for human gut health, lignocellulose degradation, global carbon recycling, soil health, and plant disease. We developed dbCAN as a web server in 2012 and actively maintain it for automated CAZyme annotation. Considering data privacy and scalability, we provide run_dbcan as a standalone software package since 2018 to allow users perform more secure and scalable CAZyme annotation on their local servers. Here, we offer a comprehensive computational protocol on automated CAZyme annotation of microbiome sequencing data, covering everything from short read pre-processing to data visualization of CAZyme and glycan substrate occurrence and abundance in multiple samples. Using a real-world metagenomic sequencing dataset, this protocol describes commands for dataset and software preparation, metagenome assembly, gene prediction, CAZyme prediction, CAZyme gene cluster (CGC) prediction, glycan substrate prediction, and data visualization. The expected results include publication-quality plots for the abundance of CAZymes, CGCs, and substrates from multiple CAZyme annotation routes (individual sample assembly, co-assembly, and assembly-free). For the individual sample assembly route, this protocol takes [~]33h on a Linux computer with 40 CPUs, while other routes will be faster. This protocol does not require programming experience from users, but it does assume a familiarity with the Linux command-line interface and the ability to run Python scripts in the terminal. The target audience includes the tens of thousands of microbiome researchers who routinely use our web server. This protocol will encourage them to perform more secure, rapid, and scalable CAZyme annotation on their local computer servers.

bioinformatics↗

bioRxiv2023

Identification of a myofibroblast differentiation program during neonatal lung development

Alveologenesis is the final stage of lung development in which the internal surface area of the lung is increased to facilitate efficient gas exchange in the mature organism. The first phase of alveologenesis involves the formation of septal ridges (secondary septae) and the second phase involves thinning of the alveolar septa. Within secondary septa, mesenchymal cells include a transient population of alveolar myofibroblasts (MyoFB) and a stable but poorly described population of lipid rich cells that have been referred to as lipofibroblasts or matrix fibroblasts (MatFB). Using a unique Fgf18CreER lineage trace mouse line, cell sorting, single cell RNA sequencing, and primary cell culture, we have identified multiple subtypes of mesenchymal cells in the neonatal lung, including an immature progenitor cell that gives rise to mature MyoFB. We also show that the endogenous and targeted ROSA26 locus serves as a sensitive reporter for MyoFB maturation. These studies identify a myofibroblast differentiation program that is distinct form other mesenchymal cells types and increases the known repertoire of mesenchymal cell types in the neonatal lung. Summary StatementDuring primary alveologenesis, alveolar myofibroblasts comprise a distinct proliferative mesenchymal lineage that matures and populates emerging secondary septa.

developmental biology↗

bioRxiv2023

In vitro antimicrobial mechanism of diacerein and potential to reverse MRSE resistance to β-lactams

Staphylococcus epidermidis is the most common pathogens causing ocular infection. With the increase of drug resistance rate, it poses a new challenge to anti-infection treatment. In this study, we analyzed the drug resistance of S. epidermidis isolated from the eye in the past 5 years to guide empirical antibiotics treatment. Then, the MIC and MBC of diacerein against MRSE were detected, and continuous induced resistance experiments confirmed that MRSE not easily induce resistance to diacerein. In addition, it was observed that diacerein induced MRSE cell lysis, increased membrane permeability and resulted in intracellular ROS accumulation. Diacerein does not have toxic effects to HCEC at effective bacteriostatic concentrations. The results of checkerboard assay indicated that the combination of diacerein and {beta}-lactams had additive effect on MRSE. We also observed that diacerein may reverse MRSE resistance to {beta}-lactam drugs by affecting the active efflux system. In conclusion, our results provide strong evidence for the high therapeutic potential of diacerein against MRSE.

microbiology↗

bioRxiv2023

Structure basis for recognition of 26RFa by the pyroglutamylated RFamide peptide receptor

The neuropeptide 26RFa, a member of the RF-amide peptide family, activates the pyroglutamylated RF-amide peptide receptor (QRFPR), a class A GPCR. The 26RFa/QRFPR system plays critical roles in energy homeostasis, making QRFPR an attractive drug target for treating obesity, diabetes, and eating disorders. However, the lack of structural information has hindered our understanding of the peptide recognition and regulatory mechanism of QRFPR, impeding drug design efforts. In this study, we determined the cryo-EM structure of the Gq-coupled QRFPR bound to 26RFa. The structure reveals a unique assembly mode of the receptor extracellular regions and the peptide N-terminus and elucidates the recognition mechanism of the C-terminal heptapeptide of 26RFa within the transmembrane binding pocket of QRFPR. The study also clarifies the similarities and distinctions in the binding pattern of the RF-amide moiety in five RF-amide peptides and the RY-amide segment in neuropeptide Y. These findings deepen our understanding of the RF-amide peptides recognition, aiding in the rational design of drugs targeting QRFPR and other RF-amide receptors.

biophysics↗

bioRxiv2023

Temporal genomic analysis of melanoma rejection identifies regulators of tumor immune evasion

Decreased intra-tumor heterogeneity (ITH) correlates with increased patient survival and immunotherapy response. However, even highly homogenous tumors may display variability in their aggressiveness, and how immunologic-factors impinge on their aggressiveness remains understudied. Here we studied the mechanisms responsible for the immune-escape of murine tumors with low ITH. We compared the temporal growth of homogeneous, genetically-similar single-cell clones that are rejected vs. those that are not- rejected after transplantation in-vivo using single-cell RNA sequencing and immunophenotyping. Non- rejected clones showed high infiltration of tumor-associated-macrophages (TAMs), lower T-cell infiltration, and increased T-cell exhaustion compared to rejected clones. Comparative analysis of rejection- associated gene expression programs, combined with in-vivo CRISPR knockout screens of candidate mediators, identified Mif (macrophage migration inhibitory factor) as a regulator of immune rejection. Mif knockout led to smaller tumors and reversed non-rejection-associated immune composition, particularly, leading to the reduction of immunosuppressive macrophage infiltration. Finally, we validated these results in melanoma patient data. Statement of significanceHere, we uncover the association of Mif expression with tumor growth and aggressiveness, specifically in low ITH tumors. These findings could facilitate the development of new strategies to treat patients with homogeneous, high-MIF expressing tumors that are unresponsive to immune checkpoint therapy.

cancer biology↗

bioRxiv2023

pH selects for distinct N2O-reducing microbiomes in tropical soil microcosms

Nitrous oxide (N2O), a greenhouse gas with ozone destruction potential, is mitigated by the microbial reduction to dinitrogen catalyzed by N2O reductase (NosZ). Bacteria with NosZ activity have been studied at circumneutral pH but the microbiology of low pH N2O reduction has remained elusive. Acidic (pH<5) tropical forest soils were collected in the Luquillo Experimental Forest in Puerto Rico, and microcosms maintained with low (0.02mM) and high (2mM) N2O assessed N2O reduction at pH 4.5 and 7.3. All microcosms consumed N2O, but long lag times of up to 7 months were observed in microcosms with 2 mM N2O. Comparative metagenome analysis revealed that Rhodocyclaceae dominated in circumneutral microcosms under both N2O feeding regimes. In acidic microcosms, Peptococcaceae dominated in high-N2O, and Hyphomicrobiaceae in low-N2O microcosms. Seventeen metagenome-assembled genomes (MAGs) recovered from these microcosms harbored nos operons, with all eight MAGs derived from acidic microcosms carrying the clade II type nosZ, lacking nitrite reductase genes (nirS, nirK). Five of these MAGs represented novel taxa indicating an unexplored N2O-reducing diversity exists in acidic tropical soils. A survey of pH 3.5-5.7 soil metagenome datasets revealed that nosZ genes commonly occur, suggesting broad distribution of N2O reduction potential in acidic soils.

microbiology↗

bioRxiv2023

In vivo affinity maturation of murine B cells reprogrammed to express human antibodies

CRISPR-edited murine B cells engineered to express human antibody variable chains proliferate, class switch, and secrete these antibodies in vaccinated mice. However, current strategies disrupt the heavy-chain locus, resulting in inefficient somatic hypermutation without functional affinity maturation. Here we show that recombined murine heavy- and kappa-variable genes can be directly and simultaneously overwritten, using Cas12a-mediated cuts at their 3-most J segments and 5 homology arms complementary to distal V segments. Cells edited in this way to express the HIV-1 broadly neutralizing antibodies 10-1074 or VRC26.25-y robustly hypermutated and generated potent neutralizing plasma in vaccinated recipient mice. 10-1074 variants isolated from these mice bound and neutralized HIV-1 envelope glycoprotein more efficiently than wild-type 10-1074 while maintaining or improving its already low polyreactivity and long in vivo half-life. We further validated this approach by generating substantially broader and more potent variants of the anti-SARS-CoV-2 antibodies ZCB11 and S309. Thus, B cells edited at their native loci affinity mature, facilitating development of broad, potent, and bioavailable antibodies and expanding the potential applications of engineered B cells.

bioengineering↗

bioRxiv2023

A conserved N-terminal motif of CUL3 mediates assembly and licenses E3 ligase activation of CRL3KLHL22

The CUL3-RING E3 ubiquitin ligases (CRL3s) play an essential role in response to extracellular nutrition and stress stimuli. The ubiquitin ligase function of CRL3s is activated through E3-E3 dimerization. However, how and why such a dimeric assembly is required for its ligase activity remains elusive. Here, we report the cryo-EM structure of the dimeric CRL3KLHL22 complex and reveal a conserved N-terminal motif in CUL3 that mediates the dimerization assembly and licenses the activation of CRL3KLHL22. Deletion of the CUL3 N-terminal motif leads to non-stochiometric assembly and impairs E3 activity of both CRL3KLHL22 and several other CRL3s. In addition, we found that the dynamics of dimeric E3-E3 superassembly generates a variable ubiquitination zone, potentially facilitating substrate recognition and ubiquitination. These findings demonstrate a CUL3 N-terminal motif-dependent E3-E3 superassembly mechanism and provide insights into the assembly and activation of CRL3s.

molecular biology↗

bioRxiv2023

Interfacial water confers transcription factors with dinucleotide specificity

Transcription factors bind to DNA by recognizing specific bases within their binding motifs. Binding to each DNA mononucleotide within the motif often contributes independently to total binding energy. However, some transcription factors (TFs) can bind to DNA more specifically than predicted by this model, by directly recognizing DNA dinucleotides. To understand this process, we have solved the structures of the basic helix-loop-helix protein MYF5, and the homeodomain protein BARHL2 together with DNA containing a set of dinucleotides that have different affinities to the proteins at high resolution (< 1 [A]). We observe that dinucleotides can be recognized either enthalpically by an extensive water network that connects the adjacent bases to the TF, or entropically by formation of a hydrophobic patch that maintains water mobility at the protein-DNA interface. The two distinct thermodynamic signatures of the two equally optimal sites also confer differential temperature sensitivity to the optimal sites, with implications for thermal regulation of gene expression. Our results uncover the enigma of how TFs can recognize more complex local features than mononucleotides, and demonstrate that water-mediated recognition is important in predicting affinities of macromolecules from their sequence.

biochemistry↗

bioRxiv2023

CXCR3-CXCL11 signaling restricts angiogenesis and promotes pericyte recruitment

Endothelial cell (EC)-pericyte interactions are known to remodel in response to hemodynamic forces, yet there is a lack of mechanistic understanding of the signaling pathways that underlie these events. Here, we have identified a novel signaling network regulated by blood flow in ECs--the chemokine receptor, CXCR3, and one of its ligands, CXCL11--that delimits EC angiogenic potential and suppresses pericyte recruitment during development through regulation of pdgfb expression in ECs. In vitro modeling of EC-pericyte interactions demonstrates that suppression of EC-specific CXCR3 signaling leads to loss of pericyte association with EC tubes. In vivo, phenotypic defects are particularly noted in the cranial vasculature, where we see a loss of pericyte association with and expansion of the vasculature in zebrafish treated with the Cxcr3 inhibitor AMG487. We also demonstrate using flow modeling platforms that CXCR3-deficient ECs are more elongated, move more slowly, and have impaired EC-EC junctions compared to their control counterparts. Together these data suggest that CXCR3 signaling in ECs drives vascular stabilization events during development.

developmental biology↗

bioRxiv2023

Permanent Neonatal Diabetes-causing Insulin Mutations have Dominant Effects on Proinsulin Processing and Beta Cell Identity

Heterozygous coding sequence mutations of the INS gene are a cause of permanent neonatal diabetes (PNDM) that results from beta cell failure. We explored the causes of beta cell failure in two PNDM patients with two distinct INS mutations. Using b and mutated hESCs, we detected accumulation of misfolded proinsulin and impaired proinsulin processing in vitro, and a dominant-negative effect of these mutations on the in vivo performance of patient-derived SC-beta cells after transplantation into NSG mice. These insulin mutations derange endoplasmic reticulum (ER) homeostasis, and result in the loss of beta-cell mass and function. In addition to anticipated apoptosis, we found evidence of beta-cell dedifferentiation, characterized by an increase of cells expressing both Nkx6.1 and ALDH1A3, but negative for insulin and glucagon. These results highlight both known and novel mechanisms contributing to the loss and functional failure of human beta cells with specific insulin gene mutations.

cell biology↗

bioRxiv2023

Intermittent fasting promotes ILC3s secreting IL-22 contributing to the beigeing of white adipose tissue

Mechanism underlying the metabolic benefit of intermittent fasting remains largely unknown. Here, we reported that intermittent fasting promoted IL-22 production by type 3 innate lymphoid cells (ILC3s) and subsequent beigeing of subcutaneous white adipose tissue. Adoptive transfer of intestinal ILC3s increased beigeing of white adipose tissue in diet-induced-obese mice. Exogenous IL-22 significantly increased the beigeing of subcutaneous white adipose tissue. Deficiency of IL-22 receptor attenuated the beigeing induced by intermittent fasting. Single-cell sequencing of sorted intestinal immune cells revealed that intermittent fasting increased aryl hydrocarbon receptor signaling in ILC3s. Analysis of cell-cell ligand receptor interactions indicated that intermittent fasting may stimulate the interaction of ILC3s with dendritic cells (DCs) and macrophages. These results establish the role of intestinal ILC3s in beigeing of white adipose tissue, suggesting that ILC3/IL-22/IL-22R axis contributes to the metabolic benefit of intermittent fasting.

physiology↗

bioRxiv2023

Probing the DCAF12 interactions with MAGEA3 and CCT5 C-terminal degrons

DCAF12 is the substrate recognition component of the CRL4 E3 ligase complex that can recognize C-terminal double-glutamic acid degrons to promote degradation of its cognate substrates via the ubiquitin proteasome system. MAGEA3 and CCT5 proteins were reported to be cellular targets of DCAF12. To further characterize the DCAF12 interactions with both MAGEA3 and CCT5, we developed a suite of biophysical and a proximity-based cellular NanoBRET assays showing that both MAGEA3 and CCT5 C-terminal degron peptides interact with DCAF12 in nanomolar affinity in vitro and in cells. Furthermore, we report here the 3.17 [A] cryo-EM structure of DCAF12-DDB1-MAGEA3 complex revealing the key DCAF12 residues involved in C-terminal degron recognition and binding. Our study provides new tools and resources to enable the discovery of small molecule handles targeting the WDR domain of DCAF12 for future PROTAC design and development.

biochemistry↗

bioRxiv2023

Neuronal wiring diagram of an adult brain

Connections between neurons can be mapped by acquiring and analyzing electron microscopic (EM) brain images. In recent years, this approach has been applied to chunks of brains to reconstruct local connectivity maps that are highly informative, yet inadequate for understanding brain function more globally. Here, we present the first neuronal wiring diagram of a whole adult brain, containing 5x107 chemical synapses between [~]130,000 neurons reconstructed from a female Drosophila melanogaster. The resource also incorporates annotations of cell classes and types, nerves, hemilineages, and predictions of neurotransmitter identities. Data products are available by download, programmatic access, and interactive browsing and made interoperable with other fly data resources. We show how to derive a projectome, a map of projections between regions, from the connectome. We demonstrate the tracing of synaptic pathways and the analysis of information flow from inputs (sensory and ascending neurons) to outputs (motor, endocrine, and descending neurons), across both hemispheres, and between the central brain and the optic lobes. Tracing from a subset of photoreceptors all the way to descending motor pathways illustrates how structure can uncover putative circuit mechanisms underlying sensorimotor behaviors. The technologies and open ecosystem of the FlyWire Consortium set the stage for future large-scale connectome projects in other species.

neuroscience↗

bioRxiv2023

A consensus cell type atlas from multiple connectomes reveals principles of circuit stereotypy and variation

The fruit fly Drosophila melanogaster combines surprisingly sophisticated behaviour with a highly tractable nervous system. A large part of the flys success as a model organism in modern neuroscience stems from the concentration of collaboratively generated molecular genetic and digital resources. As presented in our FlyWire companion paper1, this now includes the first full brain connectome of an adult animal. Here we report the systematic and hierarchical annotation of this [~]130,000-neuron connectome including neuronal classes, cell types and developmental units (hemilineages). This enables any researcher to navigate this huge dataset and find systems and neurons of interest, linked to the literature through the Virtual Fly Brain database2. Crucially, this resource includes 4,552 cell types. 3,094 are rigorous consensus validations of cell types previously proposed in the "hemibrain" connectome3. In addition, we propose 1,458 new cell types, arising mostly from the fact that the FlyWire connectome spans the whole brain, whereas the hemibrain derives from a subvolume. Comparison of FlyWire and the hemibrain showed that cell type counts and strong connections were largely stable, but connection weights were surprisingly variable within and across animals. Further analysis defined simple heuristics for connectome interpretation: connections stronger than 10 unitary synapses or providing >1% of the input to a target cell are highly conserved. Some cell types showed increased variability across connectomes: the most common cell type in the mushroom body, required for learning and memory, is almost twice as numerous in FlyWire as the hemibrain. We find evidence for functional homeostasis through adjustments of the absolute amount of excitatory input while maintaining the excitation-inhibition ratio. Finally, and surprisingly, about one third of the cell types proposed in the hemibrain connectome could not yet be reliably identified in the FlyWire connectome. We therefore suggest that cell types should be defined to be robust to inter-individual variation, namely as groups of cells that are quantitatively more similar to cells in a different brain than to any other cell in the same brain. Joint analysis of the FlyWire and hemibrain connectomes demonstrates the viability and utility of this new definition. Our work defines a consensus cell type atlas for the fly brain and provides both an intellectual framework and open source toolchain for brain-scale comparative connectomics.

neuroscience↗

bioRxiv2023

Dissecting peri-implantation development using cultured human embryos and embryo-like assembloids

Studies of cultured embryos have provided insights into human peri-implantation development. However, detailed knowledge of peri-implantation lineage developments as well as underlying mechanisms remain obscure. Using 3D-cultured human embryos, herein we report a complete cell atlas of the early post-implantation lineages and decipher cellular composition and gene signatures of the epiblast and hypoblast derivatives. In addition, we develop an embryo-like assembloid (E-assembloid) by assembling naive hESCs and extraembryonic cells. Using human embryos and E-assembloids, we reveal that WNT, BMP and Nodal signaling synergistically, but functionally differently, orchestrate human peri-implantation lineage development. Specially, we dissect mechanisms underlying extraembryonic mesoderm and extraembryonic endoderm specifications. Finally, an improved E-assembloid is developed to recapitulate the epiblast and hypoblast developments and tissue architectures in the pre-gastrulation human embryo. Our findings provide insights of the human peri-implantation development, and the E-assembloid offers a useful model to disentangle cellular behaviors and signaling interactions that drive human embryogenesis.

developmental biology↗