Biology subjects

Yan, W.

Publications and source records attributed to Yan, W..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Isoform-specific Disruption of the TP73 Gene Reveals a Critical Role for TAp73gamma in Tumorigenesis via Leptin

TP73, a member of the p53 family, is expressed as TAp73 and {Delta}Np73 along with multiple C-terminal isoforms (-{eta}). {Delta}Np73 is primarily expressed in neuronal cells and necessary for neuronal development. Interestingly, while TAp73 is a tumor suppressor and predominantly expressed in normal cells, TAp73 is found to be frequently altered in human cancers, suggesting a role of TAp73 C-terminal isoforms in tumorigenesis. To test this, the TCGA SpliceSeq database was searched and showed that exon 11 (E11) exclusion occurs frequently in several human cancers. We also found that p73 to p73{gamma} isoform switch resulting from E11 skipping occurs frequently in human prostate cancers and dog lymphomas. To determine whether p73 to p73{gamma} isoform switch plays a role in tumorigenesis, CRISPR technology was used to generate multiple cancer cell lines and a mouse model in that Trp73 E11 is deleted. Surprisingly, we found that in E11-deificient cells, p73{gamma} becomes the predominant isoform and exerts oncogenic activities by promoting cell proliferation and migration. In line with this, E11-deficient mice were more prone to obesity and B-cell lymphomas, indicating a unique role of p73{gamma} in lipid metabolism and tumorigenesis. Additionally, we found that E11-deficient mice phenocopies Trp73-deficient mice with short lifespan, infertility, and chronic inflammation. Mechanistically, Mechanistically, we showed that Leptin, a pleiotropic adipocytokine involved in energy metabolism and oncogenesis, was highly induced by p73{gamma}, necessary for p73{gamma}-mediated oncogenic activity, and associated with p73 to {gamma} isoform switch in human prostate cancer and dog lymphoma. Finally, we showed that E11-knockout promoted, whereas knockdown of p73{gamma} or Leptin suppressed, xenograft growth in mice. Our study indicates that the p73{gamma}-Leptin pathway promotes tumorigenesis and alters lipid metabolism, which may be targeted for cancer management.

cancer biology↗

bioRxiv2022

Temporal single cell atlas of non-neuronal retinal cells reveals dynamic, coordinated multicellular responses to central nervous system injury

Non-neuronal cells play key roles in the complex cellular interplay that follows central nervous system (CNS) insult. To understand this interplay at a tissue level, we generated a single-cell atlas of immune, glial and retinal pigment epithelial cells from adult mouse retina before and at multiple time points after axonal transection (optic nerve crush; ONC), identifying rare and undescribed subsets, and delineating changes in cell composition, expression programs, and interactions. Computational analysis charted an inflammatory cascade after injury with three phases. The early phase consisted of reactivation of retinal macroglia and microglia, providing chemotactic signals for immune infiltration, concurrent with infiltration of CCR2+ monocytes from the circulation. In the second phase, these differentiated to macrophage subsets resembling resident border-associated macrophages. In parallel, a multicellular interferon program, likely driven by microglia-derived type-I interferon, was synchronously activated across resident glia, expanding beyond rare interferon-responding subsets of glia unexpectedly present in the naive retina. Our findings provide insights regarding post-injury CNS tissue dynamics and a framework to decipher cellular circuitry, spatial relationships and molecular interactions following tissue injury.

immunology↗

bioRxiv2022

Arginase 1 is a key driver of immune suppression in pancreatic cancer

An extensive fibroinflammatory stroma rich in macrophages is a hallmark of pancreatic cancer. In this disease, it is well appreciated that macrophages are immunosuppressive and contribute to the poor response to immunotherapy; however, the mechanisms of immune suppression are complex and not fully understood. Immunosuppressive macrophages are classically defined by expression of the enzyme Arginase 1 (Arg1), which we demonstrated is potently expressed in pancreatic tumor associated macrophages from both human patients and mouse models. While routinely used as a polarization marker, Arg1 also catabolizes arginine, an amino acid required for T cell activation and proliferation. To investigate this metabolic function, we used a genetic and a pharmacologic approach to target Arg1 in pancreatic cancer. Genetic inactivation of Arg1 in macrophages, using a dual recombinase genetically engineered mouse model of pancreatic cancer, delayed formation of invasive disease, while increasing CD8+ T cell infiltration. Treatment of established tumors with the arginase inhibitor CB-1158 exhibited further increased CD8+ T cell infiltration, beyond that seen with the macrophage-specific knockout, and sensitized the tumors to anti-PD1 immune checkpoint blockade. Thus, our data demonstrate that Arg1 is more than simply a marker of macrophage function. Rather, Arg1 is also a driver of immune suppression and represents a promising immunotherapeutic target for pancreatic cancer.

cancer biology↗

bioRxiv2022

Bioprinting microporous functional living materials from protein-based core-shell microgels

Living materials bring together material science and biology to allow the engineering and augmenting of living systems with novel functionalities. Bioprinting promises accurate control over the formation of such complex materials through programmable deposition of cells in soft materials, but current approaches had limited success in fine-tuning cell microenvironments while generating robust macroscopic morphologies. Here, we address this challenge through the use of core-shell microgel ink to decouple cell microenvironments from the structural shell for further processing. Cells are microfluidically immobilized in the viscous core that can promote the formation of both microbial populations and mammalian cellular spheroids, followed by interparticle annealing to give covalently stabilized functional scaffolds with controlled microporosity. The results show that the core-shell strategy mitigates cell leakage while affording a favorable environment for cell culture. Furthermore, we demonstrate that different microbial consortia can be printed into scaffolds for a range of applications. By compartmentalizing microbial consortia in separate microgels, the collective bioprocessing capability of the scaffold is significantly enhanced, shedding light on strategies to augment living materials with bioprocessing capabilities.

bioengineering↗

bioRxiv2022

Low-cost genomics enable high-throughput isolate screening and strain-level microbiome profiling

Earths environments harbor complex consortia of microbial lineages that affect processes ranging from host health to biogeochemical cycles. However, understanding the evolution and function of these microbiota has been limited by an inability to isolate individual microbial constituents and assemble their complete genomes in a high-throughput manner. Here, we present a workflow for bacterial isolation and whole-genome sequencing from complex microbiota using open-source labware and the OpenTrons automated liquid handling robotics platform. Our approach circumvents the need for isolate screening (e.g., through 16S rDNA sequencing or mass spectrometry analyses) by reducing the costs of genome-sequencing to ~$10 per bacterium. Applying the workflow, we quantified genomic diversity within 45 bacterial species in the chimpanzee gut microbiota. Results revealed hotspots of recombination in bacterial genomes and elevated transmission of plasmids between distantly related bacterial species within individual chimpanzee hosts. This study develops and applies an approach for high-throughput bacterial isolation and genome sequencing, enabling population genetic analyses of bacterial strains within complex communities not currently possible with metagenomic data alone.

microbiology↗

bioRxiv2022

Boosting transformation in wheat by BBM-WUS

Agrobacterium-mediated transformation is a cost-effective and convenient way to introduce foreign genetic elements to plants. However, only limited protocols successfully generates transgenic plants with fielder, a spring wheat variety. Wheat transformation method with higher efficiency and without genotype restriction is heavily demanded. Here, a heat-shock protocol, independent of Japan Tobacco had been established. Transgenic plants can be obtained from immature embryo within only 60 days by this protocol. Morphogenic regulators Baby boom and Wuschel (BBM-WUS) was proved to promote transformation efficiency for five to six times in wheat when co-infiltrated with agrobacterium containing target construct. Notably, half of the transformants are BBM-WUS free and moreover, the BBM-WUS containing plants could be picked by florescent marker that was co-expressed with BBM-WUS. In conclusion, we managed to establish a new wheat transformation protocol with shorter duration than published protocol.

plant biology↗

bioRxiv2022

Euchromatin activity enhances segregation and compaction of heterochromatin in the cell nucleus

The large-scale organization of the genome inside the cell nucleus is critical for the cells function. Chromatin - the functional form of DNA in cells - serves as a substrate for active nuclear processes such as transcription, replication and DNA repair. Chromatins spatial organization directly affects its accessibility by ATP-powered enzymes, e.g., RNA polymerase II in the case of transcription. In differentiated cells, chromatin is spatially segregated into compartments - euchromatin and heterochromatin - the former being largely transcriptionally active and loosely packed, the latter containing mostly silent genes and densely compacted. The euchromatin/heterochromatin segregation is crucial for proper genomic function, yet the physical principles behind it are far from understood. Here, we model the nucleus as filled with hydrodynamically interacting active Zimm chains - chromosomes - and investigate how large heterochromatic regions form and segregate from euchromatin through their complex interactions. Each chromosome presents a block copolymer composed of heterochromatic blocks, capable of crosslinking that increases chromatins local compaction, and euchromatic blocks, subjected to stochastic force dipoles that capture the microscopic stresses exerted by nuclear ATPases. These active stresses lead to a dynamic self-organization of the genome, with its coherent motions driving the mixing of chromosome territories as well as large-scale heterochromatic segregation through crosslinking of distant genomic regions. We study the stresses and flows that arise in the nucleus during the heterochromatic segregation, and identify their signatures in Hi-C proximity maps. Our results reveal the fundamental role of active mechanical processes and hydrodynamic interactions in the kinetics of chromatin compartmentalization and in the emergent large-scale organization of the nucleus.

biophysics↗

bioRxiv2022

Overlapping transcriptional programs promote survival and axonal regeneration of injured retinal ganglion cells

Injured neurons in the adult mammalian central nervous system often die and seldom regenerate axons. To uncover transcriptional pathways that could ameliorate these disappointing responses we analyzed three interventions that increase survival and regeneration of mouse retinal ganglion cells (RGCs) following optic nerve crush (ONC) injury, albeit not to a clinically useful extent. We assessed gene expression in each of 46 RGC types by single cell transcriptomics following ONC and treatment. We also compared RGCs that regenerated to those that survived but did not regenerate. Each intervention enhanced survival of most RGC types, but type-independent axon regeneration required manipulation of multiple pathways. Distinct computational methods converged on separate sets of genes selectively expressed by RGCs likely to be dying, surviving, or regenerating. Overexpression of genes associated with the regeneration program enhanced axon regeneration in vivo, indicating that mechanistic analysis can be used to identify novel methods for promoting regeneration of injured neurons.

neuroscience↗

bioRxiv2022

Cell atlas of the human ocular anterior segment: Tissue-specific and shared cell types

The anterior segment of the eye consists of the cornea, iris, ciliary body, crystalline lens and aqueous humor outflow pathways. Together, these tissues are essential for the proper functioning of the eye. Disorders of vision have been ascribed to defects in all of them; some, including glaucoma and cataract, are among the most prevalent causes of blindness in the world. To characterize the cell types that comprise these tissues, we generated an anterior segment cell atlas of the human eye using high throughput single-nucleus RNA sequencing (snRNAseq). We profiled 191,992 nuclei from non-diseased anterior segment tissues from 6 human donors, identifying >60 cell types. Many of these cell types were discrete, whereas others, especially in lens and cornea, formed continua corresponding to known developmental transitions that persist in adulthood. Having profiled each tissue separately, we performed an integrated analysis of the entire anterior segment revealing that some cell types are unique to single structure whereas others are shared across tissues. The integrated cell atlas was then used to investigate cell type-specific expression patterns of more than 900 human ocular disease genes identified either through Mendelian inheritance patterns or genome-wide association studies (GWAS). SIGNIFICANCE STATEMENTSeveral of the most prevalent blinding ocular conditions worldwide, including glaucoma, cataract and uncorrected refractive error, involve structures of the anterior segment of the human eye, which consists of the cornea, iris, ciliary body, crystalline lens and aqueous humor outflow pathways. In addition to providing transcriptomic profiles of the cell types within individual tissues, this work contributes to our understanding of the relatedness and diversity of these cell types across contiguous tissues by generating an integrated anterior segment cell atlas and documenting the expression of over 900 disease-associated genes in each cell type. By allowing simultaneous interrogation of cell-type specific expression of genes across multiple tissues, the atlas may yield broad insight into normal and disease-associated anterior segment functions.

cell biology↗

bioRxiv2021

Intratumoral delivery of engineered recombinant modified vaccinia virus Ankara express-ing Flt3L and OX40L generates potent antitumor immunity through activating the cGAS/STING pathway and depleting tumor-infiltrating regulatory T cells

Intratumoral (IT) delivery of immune-activating viruses can serve as an important strategy to turn "cold" tumors into "hot" tumors, resulting in overcoming resistance to immune checkpoint blockade (ICB). Modified vaccinia virus Ankara (MVA) is a highly attenuated, non-replicative vaccinia virus that has a long history of human use. Here we report that IT recombinant MVA (rMVA), lacking E5R encoding an inhibitor of the DNA sensor cyclic GMP-AMP synthase (cGAS), expressing a dendritic cell growth factor, Fms-like tyrosine kinase 3 ligand (Flt3L), and a T cell co-stimulator, OX40L, generates strong antitumor immunity, which is dependent on CD8+ T cells, the cGAS/STING-mediated cytosolic DNA-sensing pathway, and STAT1/STAT2-mediated type I IFN signaling. Remarkably, IT rMVA depletes OX40hi regulatory T cells via OX40L/OX40 interaction and IFNAR signaling. Taken together, our study provides a proof-of-concept for improving MVA-based cancer immunotherapy, through modulation of both innate and adaptive immunity. One Sentence SummaryIntratumoral delivery of recombinant MVA for cancer immunotherapy

immunology↗

bioRxiv2021

Extrinsic KRAS signaling shapes the pancreatic microenvironment through fibroblast reprogramming.

Oncogenic KRAS is the hallmark mutation of human pancreatic cancer and a driver of tumorigenesis in genetically engineered mouse models of the disease. While the tumor cell-intrinsic effects of oncogenic Kras expression have been widely studied, its role in regulating the extensive pancreatic tumor microenvironment is less understood. Using a genetically engineered mouse model of inducible and reversible oncogenic Kras expression and a combination of approaches that include mass cytometry and single cell RNA sequencing, we have discovered that non-cell autonomous (i.e., extrinsic) oncogenic KRAS signaling reprograms pancreatic fibroblasts, activating an inflammatory gene expression program. As a result, fibroblasts become a hub of extracellular signaling, mediating the polarization and function of pro-tumorigenic myeloid cells while also preventing tissue repair. Our study provides fundamental new knowledge on the mechanisms underlying the formation of the fibroinflammatory stroma in pancreatic cancer and highlights stromal pathways with the potential to be exploited therapeutically.

cancer biology↗

bioRxiv2021

Cyclin-G-associated kinase GAK/Aux orchestrates glial autophagy via Atg9 phosphorylation in Parkinson's disease

Autophagy is a major means for the elimination of protein inclusions in neurons in neurodegenerative diseases such as Parkinsons disease (PD). Yet, the mechanism of autophagy in the other brain cell type, glia, is less well characterized and remains largely unknown. Here we present evidence that the PD risk factor Cyclin G-associated kinase (GAK)/dAuxilin (dAux) is a new component in glial autophagy. GAK/dAux directly interacts with ULK1/Atg1 via its uncoating domain. Lack of GAK/dAux increases the autophagosome number and size in adult fly glia and mouse microglia, and generally upregulates levels of components in the initiation and PI3K class III complexes including ULK1/Atg1, demonstrating that GAK/dAux regulates the onset of glial autophagy. Consistently, lack of GAK/dAux enhances Atg1 and Atg9 trafficking to autophagosomes, promoting autophagy initiation. On the other hand, lack of GAK/dAux impairs the autophagic flux and blocks substrate degradation, suggesting that GAK/dAux might play additional roles in glial autophagy. Our findings identify a new autophagy factor in glia; considering the pivotal role of glia under pathological conditions, targeting glial autophagy is potentially a therapeutic strategy for PD.

neuroscience↗

bioRxiv2021

Wall-associated Receptor Kinase and The Expression Profiles in Wheat Responding to Fungal Stress

Cell wall-associated kinases (WAKs), which are encoded by conserved gene families in plants, are crucial for development and responses to diverse stresses. However, the wheat (Triticum aestivum L.) WAKs have not been systematically classified, especially those involved in protecting plants from disease. Here, we classified 129 WAK proteins (encoded by 232 genes) and 75 WAK-Like proteins (WAKLs; encoded by 109 genes) into four groups, via a phylogenetic analysis. An examination of protein sequence alignment revealed diversity in the GUB-domain of WAKs structural organization, but it was usually characterized by a PYPFG motif followed by CxGxGCC motifs, while the EGF-domain was usually initiated with a YAC motif, and eight cysteine residues were spliced by GNPY motif. The expression profiles of WAK-encoding homologous genes varied in response to Blumeria graminis f. sp. tritici (Bgt), Puccinia striiformis f. sp. tritici (Pst) and Puccinia triticina (Pt) stress. A quantitative real-time polymerase chain reaction (qRT-PCR) analysis proved that TaWAK75 and TaWAK76b were involved in wheat resistance to Bgt. This study revealed the structure of the WAK-encoding genes in wheat, which may be useful for future functional elucidation of wheat WAKs responses to fungal infections.

plant biology↗

bioRxiv2021

A 3D gene expression atlas of the floral meristem based on spatial reconstruction of single nucleus RNA sequencing data

Identity and functions of plant cells are influenced by their precise cellular location within the plant body. Cellular heterogeneity in growth and differentiation trajectories results in organ patterning. Therefore, assessing this heterogeneity at molecular scale is a major question in developmental biology. Single-cell transcriptomics (scRNA-seq) allows to characterize and quantify gene expression heterogeneity in developing organs at unprecedented resolution. However, the original physical location of the cell is lost during the scRNA-seq procedure. To recover the original location of cells is essential to link gene activity with cellular function and morphology. Here, we reconstruct genome-wide gene expression patterns of individual cells in a floral meristem by combining single-nuclei RNA-seq with 3D spatial reconstruction. By this, gene expression differences among meristematic domains giving rise to different tissue and organ types can be determined. As a proof of principle, the data are used to trace the initiation of vascular identity within the floral meristem. Our work demonstrates the power of spatially reconstructed single cell transcriptome atlases to understand plant morphogenesis. The floral meristem 3D gene expression atlas can be accessed at http://threed-flower-meristem.herokuapp.com

plant biology↗

bioRxiv2021

A new role of low barrier hydrogen bond in mediating protein stability by small molecules

Low barrier hydrogen bond (LBHB) is a special type of hydrogen bond which occurs where two heteroatoms with similar pKa values share a single proton resulting in an unusually strong and short hydrogen bond. LBHBs in protein play important roles in enzyme catalysis and maintaining protein structural integrity but its other biochemical roles are unknown. Here we report a novel function of LBHB in selectively inducing tubulin protein degradation. A tubulin inhibitor, 3-(3-Phenoxybenzyl) amino-{beta}-carboline (PAC), promotes selective degradation of {beta}-tubulin heterodimers by binding to the colchicine site of {beta}-tubulin. Biochemical studies have revealed that PAC specifically destabilizes tubulin, making it prone to aggregation that then predisposes it to ubiquitinylation and then degradation. Structural activity analyses have indicated that the destabilization is mediated by a single hydrogen bond formed between the pyridine nitrogen of PAC and {beta}Glu198, which is identified as a LBHB. In contrast, another two tubulin inhibitors only forming normal hydrogen bonds with {beta}Glu198 exhibit no degradation effect. Thus, the LBHB accounts for the degradation. Most importantly, we screened for compounds capable of forming LBHB with {beta}Glu198 and demonstrated that BML284, a Wnt signaling activator, also promotes tubulin heterodimers degradation in a PAC-like manner as expected. Our study has identified a novel approach for designing tubulin degraders, providing a unique example of LBHB function and suggests that designing small molecules to form LBHBs with protein residues resulting in the highly specific degradation of a target protein could be a new strategy for drug development.

biochemistry↗

bioRxiv2021

Bacteroidales species are a reservoir of phase-variable antibiotic resistance genes in the human gut microbiome

Phase-variable antibiotic resistance genes (ARGs) can mitigate the fitness cost of maintaining ARGs in the absence of antibiotics and could potentially prolong the persistence of ARGs in bacterial populations. However, the origin, prevalence, and distribution of phase-variable ARGs remains poorly understood. Here, we sought to assess the threat posed by phase-variable ARGs by systematically searching for phase-variable ARGs in the human gut microbiome and examining their origin, prevalence, and distribution. Through metagenomic assembly of 2227 human gut metagenomes and genomic analysis of the Unified Human Gastrointestinal Genome (UHGG) collection, we identified phase-variable ARGs and categorized them into three classes based on the invertase regulating phase variation. In the human gut microbiome, phase-variable ARGs are commonly and exclusively distributed in Bacteroidales species. Through genomic analysis, we observed that phase-variable ARGs have convergently originated from ARG insertions into phase-variable capsule polysaccharide biosynthesis (CPS) loci at least three times. Moreover, all identified phase-variable ARGs are located within integrative conjugative elements (ICEs). Therefore, horizontal transfer via ICEs could explain the wide taxonomic distribution of phase-variable ARGs. Overall, these findings reveal that phase-variable CPS loci in Bacteroidales species are an important hotspot for the emergence of clinically-relevant phase-variable ARGs.

evolutionary biology↗

bioRxiv2021

Altered O-glycosylation Level of SARS-CoV-2 Spike Protein by Host O-glycosyltransferase Strengthens Its Trimeric Structure

The trimeric spike protein (S) mediates host-cell entry and membrane fusion of SARS-CoV-2. S protein is highly glycosylated, whereas its O-glycosylation is still poorly understood. Herein, we site-specifically examine the O-glycosylation of S protein through a mass spectrometric approach with HCD-triggered-ETD model. We identify 15 high-confidence O-glycosites and at least 10 distinct O-glycan structures on S protein. Peptide microarray assays prove that human ppGalNAc-T6 actively participates in O-glycosylation of S protein. Importantly, the upregulation of ppGalNAc-T6 expression can profoundly enhance the O-glycosylation level by generating new O-glycosites and increasing both O-glycan heterogeneity and intensities. Further molecular dynamics simulations reveal that the O-glycosylation on the protomer-interface regions, which are mainly modified by ppGalNAc-T6, can potentially stabilize the trimeric S protein structure. Our work provides deep molecular insights of how viral infection harnesses the host O-glycosyltransferases to dynamically regulate the O-glycosylation level of the viral envelope protein responsible for membrane fusion.

biochemistry↗

bioRxiv2021

Destruction of DNA-binding proteins by programmable O'PROTAC: Oligonucleotide-based PROTAC

DNA-binding proteins including transcription factors (TFs) play essential roles in gene transcription and DNA replication and repair during normal organ development and pathogenesis of diseases such as cancer, cardiovascular disease and obesity, deeming to be a large repertoire of attractive therapeutic targets. However, this group of proteins are generally considered undruggable as they lack an enzymatic catalytic site or a ligand binding pocket. PROteolysis-TArgeting Chimera (PROTAC) technology has been developed by engineering a bifunctional small molecule chimera to bring a protein of interest (POI) to the proximity of an E3 ubiquitin ligase, thus inducing the ubiquitination of POI and further degradation through proteasome pathway. Here we report the development of Oligonucleotide-based PROTAC (OPROTACs), a class of noncanonical PROTACs in which a TF-recognizing double-stranded oligonucleotide is incorporated as a binding moiety of POI. We demonstrate that OPROTACs of ERG and LEF1, two highly cancer-related transcription factors selectively promote degradation of these proteins and inhibit their transcriptional activity in cancer cells. The programmable nature of OPROTACs indicates that this approach is applicable to destruct other TFs. OPROTACs not only can serve as a research tool, but also can be harnessed as a therapeutic arsenal to target DNA binding proteins for effective treatment of diseases such as cancer.

biochemistry↗