Biology subjects

Yu, J.

Publications and source records attributed to Yu, J..

At least 451 records · Page 25Linked to original sources

bioRxiv2020

First high-quality reference genome of Amphicarpaea edgeworthii

Amphicarpaea edgeworthii, an annual twining herb, is a widely distributed species and an ideal model for studying complex flowering types and evolutionary mechanisms of species. Herein, we generated a high-quality assembly of A. edgeworthii by using a combination of PacBio, 10x Genomics libraries, and Hi-C mapping technologies. The final 11 chromosome-level scaffolds covered 90.61% of the estimated genome (343.78 Mb), which is the first chromosome-scale assembled genome of an amphicarpic plant. These data will be beneficial for the discovery of genes that control major agronomic traits, spur genetic improvement of and functional genetic studies in legumes, and supply comparative genetic resources for other amphicarpic plants.

genomics↗

bioRxiv2020

Bat and pangolin coronavirus spike glycoprotein structures provide insights into SARS-CoV-2 evolution

In recognizing the host cellular receptor and mediating fusion of virus and cell membranes, the spike (S) glycoprotein of coronaviruses is the most critical viral protein for cross-species transmission and infection. Here we determined the cryo-EM structures of the spikes from bat (RaTG13) and pangolin (PCoV_GX) coronaviruses, which are closely related to SARS-CoV-2. All three receptor-binding domains (RBDs) of these two spike trimers are in the "down" conformation, indicating they are more prone to adopt this receptor-binding inactive state. However, we found that the PCoV_GX, but not the RaTG13, spike is comparable to the SARS-CoV-2 spike in binding the human ACE2 receptor and supporting pseudovirus cell entry. Through structure and sequence comparisons, we identified critical residues in the RBD that underlie the different activities of the RaTG13 and PCoV_GX/SARS-CoV-2 spikes and propose that N-linked glycans serve as conformational control elements of the RBD. These results collectively indicate that strong RBD-ACE2 binding and efficient RBD conformational sampling are required for the evolution of SARS-CoV-2 to gain highly efficient infection.

biophysics↗

bioRxiv2020

Ex vivo therapeutic base and prime editing using chemically derived hepatic progenitors in a mouse model of tyrosinemia type 1

DNA base editors and prime editing technology capable of therapeutic base conversion enable ex vivo gene editing therapy for various genetic diseases. For such therapy, it is critical that the target cells survive well both outside the body and after transplantation. In this regard, chemically derived stem/progenitor cells are attracting attention as the most useful cell sources for clinical trials. Here, we generate chemically derived hepatic progenitors from the hereditary tyrosinemia type1 model mouse (HT1-mCdHs) and successfully correct the disease causing mutation using both adenosine base editors (ABEs) and prime editing tools. After transplantation into HT1 mice, ABE-corrected HT1-mCdHs repopulated the liver with fumarylacetoacetate hydrolase-positive cells and dramatically increased the survival rate of HT1 model mice, suggesting a safe and effective ex vivo gene editing therapy.

cell biology↗

bioRxiv2020

miR-424(322) is a molecular switch controlling pro-inflammatory vs anti-inflammatory skin DC subset differentiation by modulating TGF-β signaling

TGF-{beta} family ligands are key regulators of dendritic cell (DC) differentiation and activation. Epidermal Langerhans cells (LCs) require TGF- {beta} family signaling for their differentiation and canonical TGF-{beta}1 signaling secures a non-activated LC state. LCs reportedly control skin inflammation and are replenished from peripheral blood monocytes, which also give rise to pro-inflammatory monocyte-derived DCs (moDCs). Among all the miRNAs differentially expressed in LC vs moDCs, we observed miR-424 to be strongly induced during moDC differentiation from monocytes. We discovered that miR-424 is required for moDC differentiation from human and murine precursor cells in vitro and for inflammation-associated moDC in vivo. Mechanistically we found that low levels of miR-424 facilitate TGF-{beta}1-dependent LC differentiation at the expense of moDC differentiation. Loss of miR-424 in monocyte/DC precursors resulted in the induction of TGF-{beta} pathway. Therefore, miR-424 plays a decisive role in anti-inflammatory LC vs pro-inflammatory moDC differentiation from monocytes, and its repression allows TGF-{beta} ligands to promote LC differentiation. Short summaryMonocytes give rise to two distinct DC subsets in skin inflammation, exhibiting opposite roles in inflammation. This study identified miR-424(322) as a molecular switch controlling pro-inflammatory (moDC) vs anti-inflammatory LC subset differentiation by modulating TGF-{beta} signaling.

immunology↗

bioRxiv2020

Deficient spermiogenesis in mice lacking Rlim

The X-linked gene Rlim plays major roles in female mouse development and reproduction, where it is crucial for the maintenance of imprinted X chromosome inactivation in extraembryonic tissues of embryos. However, while females carrying a systemic Rlim knockout (KO) die around implantation, male Rlim KO mice appear healthy and are fertile, raising questions as to the pressures driving Rlim gene selection during evolution. Here we report an important role for Rlim in testis where it is highly expressed in post-meiotic round spermatids as well as in Sertoli cells. Systemic deletion of the Rlim gene leads to lower numbers of mature sperm that contains excess cytoplasm, leading to decreased sperm motility and in vitro fertilization rates. Targeting the conditional Rlim cKO specifically to the spermatogenic cell lineage largely recapitulates this phenotype. These results reveal functions of Rlim in male reproduction specifically in round spermatids during spermiogenesis with likely evolutionary implications.

developmental biology↗

bioRxiv2020

Compositional Variability and Mutation Spectra of Monophyletic SARS-CoV-2 Clades

COVID-19 and its causative pathogen SARS-CoV-2 have rushed the world into a staggering pandemic in a few months and a global fight against both is still going on. Here, we describe an analysis procedure where genome composition and its variables are related, through the genetic code, to molecular mechanisms based on understanding of RNA replication and its feedback loop from mutation to viral proteome sequence fraternity including effective sites on replicase-transcriptase complex. Our analysis starts with primary sequence information and identity-based phylogeny based on 22,051 SARS-CoV-2 genome sequences and evaluation of sequence variation patterns as mutation spectrum and its 12 permutations among organized clades tailored to two key mechanisms: strand-biased and function-associated mutations. Our findings include: (1) The most dominant mutation is C-to-U permutation whose abundant second-codon-position counts alter amino acid composition toward higher molecular weight and lower hydrophobicity albeit assumed most slightly deleterious. (2) The second abundance group includes: three negative-strand mutations U-to-C, A-to-G, G-to-A and a positive-strand mutation G-to-U generated through an identical mechanism as C-to-U. (3) A clade-associated and biased mutation trend is found attributable to elevated level of the negative-sense strand synthesis. (4) Within-clade permutation variation is very informative for associating non-synonymous mutations and viral proteome changes. These findings demand a bioinformatics platform where emerging mutations are mapped on to mostly subtle but fast-adjusting viral proteomes and transcriptomes to provide biological and clinical information after logical convergence for effective pharmaceutical and diagnostic applications. Such thoughts and actions are in desperate need, especially in the middle of the War against COVID-19.

microbiology↗

bioRxiv2020

Modular metabolite assembly in C. elegans lysosome-related organelles

Signaling molecules derived from attachment of diverse metabolic building blocks to ascarosides play a central role in the life history of C. elegans and other nematodes; however, many aspects of their biogenesis remain unclear. Using comparative metabolomics, we show that a pathway mediating formation of intestinal lysosome-related organelles (LROs) is required for biosynthesis of most modular ascarosides as well as previously undescribed modular glucosides. Similar to modular ascarosides, the modular glucosides are derived from highly selective assembly of moieties from nucleoside, amino acid, neurotransmitter, and lipid metabolism, suggesting that modular glucosides, like the ascarosides, may serve signaling functions. We further show that carboxylesterases that localize to intestinal organelles are required for the assembly of both modular ascarosides and glucosides via ester and amide linkages. Further exploration of LRO function and carboxylesterase homologs in C. elegans and other animals may reveal additional new compound families and signaling paradigms. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/262956v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@6e83cborg.highwire.dtl.DTLVardef@18e9302org.highwire.dtl.DTLVardef@1557670org.highwire.dtl.DTLVardef@1d2ca50_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Single-cell analysis identifies TCF4 and ID3 as a molecular switch of mammary epithelial stem cell differentiation.

It is well known that the expansion of the mammary epithelium during the ovarian cycles in female mammals is supported by the transient increase in mammary epithelial stem cells (MaSCs). However, dissecting the molecular mechanisms that govern MaSC function and differentiation is poorly understood due to the lack of standardized methods for their identification and isolation. The development of robust single-cell mRNA sequencing () technologies and the computational methods to analyze them provides us with novel tools to approach the challenge of studying MaSCs in a completely unbiased way without. Here, we have performed the largest scRNA-seq analysis of individual mammary epithelial cells (~70,000 cells). Our study identified a distinct cell population presenting molecular features of MaSCs. Importantly, further purification and additional in-depth single-cell analysis of these cells revealed that they are not a fully homogenous entity. Instead, we identified three subpopulations representing early stages of lineage commitment. By tracking their molecular evolution through single-cell network analysis we found that one of these subpopulations represents bipotent MaSCs from which luminal and basal lineages diverge. Importantly, we also confirmed the presence of these cells in human mammary glands. Finally, through expression and network analysis studies, we have uncovered transcription factors that are activated early during lineage commitment. These data identified E2-2 (Tcf4) and ID3 as a potential molecular switch of mammary epithelial stem cell differentiation.

developmental biology↗

bioRxiv2020

UPRmt scales mitochondrial network expansion with protein synthesis via mitochondrial import

As organisms develop, individual cells generate mitochondria to fulfill physiologic requirements. However, it remains unknown how mitochondrial network expansion is scaled to cell growth and impacted by environmental cues. The mitochondrial unfolded protein response (UPRmt) is a signaling pathway mediated by the transcription factor ATFS-1 which harbors a mitochondrial targeting sequence (MTS)1. Here, we demonstrate that ATFS-1 mediates an adaptable mitochondrial expansion program that is active throughout normal development. Developmental mitochondrial network expansion required the relatively inefficient MTS2 in ATFS-1, which allowed the transcription factor to be responsive to parameters that impact protein import capacity of the entire mitochondrial network. Increasing the strength of the ATFS-1 MTS impaired UPRmt activity throughout development due to increased accumulation within mitochondria. The insulin-like signaling-TORC13 and AMPK pathways affected UPRmt activation4,5 in a manner that correlated with protein synthesis. Manipulation to increase protein synthesis caused UPRmt activation. Alternatively, S6 kinase inhibition had the opposite effect due to increased mitochondrial accumulation of ATFS-1. However, ATFS-1 with a dysfunctional MTS6 constitutively increased UPRmt activity independent of TORC1 function. Lastly, expression of a single protein with a strong MTS, was sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent manner. We propose that mitochondrial network expansion during development is an emergent property of the synthesis of highly expressed mitochondrial proteins that exclude ATFS-1 from mitochondrial import, causing UPRmt activation. Mitochondrial network expansion is attenuated once ATFS-1 can be imported.

molecular biology↗

bioRxiv2020

Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with an endemic human CoV

A high-resolution understanding of the antibody response to SARS-CoV-2 is important for the design of effective diagnostics, vaccines and therapeutics. However, SARS-CoV-2 antibody epitopes remain largely uncharacterized, and it is unknown whether and how the response may cross-react with related viruses. Here, we use a multiplexed peptide assay ( PepSeq) to generate an epitope-resolved view of reactivity across all human coronaviruses. PepSeq accurately detects SARS-CoV-2 exposure and resolves epitopes across the Spike and Nucleocapsid proteins. Two of these represent recurrent reactivities to conserved, functionally-important sites in the Spike S2 subunit, regions that we show are also targeted for the endemic coronaviruses in pre-pandemic controls. At one of these sites, we demonstrate that the SARS-CoV-2 response strongly and recurrently cross-reacts with the endemic virus hCoV-OC43. Our analyses reveal new diagnostic and therapeutic targets, including a site at which SARS-CoV-2 may recruit common pre-existing antibodies and with the potential for broadly-neutralizing responses.

immunology↗

bioRxiv2020

A rapidly adaptable biomaterial vaccine for SARS-CoV-2

ABSTRACTThe global COVID-19 pandemic motivates accelerated research to develop safe and efficacious vaccines. To address this need, we leveraged a biomaterial vaccine technology that consists of mesoporous silica rods (MSRs) that provide a sustained release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and adjuvants to concentrate and mature antigen-presenting cells at the vaccine site. Here we explored the humoral responses resulting from the use of monophosphoryl lipid A (MPLA) as the adjuvant and SARS-CoV-2 spike proteins S1, S2, the nucleocapsid (N) protein, and receptor binding domain (RBD) as the target antigens. The dose of antigen and impact of pre-manufacturing of vaccines as versus loading antigen just-in-time was explored in these studies. Single shot MSR vaccines induced rapid and robust antibody titers to the presented antigens, even without the use of a boost, and sera from vaccinated animals demonstrated neutralizing activity against a SARS-CoV-2 pseudovirus. Overall, these results suggest the MSR vaccine system may provide potent protective immunity when utilized to present SARS-CoV-2 antigens.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2020

EXERCISE-INDUCED BENEFITS ON GLUCOSE HANDLING IN A MODEL OF DIET-INDUCED OBESITY ARE REDUCED BY CONCURRENT NICOTINAMIDE MONONUCLEOTIDE

ABSTRACTObjective Almost 40% of adults worldwide are classified as overweight or obese. Exercise is a beneficial intervention in obesity, partly due to increases in mitochondrial activity, with a potential role for the concomitant increase in nicotinamide adenine dinucleotide (NAD+). Recent studies have shown that increasing NAD+ levels through pharmacological supplementation with precursors such as nicotinamide mononucleotide (NMN) improved metabolic health in high fat diet (HFD) fed mice. We examined the combined effects of NMN and treadmill exercise on the metabolic dysregulation in HFD-induced obesity.Methods Five-week old female C57BL/6J mice were exposed to control diet or HFD. Mice fed HFD were treated with NMN in drinking water (400mg/kg; HNMN), treadmill exercise (HEx) or combined NMN and exercise (HNEx).Results Unexpectedly, NMN administration impaired several aspects of exercise-induced benefits in HFD mice, including glucose tolerance, glucose stimulated insulin secretion from islets and reduced hepatic triglyceride accumulation. Mechanistically, HNEx mice displayed increased antioxidant and reduced prooxidant gene expression in both islets and muscle, suggesting that altered redox status is associated with the loss of exercise-induced health benefits with NMN co-treatment.Conclusion Our data show that NMN treatment blocks the beneficial metabolic effects of exercise in a mouse model of diet-induced obesity in association with disturbances in redox metabolism.HighlightsNMN dampened exercise-induced benefits on glucose handling in diet-induced obesity.NMN administration in exercise enhanced ratio of antioxidants to prooxidants.We suggest NMN administration may not be beneficial when NAD+ levels are replete.Competing Interest StatementL.E.W. is an inventor on patents licensed to Metro Biotech NSW and to Jumpstart Fertility, and has received sponsored research funding from both companies. He is an advisor and shareholder in EdenRoc Sciences (Metro Biotech NSW, Metro Biotech, Liberty Biosecurity), Life Biosciences LLC and its daughter companies (Jumpstart Fertility, Continuum Biosciences, Senolytic Therapeutics, Selphagy, Animal Biosciences, Iduna) and in Intravital Pty Ltd. L.E.W. provides consulting work for Life Biosciences.View Full Text

physiology↗

bioRxiv2020

Inchworm stepping of Myc-Max heterodimer protein diffusion along DNA

Oncogenic protein Myc serves as a transcription factor to control cell metabolisms. Myc dimerizes via leucine zipper with its associated partner protein Max to form a heterodimer structure, which then binds target DNA sequences to regulate gene transcription. The regulation depends on by Myc-Max binding to DNA and searching for target sequences via diffusional motions along DNA. Here, we conduct structure-based molecular dynamics (MD) simulations to investigate the diffusion dynamics of the Myc-Max heterodimer along DNA. We found that the heterodimer protein slides on the DNA in a rotation-uncoupled manner in coarse-grained simulations, as its two helical DNA binding basic regions (BRs) alternate between open and closed conformations via inchworm stepping motions. In such motions, the two BRs of the heterodimer step across the DNA strand one by one, with step sizes up about half of a DNA helical pitch length. Atomic MD simulations of the Myc-Max heterodimer in complex with DNA have also been conducted. Hydrogen bond interactions reveal between the two BRs and two complementary DNA strands, respectively. In the non-specific DNA binding, the BR shows an onset of stepping on one association DNA strand and dissociating from the complementary strand. Overall, our simulation studies suggest that the inchworm stepping motions of the Myc-Max heterodimer can be achieved during the protein diffusion along DNA.

biophysics↗

bioRxiv2020

SARS-CoV-2 Neutralizing Antibody Responses Are More Robust in Patients with Severe Disease

We studied plasma antibody responses of 35 patients about 1 month after SARS-CoV-2 infection. Titers of antibodies binding to the viral nucleocapsid and spike proteins were significantly higher in patients with severe disease. Likewise, mean antibody neutralization titers against SARS-CoV-2 pseudovirus and live virus were higher in the sicker patients, by ~5-fold and ~7-fold, respectively. These findings have important implications for those pursuing plasma therapy, isolation of neutralizing monoclonal antibodies, and determinants of immunity.

microbiology↗

bioRxiv2020

Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs

G protein coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals1,2. Although several structures have been solved for GPCR-G protein complexes3-17, structural studies of the complex in a physiological lipid membrane environment are lacking. Additionally, most previous studies required additional antibodies/nanobodies and/or engineered G proteins for complex stabilization. In the absence of a native complex structure, the underlying mechanism of G protein activation leading to GDP/GTP exchange remains unclear. Here, we report cryo-EM structures of lipid bilayer-bound complexes of neurotensin, neurotensin receptor 1, and Gi1{beta}1{gamma}1 protein in two conformational states, resolved to 4.1 and 4.2 [A] resolution. The structures were determined without any stabilizing antibodies/nanobodies, and thus provide a native-like platform for understanding the structural basis of GPCR-G protein complex formation. Our structures reveal an extended network of protein-protein interactions at the GPCR-G protein interface compared to in detergent micelles, defining roles for the lipid membrane in modulating the structure and dynamics of complex formation, and providing a molecular explanation for the stronger interaction between GPCR and G protein in lipid bilayers. We propose a detailed allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling under near native conditions.

molecular biology↗

bioRxiv2020

LRRK2 regulates AP2M1 phosphorylation cycles to mediate endocytosis and dopaminergic neurodegeneration

Recent genetic evidence revealed endocytic pathway plays a major role in Parkinsons disease (PD). However, the molecular mechanism is poorly understood. Here we report that LRRK2, the most genetic cause of PD, binds to and phosphorylates AP2M1, the core component of endocytosis recently implicated in PD risk. Both knockout and overexpression of LRRK2 cause abnormal AP2M1 phosphorylation cycle and in turn endocytic defects. Mechanistically, knockout of LRRK2 decreases AP2M1 phosphorylation required for the initial clathrin coated vesicle (CCV) formation while LRRK2 overexpression inhibits AP2M1 uncoating for entering into a new cycle of CCV formation. Our study also uncovered a novel tissue-specific regulation of AP2M1 phosphorylation by LRRK2. Further, we found LRRK2 phosphorylation on AP2M1 mediates LRRK2-induced neuronal toxicity both in vitro and in vivo. Importantly, AP2M1 phosphorylation levels are elevated in PD patient fibroblasts. Together, our study provides a direct mechanistic link between LRRK2, AP2 and endocytosis in PD pathogenesis.

neuroscience↗

bioRxiv2020

Massively parallel quantification of CRISPR editing in cells by TRAP-seq enables better design of Cas9, ABE, CBE gRNAs of high efficiency and accuracy

The CRISPR RNA-guided endonucleases Cas9, and Cas9-derived adenine/cytosine base editors (ABE/CBE), have been used in both research and therapeutic applications. However, broader use of this gene editing toolbox is hampered by the great variability of efficiency among different target sites. Here we present TRAP-seq, a versatile and scalable approach in which the CRISPR gRNA expression cassette and the corresponding surrogate site are captured by Targeted Reporter Anchored Positional Sequencing in cells. TRAP-seq can faithfully recapitulate the CRISPR gene editing outcomes introduced to the corresponding endogenous genome site and most importantly enables massively parallel quantification of CRISPR gene editing in cells. We demonstrate the utility of this technology for high-throughput quantification of SpCas9 editing efficiency and indel outcomes for 12,000 gRNAs in human embryonic kidney cells. Using this approach, we also showed that TRAP-seq enables high throughput quantification of both ABE and CBE efficiency at 12,000 sites in cells. This rich amount of ABE/CBE outcome data enable us to reveal several novel nucleotide features (e.g. preference of flanking bases, nucleotide motifs, STOP recoding types) affecting base editing efficiency, as well as designing improved machine learning-based prediction tools for designing SpCas9, ABE and CBE gRNAs of high efficiency and accuracy (>70%). We have integrated all the 12,000 CRISPR gene editing outcomes for SpCas9, ABE and CBE into a CRISPR-centered portal: The Human CRISPR Atlas. This study extends our knowledge on CRISPR gene and base editing, and will facilitate the application and development of CRISPR in both research and therapy.

systems biology↗

bioRxiv2020

An HIV-Tat inducible mouse model system of childhood HIV-associated nephropathy.

Background Modern antiretroviral therapies (ART) have decreased the prevalence of HIV-associated nephropathy (HIVAN). Nonetheless, we continue to see children and adolescents with HIVAN all over the world. Furthermore, once HIVAN is established in children, it is difficult to revert its long-term progression, and we need better animal models of childhood HIVAN to test new treatments.Objectives To define whether the HIV-1 trans-activator (Tat) gene precipitates HIVAN in young mice, and to develop an inducible mouse model of childhood HIVAN.Design/Methods An HIV-Tat gene cloned from a child with HIVAN was used to generate recombinant adenoviral vectors (rAd-Tat). rAd-Tat and LacZ control vectors (2 × 109) were expressed in the kidney of newborn wild type and HIV-transgenic (Tg26) FVB/N mice without significant proteinuria (n = 5 - 8 per group). Mice were sacrificed 7 and 35 days later to assess their renal outcome, the expression of HIV-genes and growth factors, and markers of cell growth and differentiation by RT-qPCR, immunohistochemistry, and/or Western blots.Results HIV-Tat induced the expression of HIV-1 genes (env) and heparin binding growth factors in the kidney of HIV-Tg26 mice, and precipitated HIVAN in the first month of life. No significant renal changes were detected in wild type mice infected with rAd-Tat vectors, suggesting that HIV-Tat alone does not induce renal disease.Conclusion This new mouse model of childhood HIVAN highlights the critical role that HIV-Tat plays in the pathogenesis of HIVAN, and could be used to study the pathogenesis and treatment of HIVAN in children and adolescents.Summary statement We developed a new inducible mouse model system of childhood HIV-associated nephropathy, and demonstrated that HIV-Tat plays a critical role in this renal disease acting in synergy with other HIV-1 genes and heparin binding cytokines.View Full Text

pathology↗