Biology subjects

Chen, X.

Publications and source records attributed to Chen, X..

At least 1,261 records · Page 70Linked to original sources

bioRxiv2020

Three-dimensional ATUM-SEM reconstruction and analysis of hepatic endoplasmic reticulum-organelle interactions

The endoplasmic reticulum (ER) is a contiguous and complicated membrane network in eukaryotic cells, and membrane contact sites (MCSs) between the ER and other organelles perform vital cellular functions, including lipid homeostasis, metabolite exchange, calcium level regulation, and organelle division. Here, we establish a whole pipeline to reconstruct all ER, mitochondria, lipid droplets, lysosomes, peroxisomes, and nuclei by automated tape-collecting ultramicrotome scanning electron microscopy (ATUM-SEM) and deep-learning techniques, which generates an unprecedented 3D model for mapping liver samples. Furthermore, the morphology of various organelles is systematically analyzed. We found that the ER presents with predominantly flat cisternae and is knitted tightly all throughout the intracellular space and around other organelles. In addition, the ER has a smaller volume-to-membrane surface area ratio than other organelles, which suggests that the ER could be more suited for functions that require a large membrane surface area. Moreover, the MCSs between the ER and other organelles are explored. Our data indicate that ER-mitochondrial contacts are particularly abundant, especially for branched mitochondria. In addition, ER contacts with lipid droplets, lysosomes, and peroxisomes are also plentiful. In summary, we design an efficient method for obtaining a 3D reconstruction of biological structures at a nanometer resolution. Our study also provides the first 3D reconstruction of various organelles in liver samples together with important information fundamental for biochemical and functional studies in the liver.

cell biology↗

bioRxiv2020

Accurate Genomic Variant Detection in Single Cells with Primary Template-Directed Amplification

Improvements in whole genome amplification (WGA) would enable new types of basic and applied biomedical research, including studies of intratissue genetic diversity that require more accurate single-cell genotyping. Here we present primary template-directed amplification (PTA), a new isothermal WGA method that reproducibly captures >95% of the genomes of single cells in a more uniform and accurate manner than existing approaches, resulting in significantly improved variant calling sensitivity and precision. To illustrate the new types of studies that are enabled by PTA, we developed direct measurement of environmental mutagenicity (DMEM), a new tool for mapping genome-wide interactions of mutagens with single living human cells at base pair resolution. In addition, we utilized PTA for genome-wide off-target indel and structural variant detection in cells that had undergone CRISPR-mediated genome editing, establishing the feasibility for performing single-cell evaluations of biopsies from edited tissues. The improved precision and accuracy of variant detection with PTA overcomes the current limitations of accurate whole genome amplification, which is the major obstacle to studying genetic diversity and evolution at cellular resolution.

genomics↗

bioRxiv2020

Substrate traits govern the assembly and spatial organization of microbial community engaged in metabolic division of labor

Metabolic division of labor (MDOL) is widespread in nature, whereby a complex metabolic pathway is shared between different strains within a community for mutual benefit. However, little is known about how the mutual interactions in the microbial community engaged in MDOL are regulated. We hypothesized that when degradation of an organic compound is carried out via MDOL, the substrate traits (i.e., concentration and its toxicity) modulate the benefit allocation between the two microbial populations, thus affecting the structure of this community. We tested this hypothesis by combining mathematical modelling with experiments using engineered synthetic microbial consortia. Numerous modelling analyses suggested that the proportion of the population executing the first metabolic step can be simply estimated by Monod-like formulas governed by substrate traits. The model and the proposed formula quantitatively predicted the structure of our synthetic consortia composed of two strains degrading salicylate through MDOL. Individual-based modelling and colony pattern formation assays further indicated that our rule is also applicable to estimating community structure in spatially structured environments. Our results demonstrate that the structure of the microbial communities can be quantitatively predicted from simple environmental factors, such as substrate concentration and its toxicity, which provides novel perspectives on understanding the assembly of natural communities, as well as insights into how to manage artificial microbial systems.

microbiology↗

bioRxiv2020

Fascin1 empowers YAP mechanotransduction and promotes cholangiocarcinoma development

Mechanical forces control cell behavior, including cancer progression. Cells sense forces through actomyosin and YAP, but what regulators of actin mechanotransduction play relevant roles in vivo remains unclear. Here we identify the Fascin1 F-actin bundling protein as a key factor sustaining YAP activation in response to ECM mechanical cues. This is relevant in the mouse liver, where Fascin1 regulates YAP-dependent hepatocyte dedifferentiation. Moreover, Fascin1 is required in the AKT/NICD system and sufficient together with AKT to induce cholangiocarcinomas in mice, recapitulating genetic YAP requirements, and its expression in intrahepatic cholangiocarcinomas correlates with aggressiveness and poor patient prognosis. We propose that Fascin1 represents a pro-oncogenic mechanism that can be exploited during intrahepatic cholangiocarcinoma development to overcome a mechanical tumor-suppressive environment.

cancer biology↗

bioRxiv2020

A LAMP-based microfluidic chip for rapid detection of pathogen in Cryptococcal meningitis

Cryptococcal meningitis (CM) is a global threat with significant attributable morbidity and mortality. Information on integrated detection for CM diagnosis is still limited. This is mainly due to the presence of a large polysaccharide capsule and the tough cell wall of Cryptococcus, which makes it difficult to extract nucleic acids on the chip. In this study, we developed a LAMP-based microfluidic chip for rapid detection of pathogen in CM. We adopted 4 duplicate filtration membrane structures to improve target capture and simplify the enrichment process, and combined lyticase digestion and thermal alkaline lysisto optimize the nucleic acid extraction of Cryptococcus on the chip, and selected a portable UVA flashlight to shine the LAMP products to obtain the visual detection results which could be observed by the naked eye. This microfluidic chip, integrating sample Cryptococcus enrichment, nucleic acid extraction and LAMP detection unit, streamlined the operation process and reduced the exposure risk of directly handling cryptococcal samples. It did not require any additional instruments and demonstrated a rapid, reliable, as well as high-efficiency approach. It truly realized the "sample-to-answer" application and could be easily used for clinical cryptococcal prediagnosis.

microbiology↗

bioRxiv2020

Novel gene-specific translation mechanism of dysregulated, chronic inflammation reveals promising, multifaceted COVID-19 therapeutics

Hyperinflammation and lymphopenia provoked by SARS-CoV-2-activated macrophages contribute to the high mortality of Coronavirus Disease 2019 (COVID-19) patients. Thus, defining host pathways aberrantly activated in patient macrophages is critical for developing effective therapeutics. We discovered that G9a, a histone methyltransferase that is overexpressed in COVID-19 patients with high viral load, activates translation of specific genes that induce hyperinflammation and impairment of T cell function or lymphopenia. This noncanonical, pro-translation activity of G9a contrasts with its canonical epigenetic function. In endotoxin-tolerant (ET) macrophages that mimic conditions which render patients with pre-existing chronic inflammatory diseases vulnerable to severe symptoms, our chemoproteomic approach with a biotinylated inhibitor of G9a identified multiple G9a-associated translation regulatory pathways that were upregulated by SARS-CoV-2 infection. Further, quantitative translatome analysis of ET macrophages treated progressively with the G9a inhibitor profiled G9a-translated proteins that unite the networks associated with viral replication and the SARS-CoV-2-induced host response in severe patients. Accordingly, inhibition of G9a-associated pathways produced multifaceted, systematic effects, namely, restoration of T cell function, mitigation of hyperinflammation, and suppression of viral replication. Importantly, as a host-directed mechanism, this G9a-targeted, combined therapeutics is refractory to emerging antiviral-resistant mutants of SARS-CoV-2, or any virus, that hijacks host responses.

systems biology↗

bioRxiv2020

Notch signaling represses cone photoreceptor formation through the regulation of retinal progenitor cell states

Notch signaling is required to repress the formation of vertebrate cone photoreceptors and to maintain the proliferative potential of multipotent retinal progenitor cells. However, the mechanism by which Notch signaling controls these processes is unknown. Recently, restricted retinal progenitor cells with limited proliferation capacity and that preferentially generate cone photoreceptors have been identified. Thus, there are several potential steps during cone genesis that Notch signaling could act. Here we use cell type specific cis-regulatory elements to localize the primary role of Notch signaling in cone genesis to the formation of restricted retinal progenitor cells from multipotent retinal progenitor cells. Localized inhibition of Notch signaling in restricted progenitor cells does not alter the number of cones derived from these cells. Cell cycle promotion is not a primary effect of Notch signaling but an indirect effect on progenitor cell state transitions that leads to depletion of the multipotent progenitor cell population. Taken together, this suggests that the role of Notch signaling in cone photoreceptor formation and proliferation are both mediated by a localized function of Notch in multipotent retinal progenitor cells to repress the formation of restricted progenitor cells.

developmental biology↗

bioRxiv2020

A benchmarking study of SARS-CoV-2 whole-genome sequencing protocols using COVID-19 patient samples

The COVID-19 pandemic is a once-in-a-lifetime event, exceeding mortality rates of the flu pandemics from the 1950s and 1960s. Whole-genome sequencing (WGS) of SARS-CoV-2 plays a critical role in understanding the disease. Performance variation exists across SARS-CoV-2 viral WGS technologies, but there is currently no benchmarking study comparing different WGS sequencing protocols. We compared seven different SARS-CoV-2 WGS library protocols using RNA from patient nasopharyngeal swab samples under two storage conditions. We constructed multiple WGS libraries encompassing three different viral inputs: 1,000,000, 250,000 and 1,000 copies. Libraries were sequenced using two distinct platforms with varying sequencing depths and read lengths. We found large differences in mappability and genome coverage, and variations in sensitivity, reproducibility and precision of single-nucleotide variant calling across different protocols. We ranked the performance of protocols based on six different metrics. Our results indicated that the most appropriate protocol depended on viral input amount and sequencing depth. Our findings offer guidance in choosing appropriate WGS protocols to characterize SARS-CoV-2 and its evolution.

genomics↗

bioRxiv2020

Collagen induced inflammations

Collagen-induced arthritis (CIA) mouse model is currently the most widely used and reliable autoimmune model to study rheumatoid arthritis. In this model, we used bovine type II collagen (CII) and complete Freunds adjuvant (CFA) or incomplete Freunds adjuvant (IFA) to form emulsifier, and mice were injected intradermal to induce autoimmune arthritis (CIA). In this model, we ground bovine collagen type II (CII) with complete Freunds adjuvant (CFA) or incomplete Freunds adjuvant (IFA) to form emulsifiers, and intradermal injection in mice induced autoimmune arthritis (CIA). This article describes the mouse, CFA strains, key emulsification, anesthesia, and injection immune techniques, as well as the incidence, date of onset, score, pathological results of arthritis. The total time for preparation of reagents and immunization of 20 mice was about 2-2.5 hours. In this protocol, we induced a high incidence of CIA with DBA/1J in genetically susceptible mice and assessed the severity and pathology of the disease, at the same time we found that CII also can induced enteritis, including ileitis and colitis. The initial symptoms of arthritis appear in the 24-26 days of the experiment, that is, 3-5 days after the second immunization, the peak period of inflammation was 30-36 days, the arthritis incidence about 90-100%, at the same time, the incidence of enteritis and arthritis were the same, small intestinal inflammation was more severe, but the duration was short; while the colonic inflammation was mild, and the duration was longer than enteritis, we named it collagen induced inflammations (CIIs).

immunology↗

bioRxiv2020

Challenges in Undergraduate Synthetic Biology Training: Insights from a Canadian iGEM Student Perspective

The last two decades have seen vigorous activity in synthetic biology research and ever-increasing applications of synthetic biology technologies. However, pedagogical research on synthetic biology is scarce, especially when compared to some scientific and engineering disciplines. Within Canada, there are only three universities that formally teach synthetic biology programs; two of which are at the undergraduate level. Many Canadian undergraduate students are instead introduced to synthetic biology through participation in the annual International Genetically Engineered Machine (iGEM) competition where they work in design teams to conceive of and execute a synthetic biology project that they present at an international jamboree. We surveyed the Canadian landscape of synthetic biology education through the experience of students from the Canadian iGEM teams of 2019. Using a thematic codebook analysis, we gathered insights to generate recommendations that could empower future iGEM team operations and inform educators about best practices in teaching undergraduate synthetic biology.

scientific communication and education↗

bioRxiv2020

Cell wall damage attenuates root hair cell patterning and tissue morphogenesis mediated by the Arabidopsis receptor kinase STRUBBELIG.

Cell wall remodeling is essential for the control of growth and development as well as the regulation of stress responses. However, the underlying cell wall monitoring mechanisms remain poorly understood. Regulation of root hair fate and flower development in Arabidopsis thaliana requires signaling mediated by the atypical receptor kinase STRUBBELIG (SUB). Furthermore, SUB is involved in cell wall integrity signaling and regulates the cellular response to reduced levels of cellulose, a central component of the cell wall. Here, we show that continuous exposure to sub-lethal doses of the cellulose biosynthesis inhibitor isoxaben results in altered root hair patterning and floral morphogenesis. Genetically impairing cellulose biosynthesis also results in root hair patterning defects. We further show that isoxaben exerts its developmental effects through the attenuation of SUB signaling. Our evidence indicates that down-regulation of SUB is a multi-step process and involves changes in SUB complex architecture at the plasma membrane, enhanced removal of SUB from the cell surface, and downregulation of SUB transcript levels. The results provide molecular insight into how the cell wall regulates cell fate and tissue morphogenesis.

plant biology↗

bioRxiv2020

A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

Antibody engineering technologies face increasing demands for speed, reliability and scale. We developed CeVICA, a cell-free antibody engineering platform that integrates a novel generation method and design for camelid heavy-chain antibody VHH domain-based synthetic libraries, optimized in vitro selection based on ribosome display and a computational pipeline for binder prediction based on CDR-directed clustering. We applied CeVICA to engineer antibodies against the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike proteins and identified >800 predicted binder families. Among 14 experimentally-tested binders, 6 showed inhibition of pseudotyped virus infection. Antibody affinity maturation further increased binding affinity and potency of inhibition. Additionally, the unique capability of CeVICA for efficient and comprehensive binder prediction allowed retrospective validation of the fitness of our synthetic VHH library design and revealed direction for future refinement. CeVICA offers an integrated solution to rapid generation of divergent synthetic antibodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel antibody generation.

bioengineering↗

bioRxiv2020

A β-catenin-driven switch in TCF/LEF transcription factor binding to DNA targets sites promotes commitment of mammalian nephron progenitor cells

The canonical Wnt pathway transcriptional co-activator {beta}-catenin regulates self-renewal and differentiation of mammalian nephron progenitor cells (NPCs). We modulated {beta}-catenin levels in NPC cultures using the GSK3 inhibitor CHIR9902 (CHIR) to examine opposing developmental actions of {beta}-catenin. Low CHIR-mediated maintenance and expansion of NPCs is independent of direct engagement of TCF/LEF/ {beta}-catenin transcriptional complexes at low-CHIR dependent cell-cycle targets. In contrast, in high CHIR, TCF7/LEF1/{beta}-catenin complexes replaced TCF7L1/TCF7L2 binding on enhancers of differentiation-promoting target genes. Chromosome confirmation studies showed pre-established promoter-enhancer connections to these target genes in NPCs. High CHIR-associated de novo looping was observed in positive transcriptional feedback regulation to the canonical Wnt pathway. Thus, {beta}-catenins direct transcriptional role is restricted to the induction of NPCs where rising {beta}-catenin levels switch inhibitory TCF7L1/TCF7L2 complexes to activating LEF1/TCF7 complexes at primed gene targets poised for rapid initiation of a nephrogenic program.

developmental biology↗

bioRxiv2020

LncRNA Snhg1-driven self-reinforcing regulatory network promotes cardiomyocyte cytokinesis

Most of current cardiac regenerative approaches result in very limited cell division. Positive feedback loops are vital for cell division, but their role in CM regeneration remains unclear. We aimed to demonstrate that lncRNA Snhg1 formed a positive feedback loop with c-Myc to induce stable CM cytokinesis. We found that Snhg1 expression was increased in human and mouse fetal and myocardial infarction (MI) hearts, particularly in CMs. Snhg1 overexpression elicited stable CM proliferation and improved post-MI cardiac function. Antagonism of Snhg1 in neonatal mice inhibited CM proliferation and impaired cardiac repair after MI. Proliferative effect was confirmed using cardiac-specific transgenic mice. RNA pull-down assays showed that Snhg1 directly bound to PTEN and activated PI3K-Akt pathway, resulting in c-Myc activation. Chromatin immunoprecipitation experiments showed that Snhg1 expression was upregulated by c-Myc binding to the Snhg1 promoter region, indicating a positive feedback loop between c-Myc and Snhg1. In conclusion, c-Myc/Snhg1/PI3k-Akt positive feedback loop drove sustained activation of cell cycle re-entry and induced stable CM cytokinesis, and thus may be an attractive strategy for promoting heart regenerative response. Clinical PerspectivesMost of the current cardiac regenerative approaches result in very limited cell division and little new cardiomyocyte (CM) mass. Positive feedback loops are vital for cell division, but their role in CM regeneration remain unclear. Here, we identified the long noncoding RNA Snhg1 as a driver to induce stable CM division and improve cardiac function after myocardial infarction (MI) by forming a positive feedback loop to sustain PI3K-Akt signaling activation. This finding might provide a novel therapeutic of Snhg1 as a promising regenerative approach to improve the prognosis of patients with heart failure.

developmental biology↗

bioRxiv2020

Synergistic Combination of Cytotoxic Chemotherapy and Cyclin Dependent Kinase 4/6 Inhibitors in Biliary Tract Cancers

Biliary tract cancers (BTCs) are uncommon but highly lethal gastrointestinal malignancies. Gemcitabine/cisplatin is a standard-of-care (SOC) systemic therapy, but has a modest impact on survival and harbor toxicities including myelosuppression, nephropathy, neuropathy and ototoxicity. While BTCs are characterized by aberrations activating the cyclinD1-CDK4/6-CDKN2A-RB pathway, clinical use of CDK4/6 inhibitors as monotherapy is limited by lack of validated biomarkers, diffident pre-clinical efficacy and development of acquired drug resistance. Emerging studies have explored therapeutic strategies to enhance the anti-tumor efficacy of CDK4/6 inhibitors by combination with chemotherapy-regimens but their mechanism of action remains elusive. Here, we report in vitro and in vivo synergy in BTC models, showing enhanced efficacy, reduced toxicity and better survival with a combination comprising gemcitabine/cisplatin and CDK4/6 inhibitors. Furthermore, we demonstrated that abemaciclib monotherapy had only modest efficacy due to autophagy induced resistance. Notably, triplettherapy was able to potentiate efficacy through elimination of the autophagic flux. Correspondingly, abemaciclib potentiated RRM1 reduction, resulting in sensitization to gemcitabine. Conclusions: As such, these data provide robust pre-clinical mechanistic evidence of synergy between gemcitabine/cisplatin and CDK4/6 inhibitors, and delineate a path forward for translation of these findings to preliminary clinical studies in advanced BTC patients.

cancer biology↗

bioRxiv2020

Spaceflight decelerates the epigenetic clock orchestrated with a global alteration in DNA methylome and transcriptome in the mouse retina

Astronauts exhibit an assortment of clinical abnormalities in their eyes during long-duration spaceflight. The purpose of this study was to determine whether spaceflight induces epigenomic and transcriptomic reprogramming in the retina or alters the epigenetic clock. The mice were flown for 37 days in animal enclosure modules on the International Space Station; ground-based control animals were maintained under similar housing conditions. Mouse retinas were isolated and both DNA methylome and transcriptome were determined by deep sequencing. We found that a large number of genes were differentially methylated with spaceflight, whereas there were fewer differentially expressed genes at the transcriptome level. Several biological pathways involved in retinal diseases such as macular degeneration were significantly altered. Our results indicated that spaceflight decelerated the retinal epigenetic clock. This study demonstrates that spaceflight impacts the retina at the epigenomic and transcriptomic levels, and such changes could be involved in the etiology of eye-related disorders among astronauts.

genomics↗

bioRxiv2020

Stretch and Twist of HEAT Repeats Leads to Activation of DNA-PK Kinase

Phosphatidylinositol 3-kinase-related kinases (PIKKs) are composed of conserved FAT and kinase domains (FATKIN) along with varied solenoid structures made of HEAT repeats. These kinases are activated in response to cellular stress signals, but the mechanisms governing activation and regulation remain unresolved. For DNA-dependent protein kinase (DNA-PK), all existing structures represent inactive states with resolution limited to 4.3 [A] at best. Here we report the cryoEM structures of DNA-PKcs (catalytic subunit) bound to a DNA end, or complexed with Ku70/80 and DNA, in both inactive and activated forms at resolutions of 3.7 [A] overall, and 3.2 [A] for FATKIN. These structures reveal the sequential transition of DNA-PK from inactive to activated forms. Most notably, activation of the kinase involves previously unknown stretching and twisting within individual solenoid segments and coordinated shifts of neighboring segments in opposite directions. This unprecedented structural plasticity of helical repeats may be a general feature of HEAT-repeat proteins.

biochemistry↗

bioRxiv2020

Satellite subgenomic particles are key regulators of adeno-associated virus life cycle

Historically, AAV defective interfering particles (DI) were known as abnormal virions arising from natural replication and encapsidation errors. Through single virion genome analysis, we revealed that a major category of DI particles contains a double stranded DNA genome in a "snapback" configuration (SBG). The 5-SBGs include the P5 promoters and partial rep gene sequences. The 3-SBGs contains the capsid region. The molecular configuration of 5-SBGs allowed double stranded RNA transcription in their dimer configuration, which in turn regulate AAV rep expression and may improve AAV packaging. In contrast, 3-SBGs at its dimer configuration increased levels of cap protein. The generation and accumulation of 5-SBGs and 3-SBGs appears to be coordinated to balance the viral gene expression level. Therefore, the functions of 5-SBGs and 3-SBGs may help maximize the yield of AAV progenies. We postulate that AAV virus population behaved as a colony and utilizes its subgenomic particles to overcome the size limit of viral genome and encodes additional essential functions.

molecular biology↗