Biology subjects

Xu, C.

Publications and source records attributed to Xu, C..

At least 361 records · Page 20Linked to original sources

bioRxiv2020

Mycobacterium tuberculosis Rv3194c efficiently facilitates mycobacterial-lung epithelial interaction through its HA-binding site

Mycobacterium tuberculosis adhesins are surface-exposed molecules that mediate pathogen-host interaction, a fundamental step towards host infection. Here we show that serine protease (Rv3194c) promotes mycobacterial infection to lung epithelial through its hyaluronic acid (HA)-binding site. Both enzyme-linked immunosorbent assay and surface plasmon resonance analysis revealed that Rv3194c bound to HA. Utilizing synthetic peptides, we next defined HA-binding site of 20 amino acids from 91 to 110 of Rv3194c (P91-110). Immunofluorescence assay and an FACScan showed that Rv3194c was interacted with A549 cells (human lung epithelial cells), and its interaction was abolished by the addition of hyaluronidase or P91-110. Experimental infection in Vitro revealed that Rv3194c participates in attachment of recombinant Mycobacterium smegmatis (Rv3194c/MS) to A549 cells, and P91-110 treatment of A549 cells almost inhibited Rv3194c/MS-A549 cells interaction. To provide in vivo evidence, we constructed a reporter strain of M. smegmatis expressed a derivative of the firefly luciferase that is shifted to red (FFlucRT) in combination with Rv3194c (Rv3194c+FFlucRT/MS) to infect the rodents and monitor the progression of the disease. Using bioluminescence imaging and bacterial counts in lung tissue confirmed that Rv3194c dramatically enhanced the persistence of M. smegmatis. In addition, treatment of intratracheal Rv3194c+FFlucRT/MS-infected mice with P91-110 significantly suppressed the growth of Rv3194c+FFlucRT/MS in vivo. Taken together, these results demonstrate that Rv3194c was identified as a HA-binding adhesin, and P91-110 as anti-adhesion agents has potential for therapeutic and prophylactic interventions in mycobacterial infection.

microbiology↗

bioRxiv2020

Mitochondrial Proteostasis Requires Genes Encoded in a Neurodevelopmental Syndrome Locus that are Necessary for Synapse Function

Eukaryotic cells maintain proteostasis through mechanisms that require cytoplasmic and mitochondrial translation. Genetic defects affecting cytoplasmic translation perturb synapse development, neurotransmission, and are causative of neurodevelopmental disorders such as Fragile X syndrome. In contrast, there is little indication that mitochondrial proteostasis, either in the form of mitochondrial protein translation and/or degradation, is required for synapse development and function. Here we focus on two genes deleted in a recurrent copy number variation causing neurodevelopmental disorders, the 22q11.2 microdeletion syndrome. We demonstrate that SLC25A1 and MRPL40, two genes present in this microdeleted segment and whose products localize to mitochondria, interact and are necessary for mitochondrial protein translation and proteostasis. Our Drosophila studies show that mitochondrial ribosome function is necessary for synapse neurodevelopment, function, and behavior. We propose that mitochondrial proteostasis perturbations, either by genetic or environmental factors, are a novel pathogenic mechanism for neurodevelopmental disorders.

neuroscience↗

bioRxiv2020

CoolMPS™: Advanced massively parallel sequencing using antibodies specific to each natural nucleobase

Massively parallel sequencing (MPS) on DNA nanoarrays provides billions of reads at relatively low cost and enables a multitude of genomic applications. Further improvement in read length, sequence quality and cost reduction will enable more affordable and accurate comprehensive health monitoring tests. Currently the most efficient MPS uses dye-labeled reversibly terminated nucleotides (RTs) that are expensive to make and challenging to incorporate. Furthermore, a part of the dye-linker (scar) remains on the nucleobase after cleavage and interferes with subsequent sequencing cycles. We describe here the development of a novel MPS chemistry (CoolMPS) utilizing unlabeled RTs and four natural nucleobase-specific fluorescently labeled antibodies with fast (30 sec) binding. We implemented CoolMPS on MGIs PCR-free DNBSEQ MPS platform using arrays of 200nm DNA nanoballs (DNBs) generated by rolling circle replication and demonstrate 3-fold improvement in signal intensity and elimination of scar interference. Single-end 100-400 base and pair-end 2x150 base reads with high quality were readily generated with low out-of-phase incorporation. Furthermore, DNBs with less than 50 template copies were successfully sequenced by strong-signal CoolMPS with 3-times higher accuracy than in standard MPS. CoolMPS chemistry based on natural nucleobases has potential to provide longer, more accurate and less expensive MPS reads, including highly accurate "4-color sequencing" on the most efficient dye-crosstalk-free 2-color imagers with an estimated sequencing error rate of 0.00058% (one error in 170,000 base calls) in a proof-of-concept demonstration.

genomics↗

bioRxiv2020

Modulation of metabolic functions through Cas13d-mediated gene knockdown in liver

RNA knockdown in vivo carries significant potential for disease modelings and therapies. Despite the emerging approaches of CRISPR/Cas9-mediated permanent knock out of targeted genes, strategies targeting RNA for disruption are advantageous in the treatment of acquired metabolic disorders when permanent modification of the genome DNA is not appropriate, and RNA virus infection diseases when pathogenic DNA is not available (such as SARS-Cov-2 and MERS infections). Recently, Cas13d, a family of RNA-targeting CRISPR effectors, has been shown to accomplish robust down-regulation of cellular RNAs in mammalian cells in vitro. Among the various Cas13d subtypes, CasRx (RfxCas13d) showed the most potent RNA knockdown efficiency in HEK293T cells. However, the RNA-targeting activity of Cas13d still needs to be verified in vivo. In this study, the CasRx system was demonstrated to efficiently and functionally knock down genes related to metabolism functions, including Pten, Pcsk9 and lncLstr, in mouse hepatocytes. CasRx-mediated simultaneous knockdown of multiple genes was also achieved by sgRNA arrays, providing a useful strategy to modulate complex metabolism networks. Moreover, the AAV (adeno-associated virus)-mediated delivery of CasRx and Pcsk9 sgRNAs into mouse liver successfully decreased serum PCSK9, resulting in significant reduction of serum cholesterol levels. Importantly, CasRx-mediated knockdown of Pcsk9 is reversible and Pcsk9 could be repeatedly down-regulated, providing an effective strategy to reversibly modulate metabolic genes. The present work supplies a successful proof-of-concept trial that suggests efficient and regulatory knockdown of target metabolic genes for a designed metabolism modulation in the liver.

genomics↗

bioRxiv2020

Molecular Diversity Among Adult Hippocampal and Entorhinal Cells

The hippocampal-entorhinal system is comprised of functionally distinct subregions collectively critical for cognition, and selectively vulnerable to aging and pathological processes. To gain insights into neuronal and non-neuronal populations within this system, we performed single-nucleus transcriptional profiling from five human hippocampal-entorhinal subregions. We found that transcriptomic diversity of excitatory neurons across these subregions reflected the molecular transition from three-layered archicortex to six-layered neocortex. Additionally, mRNA and protein for DCX, an immature neuron marker, were clearly detected in some cells, but not in dentate granule cells, the cell-type predicted to be generated in adult neurogenesis. We also found that previously functionally uncharacterized METTL7B was enriched in human and non-human primate neuronal subtypes less vulnerable to initial Alzheimers disease pathology. Proteomic and biochemical assays revealed METTL7B interacts with Alzheimers disease-related proteins, including APP, and its overexpression reduced amyloid-beta generation. These results reveal cell type-specific molecular properties relevant for hippocampal-entorhinal physiology and dysfunction.

neuroscience↗

bioRxiv2019

Advanced Whole Genome Sequencing Using a Complete PCR-free Massively Parallel Sequencing (MPS) Workflow

BackgroundSystematic errors can be introduced from DNA amplification during massively parallel sequencing (MPS) library preparation and sequencing array formation. Polymerase chain reaction (PCR)-free genomic library preparation methods were previously shown to improve whole genome sequencing (WGS) quality on the Illumina platform, especially in calling insertions and deletions (InDels). We hypothesized that substantial InDel errors continue to be introduced by the remaining PCR step of DNA cluster generation. In addition to library preparation and sequencing, data analysis methods are also important for the accuracy of the output data.In recent years, several machine learning variant calling pipelines have emerged, which can correct the systematic errors from MPS and improve the data performance of variant calling. ResultsHere, PCR-free libraries were sequenced on the PCR-free DNBSEQ arrays from MGI Tech Co., Ltd. (referred to as MGI) to accomplish the first true PCR-free WGS which the whole process is truly not only PCR-free during library preparation but also PCR-free during sequencing. We demonstrated that PCR-based WGS libraries have significantly (about 5 times) more InDel errors than PCR-free libraries.Furthermore, PCR-free WGS libraries sequenced on the PCR-free DNBSEQ platform have up to 55% less InDel errors compared to the NovaSeq platform, confirming that DNA clusters contain PCR-generated errors.In addition, low coverage bias and less than 1% read duplication rate was reproducibly obtained in DNBSEQ PCR-free using either ultrasonic or enzymatic DNA fragmentation MGI kits combined with MGISEQ-2000. Meanwhile, variant calling performance (single-nucleotide polymorphisms (SNPs) F-score>99.94%, InDels F-score>99.6%) exceeded widely accepted standards using machine learning (ML) methods (DeepVariant or DNAscope). ConclusionsEnabled by the new PCR-free library preparation kits, ultra high-thoughput PCR-free sequencers and ML-based variant calling, true PCR-free DNBSEQ WGS provides a powerful solution for improving WGS accuracy while reducing cost and analysis time, thus facilitating future precision medicine, cohort studies, and large population genome projects.

genomics↗

bioRxiv2019

Signature activities of 20S proteasome include degradation of the ubiquitin-tag with the protein under hypoxia

Careful removal of unwanted proteins is necessary for cell survival. The primary constitutive intracellular protease is the 26S proteasome complex, often found in equilibrium with its free catalytic subcomplex- the 20S core particle. Protein degradation by 26S is tightly regulated by prior ubiquitination of substrates, whereas 20S is amenable to substrates with an unstructured segment. Differentiating their contributions to intracellular proteolysis is challenging due to their common catalytic sites. Here, by chemically synthesizing a synoptic set of homogenous ubiquitinated proteins, we ascribe signature features to 20S function and demonstrate a unique property: degrading the ubiquitin-tag along with the target protein. Cryo-EM confirms that a ubiquitinated substrate can induce asymmetric conformational changes to 20S. Mass-spectrometry of intracellular peptidome under hypoxia and in human failing heart identifies the signature properties of 20S in cells. Moreover, the ability of 20S proteasome to clear toxic proteins rapidly, contributes to better survival under these conditions.

biochemistry↗

bioRxiv2019

Bmal1 integrates mitochondrial metabolism and macrophage activation

Metabolic pathways and inflammatory processes are under circadian regulation. While rhythmic immune cell recruitment is known to impact infection outcomes, whether the circadian clock modulates immunometabolism remains unclear. We find the molecular clock Bmal1 is induced by inflammatory stimulants, including Ifn-{gamma}/lipopolysaccharide (M1) and tumor conditioned medium, to maintain mitochondrial metabolism under these metabolically stressed conditions in mouse macrophages. Upon M1 stimulation, myeloid-specific Bmal1 knockout (M-BKO) renders macrophages unable to sustain mitochondrial function, enhancing succinate dehydrogenase (SDH)-mediated mitochondrial ROS production and Hif-1-dependent metabolic reprogramming and inflammatory damage. In tumor-associated macrophages, the aberrant Hif-1 activation and metabolic dysregulation by M-BKO contribute to an immunosuppressive tumor microenvironment. Consequently, M-BKO increases melanoma tumor burden, while administrating an SDH inhibitor dimethyl malonate suppresses tumor growth. Therefore, Bmal1 functions as a metabolic checkpoint integrating macrophage mitochondrial metabolism, redox homeostasis and effector functions. This Bmal1-Hif-1 regulatory loop may provide therapeutic opportunities for inflammatory diseases and immunotherapy.

cell biology↗

bioRxiv2019

Single-cell RNA-seq data reveals TNBC tumor heterogeneity through characterizing subclone compositions and proportions

Understanding subclonal architecture and their biological functions poses one of the key challenges to deeply portray and investigative the cause of triple-negative breast cancer (TNBC). Here we combine single-cell and bulk sequencing data to analyze tumor heterogeneity through characterizing subclone compositions and proportions. Based on sing-cell RNA-seq data (GSE118389) we identified five distinct cell subpopulations and characterized their biological functions based on their gene markers. According to the results of functional annotation, we found that C1 and C2 are related to immune functions, while C5 is related to programmed cell death. Then based on subclonal basis gene expression matrix, we applied deconvolution algorithm on TCGA tissue RNA-seq data and observed that microenvironment is diverse among TNBC subclones, especially C1 is closely related to T cells. Whats more, we also found that high C5 proportions would led to poor survival outcome, log-rank test p-value and HR [95%CI] for five years overall survival in GSE96058 dataset were 0.0158 and 2.557 [1.160-5.636]. Collectively, our analysis reveals both intra-tumor and inter-tumor heterogeneity and their association with subclonal microenvironment in TNBC (subclone compositions and proportions), and uncovers the organic combination of subclones dictating poor outcomes in this disease. HighlightsWe applied deconvolution algorithm on subclonal basis gene expression matrix to link single cells and bulk tissue together.

cancer biology↗

bioRxiv2019

The microRNA476a/RFL module regulates adventitious root formation through a mitochondria-dependent pathway in Populus

Adventitious root (AR) formation at the base of stem cuttings determines the efficiency of clonal propagation for woody plants. Many endogenous and environmental factors influence AR formation. However, our knowledge about the regulation of AR development by mitochondrial metabolism in plants is very limited. Here we identified Populus-specific miR476a as a novel regulator of wound-induced adventitious rooting via orchestrating mitochondrial homeostasis in poplar. MiR476a exhibited inducible expression during AR formation and directly targets several Restorer of Fertility like (RFL) genes encoding mitochondrion-localized pentatricopeptide repeat proteins. Genetic modification of miR476-RFL expression revealed the miR476/RFL-mediated dynamic regulation of mitochondrial homeostasis on AR formation in transgenic poplar. Furthermore, mitochondrial perturbation via exogenous chemical inhibitor validated that the miR476a/RFL-directed AR formation depended on mitochondrial regulation though modulating the auxin pathway. Our results established a miRNA-directed mitochondrion-auxin signaling cascade required for AR development, providing novel insights into the understanding of mitochondrial regulation on plant developmental plasticity.

plant biology↗

bioRxiv2019

Quantitative analysis of 1300-nm three-photon calcium imaging in the mouse brain

1300-nm three-photon calcium imaging has emerged as a useful technique to allow calcium imaging in deep brain regions. Application to large-scale neural activity imaging entails a careful balance between recording fidelity and tissue heating. We calculated and experimentally verified the excitation pulse energy to achieve the minimum photon count required for the detection of calcium transients in GCaMP6s-expressing neurons for 920-nm two-photon and 1320-nm three-photon excitation, respectively. Brain tissue heating by continuous three-photon imaging was simulated with Monte Carlo method and experimentally validated with immunohistochemistry. We observed increased immunoreactivity with 150 mW excitation power at 1.0- and 1.2-mm imaging depths. Based on the data, we explained how three-photon excitation achieves better calcium imaging fidelity than two-photon excitation in the deep brain and quantified the imaging depth where three-photon microscopy should be applied. Our analysis presents a translatable model for the optimization of three-photon calcium imaging based on experimentally tractable parameters.

neuroscience↗

bioRxiv2019

AutoGenome: An AutoML Tool for Genomic Research

Deep learning have made great successes in traditional fields like computer vision (CV), natural language processing (NLP) and speech processing. Those achievements greatly inspire researchers in genomic study and make deep learning in genomics a very hot topic. Convolutional neural network (CNN) and recurrent neural network (RNN) are frequently used for genomic sequence prediction problems; multiple layer perception (MLP) and auto-encoders (AE) are frequently used for genomic profiling data like RNA expression data and gene mutation data. Here, we introduce a new neural network architecture, named residual fully-connected neural network (RFCN) and demonstrate its advantage for modeling genomic profiling data. We further incorporate AutoML algorithms and implement AutoGenome, an end-to-end automated genomic deep learning framework. By utilizing the proposed RFCN architectures, automatic hyper-parameter search and neural architecture search algorithms, AutoGenome can train high-performance deep learning models for various kinds of genomic profiling data automatically. To make researchers better understand the trained models, AutoGenome can assess the feature importance and export the most important features for supervised learning tasks, and the representative latent vectors for unsupervised learning tasks. We envision AutoGenome to become a popular tool in genomic studies.

genomics↗

bioRxiv2019

Producing Human Amniotic Epithelial Cells-only Membrane for Transplantation

Human amniotic epithelial cells (hAECs), as pluripotent stem cells, have characteristics of immune privilege and great clinical potential. Here, we produced hAECs membrane consisting of single-layer hAECs and basal membrane (BM) of human amniotic membrane (hAM). In conventional methods, hAECs were isolated from hAM by repeated trypsin digestion. In this study, collagenase I and cell scraper were used to remove human amniotic mesenchymal stem cells (hAMSCs) from hAM and hAECs-only membranes were produced. These hAECs on the membranes were evaluated by surface biomarkers including epithelial cell adhesion molecule (EpCAM), stage-specific embryonic antigen 4 (SSEA4) and endoglin (CD105), transcriptional level of biomarkers including POU class 5 homeobox 1 (OCT4), sex determining region Y-box 2 (SOX2), fibroblast growth factor 4 (FGF4), immunofluorescence of cytokeratin-8 (CK-8), alpha smooth muscle actin (-SMA) and collagen type I alpha 1 chain (ColA1). Finally, the hAECs membrane were transplanted on skin-removed mice to evaluate its effect on wound healing. In comparison to the hAECs isolated by the conventional methods, the cells isolated by this proposed method had higher purity of hAECs, expressed higher in pluripotency related genes, and maintained an epithelium construction in a long-term culture. In mice application, the hAECs membrane effectively improved the skin wound healing. An efficient method was successfully established to produce hAECs membrane in this work which not only held promise to obtain hAECs in higher purity and quality, but also showed practical clinical potential.

bioengineering↗

bioRxiv2019

DNB-Based On-Chip Motif Finding (DocMF): a High-Throughput Method to Profile Different Types of Protein-DNA Interactions

Here we report a highly sensitive DNB-based on-chip Motif Finding (DocMF) system that utilizes high throughput next-generation-sequencing (NGS) chips to profile protein binding or cleaving activity. Using DocMF, we successfully identified a variety of endonuclease recognition sites and the protospacer-adjacent-motif (PAM) sequences of different CRISPR systems. Our DocMF platform can simultaneously screen both 5 and 3 PAM regions with high coverage using the same NGS library/chip. For the well-studied SpCas9, our DocMF platform identified a small proportion of noncanonical 5-NAG-3 ([~]5%) and 5-NGA-3 ([~]1.6%), in addition to its common PAMs, 5-NGG-3 ([~]89.9%). We also used the DocMF to assay two uncharacterized Cas endonucleases, VeCas9 and BvCpf1. VeCas9 PAMs were not detected by the conventional PAM depletion method. However, DocMF discovered that both VeCas9 and BvCpf1 required broader and more complicated PAM sequences for target recognition. VeCas9 preferred the R-rich motifs, whereas BvCpf1 used the T-rich PAMs. Moreover, after slightly changing the experimental protocol, we observed that dCas9, a DNA-binding protein lacking endonuclease activity, preferably binded to the previously reported PAMs 5-NGG-3. In summary, our studies demonstrate that DocMF is the first tool with the capacity to exhaustively assay both the binding and the cutting properties of different DNA-binding proteins.

molecular biology↗

bioRxiv2019

Cell-Surface Proteomic Profiling in the Fly Brain Uncovers New Wiring Regulators

Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues, and thus govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally-resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed a global down-regulation of wiring molecules and an up-regulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in vivo screen identified 20 new cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally-resolved in situ cell-surface proteomic profiling in discovering new regulators of brain wiring.

neuroscience↗

bioRxiv2019

Erosion of the Epigenetic Landscape and Loss of Cellular Identity as a Cause of Aging in Mammals

All living things experience entropy, manifested as a loss of inherited genetic and epigenetic information over time. As budding yeast cells age, epigenetic changes result in a loss of cell identity and sterility, both hallmarks of yeast aging. In mammals, epigenetic information is also lost over time, but what causes it to be lost and whether it is a cause or a consequence of aging is not known. Here we show that the transient induction of genomic instability, in the form of a low number of non-mutagenic DNA breaks, accelerates many of the chromatin and tissue changes seen during aging, including the erosion of the epigenetic landscape, a loss of cellular identity, advancement of the DNA methylation clock and cellular senescence. These data support a model in which a loss of epigenetic information is a cause of aging in mammals.\n\nOne Sentence SummaryThe act of repairing DNA breaks induces chromatin reorganization and a loss of cell identity that may contribute to mammalian aging

molecular biology↗

bioRxiv2019

Inference of Single-Cell Phylogenies from Lineage Tracing Data

The pairing of CRISPR/Cas9-based gene editing with massively parallel single-cell readouts now enables large-scale lineage tracing. However, the rapid growth in complexity of data from these assays has outpaced our ability to accurately infer phylogenetic relationships. To address this, we provide three resources. First, we introduce Cassiopeia - a suite of scalable and theoretically grounded maximum parsimony approaches for tree reconstruction. Second, we provide a simulation framework for evaluating algorithms and exploring lineage tracer design principles. Finally, we generate the most complex experimental lineage tracing dataset to date - consisting of 34,557 human cells continuously traced over 15 generations, 71% of which are uniquely marked - and use it for benchmarking phylogenetic inference approaches. We show that Cassiopeia outperforms traditional methods by several metrics and under a wide variety of parameter regimes, and provide insight into the principles for the design of improved Cas9-enabled recorders. Together these should broadly enable large-scale mammalian lineage tracing efforts. Cassiopeia and its benchmarking resources are publicly available at www.github.com/YosefLab/Cassiopeia.

bioinformatics↗

bioRxiv2019

Non-invasive multiphoton imaging of neural structure and activity in Drosophila

We developed a multiphoton imaging method to capture neural structure and activity in behaving flies through the intact cuticles. Our measurements show that the fly head cuticle has surprisingly high transmission at wavelengths > 900 nm, and the difficulty of through-cuticle imaging is due to the air sacs and/or fat tissue underneath the head cuticle. By compressing the air sacs, we performed deep multiphoton imaging of fly brains through the intact cuticle. Our anatomical and functional imaging results show that 2- and 3-photon imaging are comparable in superficial regions such as the mushroom body, but 3-photon imaging is superior in deeper regions such as the central complex and beyond. We further demonstrated 2-photon through-cuticle functional imaging of odor-evoked calcium responses from the mushroom body {gamma}-lobes in behaving flies short-term and long-term (12 consecutive hours). The through-cuticle imaging method developed here extends the time limits of in vivo imaging in flies, and opens up new ways to capture neural structure and activity from the intact fly brain.

neuroscience↗