Biology subjects

Xu, M.

Publications and source records attributed to Xu, M..

At least 253 records · Page 14Linked to original sources

bioRxiv2020

Identification of SARS-CoV-2 3CL Protease Inhibitors by a Quantitative High-throughput Screening

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emphasized the urgency to develop effective therapeutics. Drug repurposing screening is regarded as one of the most practical and rapid approaches for the discovery of such therapeutics. The 3C like protease (3CLpro), or main protease (Mpro) of SARS-CoV-2 is a valid drug target as it is a specific viral enzyme and plays an essential role in viral replication. We performed a quantitative high throughput screening (qHTS) of 10,755 compounds consisting of approved and investigational drugs, and bioactive compounds using a SARS-CoV-2 3CLpro assay. Twenty-three small molecule inhibitors of SARS-CoV-2 3CLpro have been identified with IC50s ranging from 0.26 to 28.85 M. Walrycin B (IC50 = 0.26 {micro}M), Hydroxocobalamin (IC50 = 3.29 {micro}M), Suramin sodium (IC50 = 6.5 {micro}M), Z-DEVD-FMK (IC50 = 6.81 {micro}M), LLL-12 (IC50 = 9.84 {micro}M), and Z-FA-FMK (IC50 = 11.39 {micro}M) are the most potent 3CLpro inhibitors. The activities of anti-SARS-CoV-2 viral infection was confirmed in 7 of 23 compounds using a SARS-CoV-2 cytopathic effect assay. The results demonstrated a set of SARS-CoV-2 3CLpro inhibitors that may have potential for further clinical evaluation as part of drug combination therapies to treating COVID-19 patients, and as starting points for chemistry optimization for new drug development.

pharmacology and toxicology↗

bioRxiv2020

Targeted Genome Editing of Bacteria Within Microbial Communities

Knowledge of microbial gene functions comes from manipulating the DNA of individual species in isolation from their natural communities. While this approach to microbial genetics has been foundational, its requirement for culturable microorganisms has left the majority of microbes and their interactions genetically unexplored. Here we describe a generalizable methodology for editing the genomes of specific organisms within a complex microbial community. First, we identified genetically tractable bacteria within a community using a new approach, Environmental Transformation Sequencing (ET-Seq), in which non-targeted transposon integrations were mapped and quantified following community delivery. ET-Seq was repeated with multiple delivery strategies for both a nine-member synthetic bacterial community and a ~200-member microbial bioremediation community. We achieved insertions in 10 species not previously isolated and identified natural competence for foreign DNA integration that depends on the presence of the community. Second, we developed and used DNA-editing All-in-one RNA-guided CRISPR-Cas Transposase (DART) systems for targeted DNA insertion into organisms identified as tractable by ET-Seq, enabling organism- and locus-specific genetic manipulation within the community context. These results demonstrate a strategy for targeted genome editing of specific organisms within microbial communities, establishing a new paradigm for microbial manipulation relevant to research and applications in human, environmental, and industrial microbiomes.

microbiology↗

bioRxiv2020

Targeting heparan sulfate proteoglycan-assisted endocytosis as a COVID-19 therapeutic option

The cell entry of SARS-CoV-2 has emerged as an attractive drug repurposing target for COVID-19. Here we combine genetics and chemical perturbation to demonstrate that ACE2-mediated entry of SARS-CoV and CoV-2 requires the cell surface heparan sulfate (HS) as an assisting cofactor: ablation of genes involved in HS biosynthesis or incubating cells with a HS mimetic both inhibit Spike-mediated viral entry. We show that heparin/HS binds to Spike directly, facilitates the attachment of viral particles to the cell surface to promote cell entry. We screened approved drugs and identified two classes of inhibitors that act via distinct mechanisms to target this entry pathway. Among the drugs characterized, Mitoxantrone is a potent HS inhibitor, while Sunitinib and BNTX disrupt the actin network to indirectly abrogate HS-assisted viral entry. We further show that drugs of the two classes can be combined to generate a synergized activity against SARS-CoV-2-induced cytopathic effect. Altogether, our study establishes HS as an attachment factor that assists SARS coronavirus cell entry, and reveals drugs capable of targeting this important step in the viral life cycle.

cell biology↗

bioRxiv2020

RNF219 interacts with CCR4-NOT in regulating stem cell differentiation

Regulation of RNA stability plays a crucial role in gene expression control. Deadenylation is the initial rate-limiting step for the majority of RNA decay events. Here, we show that RING Finger Protein 219 (RNF219) interacts with the CCR4-NOT deadenylase complex. RNF219-CCR4-NOT exhibits deadenylation activity in vitro. RNA-seq analyses identify some of the 2-cell specific genes and the neuronal genes significantly down-regulated upon RNF219 knockdown, while up-regulated after depletion of the CCR4-NOT subunit CNOT10 in mouse embryonic stem (ES) cells. RNF219 depletion leads to impaired neuronal lineage commitment during ES cell differentiation. Our study suggests that RNF219 is a novel interacting partner of CCR4-NOT, and required for maintenance of ES cell pluripotency.View Full Text

molecular biology↗

bioRxiv2020

A Simple Procedure for Bacterial Expression and Purification of the Fragile X Protein Family

The fragile X protein family consists of three RNA-binding proteins involved in translational regulation. Fragile X mental retardation protein (FMRP) is well-studied, as its loss leads to fragile X syndrome, a neurodevelopmental disorder which is the most prevalent form of inherited mental retardation and the primary monogenetic cause of autism. Fragile X related proteins 1 and 2 (FXR1P & FXR2P) are autosomal paralogs of FMRP that are involved in promoting muscle development and neural development, respectively. There is great interest in studying this family of proteins, yet researchers have faced much difficulty in expressing and purifying the full-length versions of these proteins in sufficient quantities. We have developed a simple, rapid, and inexpensive procedure that allows for the recombinant expression and purification of full-length human FMRP, FXR1P, and FXR2P from Escherichia coli in high yields, free of protein and nucleic acid contamination. In order to assess the proteins function after purification, we confirmed their binding to pseudoknot and G-quadruplex forming RNAs.

biochemistry↗

bioRxiv2020

Spontaneous variability predicts adaptive motor response in vocal pitch control

Our motor system uses sensory feedback to keep behavioral performance in desired status. From this view, motor fluctuation is not simply noise inevitably caused in the nervous system, but should provide a role in generating variations to explore better outcomes via their sensory feedback. Vocal control system offers a good model to investigate such adaptive sensory-motor interactions. The pitch, or fundamental frequency (FF), of voice is adaptively regulated by hearing its auditory feedback to compensate FF deviations. Animal studies, particularly for songbirds, have demonstrated that the variability in vocal features contributes to the adaptive control, although the same issue in human vocalizations has remained unclear. Here, we tested whether and how the motor variability contributes to adaptive control of vocal FF in humans. We measured the amount of compensatory vocal responses against FF shifts in the auditory feedback, and quantified the motor variability as amplitudes of spontaneous FF fluctuations during no shift vocalizations. The result showed a positive correlation between the ratio of compensation and the spontaneous vocal variability. Further analysis indicated that this correlation was due to slowly fluctuating components (<5 Hz) of the variability, but not fast fluctuations (6-30 Hz), which is likely to reflect controllability from the central nervous system. Moreover, the compensatory responses consisted of the same frequency range with the slow component in the spontaneous variability. These findings consistently demonstrated that the spontaneous motor variability predicts the adaptive control in vocal FF, supporting the motor exploration hypothesis. Significance statementWe regulate our own vocalization by hearing own voice. This fact is typically observed as canceling-out (compensatory) responses in vocalized pitch when artificial pitch shifts were induced in the auditory feedback of own voice. Interestingly, the amount of such compensation widely ranges among talkers from perfect cancellation to almost nothing. Here we demonstrated that participants who spontaneously exhibited larger fluctuations showed greater amounts of the compensation against feedbacked pitch shifts. Our in-depth analyses showed that slowly fluctuating components in spontaneous pitch variability are specifically correlated with the compensation ratios, and was shared in the compensatory response as a dominant component. These findings support the idea that such variability contributes to generating motor explorations to find better outcomes in motor controls.

neuroscience↗

bioRxiv2020

An OpenData portal to share COVID-19 drug repurposing data in real time

The National Center for Advancing Translational Sciences (NCATS) has developed an online open science platform - named the NCATS OpenData Portal (ODP) - for quickly and freely sharing complete NCATS translational datasets via an open-access, user-friendly interface. This paper describes the establishment of the ODP, initially deployed during the COVID-19 crisis, and provides a detailed analysis of COVID-19 drug repurposing screening datasets that served as the first large-scale use case for the platform. Over 10,000 compounds were tested across 17 quantitative high-throughput assays, covering a wide spectrum of the SARS-CoV-2 life cycle. In total, over 87,000 concentration-response curves and 426,000 data points were made publicly available on ODP in near real-time, enabling immediate access to complete datasets. The resource is flexible in accommodating various types and structures of data, and it has already expanded since its launch to host additional datasets for COVID-19, other viruses of pandemic potential, and beyond. The OpenData Portal has been designed as a scalable platform for real-time data sharing across drug discovery campaigns, regardless of disease area, with the overarching goal of accelerating discovery at NCATS, the NIH, and the greater scientific community.

microbiology↗

bioRxiv2020

Haplotype-Resolved Assembly for Synthetic Long Reads Using a Trio-Binning Strategy

The accuracy and completeness of genome haplotyping are crucial for characterizing the relationship between human disease susceptibility and genetic variations, especially for the heterozygous variations. However, most of current variations are unphased genotypes, and the construction of long-range haplotypes remains challenging. We introduced a de novo haplotype-resolved assembly tool, HAST that exports two haplotypes of a diploid species for synthetic long reads with trio binning. It generates parental distinguishing k-mer libraries, partitions reads from the offspring according to the unique markers, and individually assembles them to resolve the haplotyping problem. Based on the stLFR co-barcoding data of an Asian as well as his parental massive parallel sequencing data, we utilized HAST to recover both haplotypes with a scaffold N50 of >11 Mb and an assembly accuracy of 99.99995% (Q63). The complete and accurate employment of long-range haplotyping information provided sub-chromosome level phase blocks (N50 [~]13 Mb) with 99.6% precision and 94.1% recall on average. We suggest that the accurate and efficient approach accomplishes the regeneration of the haplotype chromosomes with trio binning, thus promoting the determination of haplotype phase, the heterosis of crossbreeding, and the formation of autopolyploid and allopolyploid.

bioinformatics↗

bioRxiv2020

Systematic characterization of somatic mutation-mediated microRNA regulatory network perturbations

Somatic mutations are a major source of cancer development. Many driver mutations have been identified in protein coding regions. However, the function of mutations located in microRNAs (miRNAs) and their target binding sites along the human genome remains largely unknown. Here, we built comprehensive cancer-specific miRNA regulatory networks across 30 cancer types to systematically analyze the effect of mutations on miRNA related pathways. 3,518,261 mutations from 9,819 samples were mapped to miRNA-gene interactions (mGI), and mutations in miRNAs versus in their target genes show a mutually exclusive pattern in almost all cancer types. Using a linear regression method, we further identified 89 driver mutations in 14 cancer types that can significantly perturb miRNA regulatory networks. We find that driver mutations play their roles by altering RNA binding energy and the expression of target genes. Finally, we demonstrate that mutated driver gene targets are significantly down-regulated in cancer and function as tumor suppressors during cancer progression, suggesting potential miRNA candidates with significant clinical implications. We provide this data resource (CanVar-mGI) through a user-friendly, open-access web portal. Together, our results will facilitate novel non-coding biomarker identification and therapeutic drug design.

bioinformatics↗

bioRxiv2020

Cascade Diversification Directs the Generation of Neuronal Diversity in Hypothalamus

The hypothalamus contains an astounding heterogeneity of neurons to achieve its role in regulating endocrine, autonomic and behavioral functions. Despite previous progress in deciphering the gene regulatory programs linked to hypothalamus development, its molecular developmental trajectory and origin of neuronal diversity remain largely unknown. Here we combine transcriptomic profiling of 43,261 cells derived from Rax+ hypothalamic neuroepithelium with lineage tracing to map a developmental landscape of mouse hypothalamus and delineate the developmental trajectory of radial glial cells (RGCs), intermediate progenitor cells (IPCs), nascent neurons and peptidergic neurons in the lineage hierarchy. We show that RGCs adopt a conserved strategy for multipotential differentiation but generate both Ascl1+ and Neurog2+ IPCs, which display regionally differential origins in telencephalon. As transit-amplifying cells, Ascl1+ IPCs differ from their telencephalic counterpart by displaying fate bifurcation to produce both glutamatergic and GABAergic neurons. After classifying the developing neurons into 29 subtypes coded by diverse transcription factors, neurotransmitters and neuropeptides, we identified their molecular determinants via regulon analysis and further found that postmitotic neurons at nascent state possess the potential to resolve into more diverse subtypes of peptidergic neurons. Together, our study offers a single-cell framework for hypothalamus development and reveals that multiple cell types along the order of lineage hierarchy contribute to the fate diversification of hypothalamic neurons in a stepwise fashion, suggesting that a cascade diversifying model can deconstruct the origin of neuronal diversity.

developmental biology↗

bioRxiv2020

SCAN-ATAC Sim: a scalable and efficient method to simulate single-cell ATAC-seq from bulk-tissue experiments

SummaryscATAC-seq is a powerful approach for characterizing cell-type-specific regulatory landscapes. However, it is difficult to benchmark the performance of various scATAC-seq analysis techniques (such as clustering and deconvolution) without having a priori a known set of gold-standard cell types. To simulate scATAC-seq experiments with known cell-type labels, we introduce an efficient and scalable scATAC-seq simulation method (SCAN-ATAC-Sim) that down-samples bulk ATAC-seq data (e.g., from representative cell lines or tissues). Our protocol uses a consistent but tunable signal-to-noise ratio across cell types in a scATAC-seq simulation for integrating bulk experiments with different levels of background noise, and it independently samples twice without replacement to account for the diploid genome. Because it uses an efficient weighted reservoir sampling algorithm and is highly parallelizable with OpenMP, our implementation in C++ allows millions of cells to be simulated in less than an hour on a laptop computer. AvailabilitySCAN-ATAC-Sim is available at scan-atac-sim.gersteinlab.org. Contactpi@gersteinlab.org Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2020

The SARS-CoV-2 cytopathic effect is blocked with autophagy modulators

SARS-CoV-2 is a new type of coronavirus capable of rapid transmission and causing severe clinical symptoms; much of which has unknown biological etiology. It has prompted researchers to rapidly mobilize their efforts towards identifying and developing anti-viral therapeutics and vaccines. Discovering and understanding the virus’ pathways of infection, host-protein interactions, and cytopathic effects will greatly aid in the design of new therapeutics to treat COVID-19. While it is known that chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including inhibiting autophagy, there are also dose-limiting toxicities in patients that make clearly establishing their potential mechanisms-of-action problematic. Therefore, we evaluated a range of other autophagy modulators to identify an alternative autophagy-based drug repurposing opportunity. In this work, we found that 6 of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero-E6 cells with EC50 values ranging from 2.0 to 13 µM and selectivity indices ranging from 1.5 to >10-fold. Immunofluorescence staining for LC3B and LysoTracker dye staining assays in several cell lines indicated their potency and efficacy for inhibiting autophagy correlated with the measurements in the SARS-CoV-2 cytopathic effect assay. Our data suggest that autophagy pathways could be targeted to combat SARS-CoV-2 infections and become an important component of drug combination therapies to improve the treatment outcomes for COVID-19.One Sentence Summary Blocking SARS-CoV-2 cytopathic effects with selective autophagy inhibitors underlying the clinical benefits of chloroquine and hydroxychloroquine.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2020

A GRAB sensor reveals activity-dependent non-vesicular somatodendritic adenosine release

The purinergic signaling molecule adenosine (Ado) modulates many physiological and pathological brain functions,but its spatiotemporal release dynamics in the brain remains largely unknown. We developed a genetically encoded GPCR-Activation-Based Ado sensor (GRABAdo) in which Ado-induced changes in the human A2A receptor are reflected by fluorescence increases. This GRABAdo revealed that neuronal activity-induced extracellular Ado elevation was due to direct Ado release from somatodendritic regions of the neuron, requiring calcium influx through L-type calcium channels, rather than the degradation of extracellular ATP. The Ado release was slow ([~]30 s) and depended on equilibrative nucleoside transporters (ENTs) rather than conventional vesicular release mechanisms. Thus, GRABAdo reveals an activity-dependent slow Ado release from somatodendritic region of the neuron, potentially serving modulating functions as a retrograde signal.

neuroscience↗

bioRxiv2020

Cancer SIGVAR: A semi-automated interpretation tool for germline variants of hereditary cancer-related genes

The American College of Medical Genetics and Genomics and the Association for Molecular Pathology published guidelines in 2015 for the clinical interpretation of Mendelian disorder sequence variants based on 28 criteria. ClinGen Sequence Variant Interpretation (SVI) Working Groups have developed many adaptations or refinements of these guidelines to improve the consistency of interpretation. We combined the most recent adaptations to expand the criteria from 28 to 48 and developed a tool called Cancer SIGVAR to help healthcare workers and genetic counselors interpret the clinical significance of cancer germline variants, which is critical for the clinical diagnosis and treatment of hereditary cancer. Our tool can accept VCF files as input and realize fully automated interpretation based on 21 criteria and semi-automated interpretation based on 48 criteria. We validated our tool on the ClinVar and CLINVITAE benchmark databases for the accuracy of fully automated interpretation, achieving an average consistency for pathogenic and benign assessment up to 93.40% and 82.54%, respectively. We compared Cancer SIGVAR with a similar tool, InterVar, and analyzed the main differences in criteria and implementation. In addition, to verify the performance of semi-automated interpretation based on 48 criteria, we selected 911 variants from two benchmark databases and reached an average classification consistency of 98.35%. Our findings highlight the need to optimize automated interpretation tools based on constantly updated guidelines.

genetics↗

bioRxiv2020

Clinical Characteristics and Risk of Second Primary Lung Cancer After Cervix Cancer: A Population-Based Study

BackgroundLung cancer as a second primary malignancy is increasingly common, but the clinical characteristics of second primary non-small cell lung cancer after cervix cancer (CC-NSCLC) in comparison with first primary non-small cell lung cancer (NSCLC1) is unknown. MethodsThe Surveillance, Epidemiology and EndResults (SEER) cancer registry between 1998 and 2010 was used to conduct a large population-based cohort analysis. Demographic and clinical characteristics as well as prognostic data were systematically analyzed. We further compared overall survival (OS) in the two cohorts. Risk factors of secondary primary lung cancer in cervical cancer patients were also analyzed. Results557 (3.52%) had developed second primary lung cancer after cervix cancer and 451 were eligible for inclusion in the final analyses. In comparison to NSCLC1, patients with CC-NSCLC had a higher rate of squamous cell carcinoma (SCC) (36.59% vs. 19.07%, p<0.01). Median OS was longer for CC-NSCLC than for NSCLC1 before propensity score matching (PSM) (16 vs. 13months) but there was no significant difference after PSM. High-risk factors in cervical cancer to developing CC-NSCLC include: 50-79years old, black race (OR 1.417; 95%CI 1.095-1.834; p<0.05)and history of radiotherapy (OR 1.392; 95%CI 1.053-1.841; p<0.05). Conclusion50-79years old, black race and history of radiotherapy were independent risk factors of second primary lung cancer in cervical cancer patient. CC-NSCLC patients had distinctive clinical characteristics and a better prognosis compared with NSCLC1 patients.

cancer biology↗

bioRxiv2020

A temperature-dependent molecular switch to adjust behavior to changing environmental conditions

Daily temporal organisation of behavioural and physiological functions offers a fitness advantage for most animals. Optimized temporal niches are determined by an interplay between external environmental rhythms and internal circadian clocks. While daily light:dark cycles serve as a robust time cue (Zeitgeber) to synchronise circadian clocks, it is not clear how animals experiencing only weak environmental cues deal with this problem. Like humans, flies of the genus Drosophila originate in sub-Saharan Africa and spread North in Europe up to the polar circle where they experience extremely long days in the summer or even constant light (LL). LL is known to disrupt clock function, due to constant activation of the deep brain photoreceptor CRYPTOCHROME (CRY), which induces constant degradation of the clock protein TIMELESS (TIM). Temperature cycles are able to overcome these arrhythmia inducing effects of LL, reinstating clock protein oscillations and rhythmic behaviour. We show here that for this to occur a recently evolved natural allele (ls-tim) of the timeless gene is required, whereby the presence of this allele within the central clock neurons is sufficient. The ls-tim allele encodes a longer, less-light sensitive form of TIM (L-TIM) in addition to the shorter (S-TIM) form, the only form encoded by the ancient s-tim allele. Only after blocking light-input by removing functional CRY, s-tim flies are able to synchronise molecular and behavioural rhythms to temperature cycles in LL. Additional removal of light input from the visual system results in a phase advance of molecular and behavioural rhythms, showing that the visual system contributes to temperature synchronization in LL. We show that ls-tim, but not s-tim flies can synchronise their behavioural activity to semi-natural LL and temperature cycle conditions reflecting long Northern Europe summer days, the season when Drosophila populations massively expand. Our observations suggest that this functional gain associated with ls-tim is driving the Northern spread of this allele by directional selection.

neuroscience↗

bioRxiv2020

Comparison of long read methods for sequencing and assembly of a plant genome

Sequencing technologies have advanced to the point where it is possible to generate high accuracy, haplotype resolved, chromosome scale assemblies. Several long read sequencing technologies are available on the market and a growing number of algorithms have been developed over the last years to assemble the reads generated by those technologies. When starting a new genome project, it is therefore challenging to select the most cost-effective sequencing technology as well as the most appropriate software for assembly and polishing. For this reason, it is important to benchmark different approaches applied to the same sample. Here, we report a comparison of three long read sequencing technologies applied to the de novo assembly of a plant genome, Macadamia jansenii. We have generated sequencing data using Pacific Biosciences (Sequel I), Oxford Nanopore Technologies (PromethION) and BGI (single-tube Long Fragment Read) technologies for the same sample. Several assemblers were benchmarked in the assembly of PacBio and Nanopore reads. Results obtained from combining long read technologies or short read and long read technologies are also presented. The assemblies were compared for contiguity, accuracy and completeness as well as sequencing costs and DNA material requirements. Overall, the three long read technologies produced highly contiguous and complete genome assemblies of Macadamia jansenii. At the time of sequencing, the cost associated with each method was significantly different but continuous improvements in technologies have resulted in greater accuracy, increased throughput and reduced costs. We propose updating this comparison regularly with reports on significant iterations of the sequencing technologies.

genomics↗

bioRxiv2020

Laser capture microdissection-based single-cell RNA sequencing reveals optic nerve crush-related early mRNA alterations in retinal ganglion cells

Retinal ganglion cells (RGC) are the primary cell type injured in a variety of diseases of the optic nerve, and the early changes of RGCs RNA profiling may be important to understand the mechanism of optic nerve injury and axon regeneration. Here we employed the optic nerve crush (ONC) model to explore early mRNA alterations in RGCs using laser capture microdissection (LCM) and single-cell RNA sequencing. We successfully established an optimal LCM protocol using 30 m-thick retinal tissue sections mounted on glass slides and laser pressure catapulting (LPC) to collect RGCs and obtain high-quality RNA for single-cell sequencing. Based on our protocol, we identified 8744 differentially expressed genes that were involved in ONC-related early mRNA alterations in RGCs. Candidate genes included Atf3, Lgals3, LOC102551701, Plaur, Tmem140 and Maml1. The LCM-based single-cell RNA sequencing allowed new insights into the early mRNA changes in RGCs, highlighting new molecules associated with ONC.

neuroscience↗