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Xu, J.

Publications and source records attributed to Xu, J..

At least 757 records · Page 42Linked to original sources
bioRxiv2020

Coupling metabolic addiction with negative autoregulation to improve strain stability and pathway yield

Metabolic addiction, an organism that is metabolically addicted with a compound to maintain its growth fitness, is an underexplored area in metabolic engineering. Microbes with heavily engineered pathways or genetic circuits tend to experience metabolic burden leading to degenerated or abortive production phenotype during long-term cultivation or scale-up. A promising solution to combat metabolic instability is to tie up the end-product with an intermediary metabolite that is essential to the growth of the producing host. Here we present a simple strategy to improve both metabolic stability and pathway yield by coupling chemical addiction with negative autoregulatory genetic circuits. Naringenin and lipids compete for the same precursor with inversed pathway yield in oleaginous yeast. Negative autoregulation of the lipogenic pathways, enabled by CRISPRi and fatty acid-inducible promoters, repartitioned malonyl-CoA to favor flavonoid synthesis and increased naringenin production by 74.8%. With flavonoid-sensing hybrid promoters to control leucine synthesis, this flavonoid addiction phenotype confers a selective growth advantage to the naringenin-producing cell. The engineered yeast persisted 90.9% of naringenin titer up to 324 generations. Cells without flavonoid addiction regained growth fitness but lost 94.5% of the naringenin titer after cell passage beyond 300 generations. Metabolic addiction and negative autoregulation may be generalized as basic tools to eliminate metabolic heterogeneity, improve strain stability and pathway yield.

synthetic biology↗
bioRxiv2020

Susceptibility of tree shrew to SARS-CoV-2 infection

Since SARS-CoV-2 became a pandemic event in the world, it has not only caused huge economic losses, but also a serious threat to global public health. Many scientific questions about SARS-CoV-2 and COVID-19 were raised and urgently need to be answered, including the susceptibility of animals to SARS-CoV-2 infection. Here we tested whether tree shrew, an emerging experimental animal domesticated from wild animal, is susceptible to SARS-CoV-2 infection. No clinical signs were observed in SARS-CoV-2 inoculated tree shrews during this experiment except the increasing body temperature (above 39{degrees} C) particular in female animals during infection. Low levels of virus shedding and replication in tissues occurred in all three age groups, each of which showed his own characteristics. Histopathological examine revealed that pulmonary abnormalities were mild but the main changes although slight lesions were also observed in other tissues. In summary, tree shrew is not susceptible to SARS-CoV-2 infection and may not be a suitable animal for COVID-19 related researches.

microbiology↗
bioRxiv2020

Effect of storage conditions on peripheral leukocytes transcriptome

IntroductionPeripheral blood leukocytes are essential components of the innate and adaptive immune responses. Their transcriptome reflects an individuals physiological and pathological state, consequently bulk and single-cell RNA sequencing have been used to assess health and disease. As RNA is dynamic and could be affected by ex vivo conditions before RNA stabilization, we have assessed the influence of temporary storage on the transcriptome. MethodsWe collected peripheral blood from six healthy donors and processed it immediately or stored it either at 4{square} or room temperature (RT, 18-22{square}) for 2h, 6h and 24h. Total cellular RNA was extracted from leukocytes after red blood cells lysis and the transcriptome analyzed using RNA sequencing. ResultsWe identified 152 up-regulated and 12 down-regulated coding genes in samples stored at 4{square} for 24h with most of the up-regulated genes related to nucleosome assembly. More coding genes changed expression at RT, with 1218 increased and 1480 decreased. The increased genes were particularly related to mRNA processing and apoptosis, while the decreased genes were associated with neutrophil activation and cytokine production, implying a reduced proportion of neutrophils, which was confirmed by leukocyte subsets analysis. Most house-keeping genes changed expression during storage, but genes such as TUBB and C1orf43 were relatively stable and could serve as reference genes. ConclusionTemporary storage conditions profoundly affect leukocyte gene expression profiles and change leukocyte subset proportions. Blood samples stored at 4{square} for 6h largely maintain their original transcriptome.

molecular biology↗
bioRxiv2020

K-means Based Unsupervised Feature Selection to Prioritize Biomarkers of Different Disease Clinical Phases

Huntingtons disease is caused by a single gene mutation, which is potentially a good model for development of biomarkers corresponding to different disease phase and clinical phenotypes. Hypothesis-driven and omics discovery approaches have not yet identified effective candidate biomarkers in HD. So, it is urgent to develop engagement and disease-phase specific biomarkers. The advanced sequencing technology makes it possible to develop data-driven methods for biomarkers discovery. Therefore, in this study, we designed k-means based unsupervised feature selection (KFS) method to prioritize biomarkers of different disease clinical phases. KFS first conducts k-means clustering on the samples with gene expression data, then it conducts feature selection based on the feature selection matrix to prioritize biomarkers of different samples. By conducting alternative iteration of clustering and feature selection to screen key genes which corresponding to the complex clinical phenotypes of different disease phases. Further gene ontology and enrichment analysis highlight potential molecular mechanisms of HD. Our experimental analyses have uncovered new disease-related genes and disease-associated pathways, which in turn have provided insight into the molecular mechanisms during the disease progression.

bioinformatics↗
bioRxiv2020

Inhibition of Striatal-Enriched Protein Tyrosine Phosphatase (STEP) Activity Reverses Behavioral Deficits in a Rodent Model of Autism.

Autism spectrum disorders (ASDs) are highly prevalent childhood illnesses characterized by impairments in communication, social behavior, and repetitive behaviors. Studies have found aberrant synaptic plasticity and neuronal connectivity during the early stages of brain development and have suggested that these contribute to an increased risk for ASD. STEP is a protein tyrosine phosphatase that regulates synaptic plasticity and is implicated in several cognitive disorders. Here we test the hypothesis that STEP may contribute to some of the aberrant behaviors present in the VPA-induced mouse model of ASD. In utero VPA exposure of pregnant dams results in autistic-like behavior in the pups, which is associated with a significant increase in the STEP expression in the prefrontal cortex. The elevated STEP protein levels are correlated with increased dephosphorylation of STEP substrates GluN2B, Pyk2 and ERK, suggesting upregulated STEP activity. Moreover, pharmacological inhibition of STEP rescues the sociability, repetitive and abnormal anxiety phenotypes commonly associated with ASD. These data suggest that STEP may play a role in the VPA model of ASD and STEP inhibition may have a potential therapeutic benefit in this model.

neuroscience↗
bioRxiv2020

Comparison of SARS-CoV-2 infections among 3 species of non-human primates

COVID-19, caused by SARS-CoV-2 infection, has recently been announced as a pandemic all over the world. Plenty of diagnostic, preventive and therapeutic knowledges have been enriched from clinical studies since December 2019. However, animal models, particularly non-human primate models, are urgently needed for critical questions that could not be answered in clinical patients, evaluations of anti-viral drugs and vaccines. In this study, two families of non-human primates, Old world monkeys (12 Macaca mulatta, 6 Macaca fascicularis) and New world monkeys (6 Callithrix jacchus), were experimentally inoculated with SARS-CoV-2. Clinical signs were recorded. Samples were collected for analysis of viral shedding, viremia and histopathological examination. Increased body temperature was observed in 100% (12/12) M. mulatta, 33.3% (2/6) M. fascicularis and none (0/6) of C. jacchus post inoculation of SARS-CoV-2. All of M. mulatta and M. fascicularis showed chest radiographic abnormality. Viral genomes were detected in nasal swabs, throat swabs, anal swabs and blood from all 3 species of monkeys. Viral shedding from upper respiratory samples reached the peak between day 6 and day 8 post inoculation. From necropsied M. mulatta and M. fascicularis, the tissues showing virus positive were mainly lung, weasand, bronchus and spleen. No viral genome was seen in any of tissues from 2 necropsied C. jacchus. Severe gross lesions and histopathological changes were observed in lung, heart and stomach of SARS-CoV-2 infected animals. In summary, we have established a NHP model for COVID-19, which could be used to evaluate drugs and vaccines, and investigate viral pathogenesis. M. mulatta is the most susceptible to SARS-CoV-2 infection, followed by M. fascicularis and C. jacchus. One Sentence SummaryM. mulatta is the most susceptible to SARS-CoV-2 infection as compared to M. fascicularis and C. jacchus.

microbiology↗
bioRxiv2020

A Systematic Analysis of Mosquito-Microbiome Biosynthetic Gene Clusters Reveals Antimalarial Siderophores that Reduce Mosquito Reproduction Capacity

Advances in infectious disease control strategies through genetic manipulation of insect microbiomes have heightened interest in microbially produced small molecules within mosquitoes. Herein, 33 mosquito-associated bacterial genomes were mined and over 700 putative biosynthetic gene clusters (BGCs) were identified, 135 of which belong to known classes of BGCs. After an in-depth analysis of the 135 BGCs, iron-binding siderophores were chosen for further investigation due to their high abundance and well-characterized bioactivities. Through various metabolomic strategies, eight siderophore scaffolds were identified in six strains of mosquito-associated bacteria. Among these, serratiochelin A and pyochelin were found to reduce female Anopheles gambiae overall fecundity likely by lowering their blood feeding rate. Serratiochelin A and pyochelin were further found to inhibit the Plasmodium parasite asexual blood and liver stages in vitro. Our work supplies a bioinformatic resource for future mosquito microbiome studies and highlights an understudied source of bioactive small molecules.

microbiology↗
bioRxiv2020

Single-cell atlas of a non-human primate reveals new pathogenic mechanisms of COVID-19

Stopping COVID-19 is a priority worldwide. Understanding which cell types are targeted by SARS-CoV-2 virus, whether interspecies differences exist, and how variations in cell state influence viral entry is fundamental for accelerating therapeutic and preventative approaches. In this endeavor, we profiled the transcriptome of nine tissues from a Macaca fascicularis monkey at single-cell resolution. The distribution of SARS-CoV-2 facilitators, ACE2 and TMRPSS2, in different cell subtypes showed substantial heterogeneity across lung, kidney, and liver. Through co-expression analysis, we identified immunomodulatory proteins such as IDO2 and ANPEP as potential SARS-CoV-2 targets responsible for immune cell exhaustion. Furthermore, single-cell chromatin accessibility analysis of the kidney unveiled a plausible link between IL6-mediated innate immune responses aiming to protect tissue and enhanced ACE2 expression that could promote viral entry. Our work constitutes a unique resource for understanding the physiology and pathophysiology of two phylogenetically close species, which might guide in the development of therapeutic approaches in humans. Bullet pointsO_LIWe generated a single-cell transcriptome atlas of 9 monkey tissues to study COVID-19. C_LIO_LIACE2+TMPRSS2+ epithelial cells of lung, kidney and liver are targets for SARS-CoV-2. C_LIO_LIACE2 correlation analysis shows IDO2 and ANPEP as potential therapeutic opportunities. C_LIO_LIWe unveil a link between IL6, STAT transcription factors and boosted SARS-CoV-2 entry. C_LI

cell biology↗
bioRxiv2020

Room-temperature-storable PCR Mixes for SARS-CoV-2 Detection

A novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) emerged in late 2019, causing an outbreak of pneumonia [coronavirus disease 2019 (COVID-19)] in Wuhan, China, which then rapidly spread globally. Although the use of ready-made reaction mixes can enable more rapid PCR-based diagnosis of COVID-19, the need to transport and store these mixes at low temperatures presents challenges to already overburdened logistics networks. Here, we present an optimized freeze-drying procedure that allows SARS-CoV-2 PCR mixes to be transported and stored at ambient temperatures, without loss of activity. Additive-supplemented PCR mixes were freeze-dried. The residual moisture of the freeze-dried PCR mixes was measured by Karl-Fischer titration. We found that freeze-dried PCR mixes with [~]1.2% residual moisture are optimal for storage, transport, and reconstitution. The sensitivity, specificity, and repeatability of the freeze-dried reagents were similar to those of freshly prepared, wet reagents. The freeze-dried mixes retained activity at room temperature (18[~]25{degrees}C) for 28 days, and for 14 and 10 days when stored at 37{degrees}C and 56{degrees}C, respectively. The uptake of this approach will ease logistical challenges faced by transport networks and make more cold storage space available at diagnosis and hospital laboratories. This method can also be applied to the generation of freeze-dried PCR mixes for the detection of other pathogens.

microbiology↗
bioRxiv2020

Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

The global pandemic of Coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable in vitro neutralization assay is very important for the development of neutralizing antibodies, vaccines and other inhibitors. In this study, G protein-deficient vesicular stomatitis virus (VSVdG) bearing full-length and truncated spike (S) protein of SARS-CoV-2 were evaluated. The virus packaging efficiency of VSV-SARS-CoV-2-Sdel18 (S with C-terminal 18 amino acid truncation) is much higher than VSV-SARS-CoV-2-S. A neutralization assay for antibody screening and serum neutralizing titer quantification was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and human angiotensin-converting enzyme 2 (ACE2) overexpressed BHK21 cell (BHK21-hACE2). The experimental results can be obtained by automatically counting EGFP positive cell number at 12 hours after infection, making the assay convenient and high-throughput. The serum neutralizing titer of COVID-19 convalescent patients measured by VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with live SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting receptor binding domain (RBD) of SARS-CoV-2-S were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.

microbiology↗
bioRxiv2020

Convergence within divergence: insights of wheat adaptation from Triticum population sequencing

Bread wheat expanded its habitats from a small core area of the Fertile Crescent to global environments within [~]10,000 years. Genetic mechanisms of this remarkable evolutionary success are not well understood. By whole-genome sequencing of populations from 25 subspecies within genera Triticum and Aegilops, we identified composite introgression from these wild populations contributing 13%[~]36% of the bread wheat genome, which tremendously increased the genetic diversity of bread wheat and allowed its divergent adaptation. Meanwhile, convergent adaption to human selection showed 2- to 16-fold enrichment relative to random expectation in Triticum species despite their drastic differences in ploidy levels and growing zones, indicating the vital importance of adaptive constraints in the success of bread wheat. These results showed the genetic necessities of wheat as a global crop and provided new perspectives on leveraging adaptation success across species for crop improvement.

genetics↗
bioRxiv2020

A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing

An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.

systems biology↗
bioRxiv2020

Single-cell transcriptome profiling an adult human cell atlas of 15 major organs

BackgroundAs core units of organ tissues, cells of various types play their harmonious rhythms to maintain the homeostasis of the human body. It is essential to identify the characteristics of cells in human organs and their regulatory networks for understanding the biological mechanisms related to health and disease. However, a systematic and comprehensive single-cell transcriptional profile across multiple organs of a normal human adult is missing. ResultsWe perform single-cell transcriptomes of 84,363 cells derived from 15 tissue organs of one adult donor and generate an adult human cell atlas. The adult human cell atlas depicts 252 subtypes of cells, including major cell types such as T, B, myeloid, epithelial, and stromal cells, as well as novel COCH+ fibroblasts and FibSmo cells, each of which is distinguished by multiple marker genes and transcriptional profiles. These collectively contribute to the heterogeneity of major human organs. Moreover, T cell and B cell receptor repertoire comparisons and trajectory analyses reveal direct clonal sharing of T and B cells with various developmental states among different tissues. Furthermore, novel cell markers, transcription factors and ligand-receptor pairs are identified with potential functional regulations in maintaining the homeostasis of human cells among tissues. ConclusionsThe adult human cell atlas reveals the inter- and intra-organ heterogeneity of cell characteristics and provides a useful resource in uncovering key events during the development of human diseases in the context of the heterogeneity of cells and organs.

cell biology↗
bioRxiv2020

Integrated lipidomics and proteomics network analysis highlights lipid and immunity pathways associated with Alzheimer's disease

INTRODUCTIONThere is an urgent need to understand the molecular mechanisms underlying Alzheimers Disease (AD) to enable early diagnosis and develop effective treatments. Here we aim to investigate Alzheimers dementia using an unsupervised lipid, protein and gene multi-omic integrative approach. METHODSA lipidomics dataset (185 AD, 40 MCI and 185 controls) and a proteomics dataset (201 AD patients, 104 MCI individuals and 97 controls) were utilised for weighted gene co-expression network analyses (WGCNA). An additional proteomics dataset (94 AD, 55 MCI and 100 controls) was included for external proteomics validation. Modules created within each modality were correlated with clinical AD diagnosis, brain atrophy measures and disease progression, as well as with each other. Gene Ontology (GO) enrichment analysis was employed to examine the biological processes and molecular and cellular functions for protein modules associated with AD phenotypes. Lipid species were annotated in the lipid modules associated with AD phenotypes. Associations between established AD risk loci and lipid/protein modules that showed high correlation with AD phenotypes were also explored. RESULTSFive of the 20 identified lipid modules and five of the 17 identified protein modules were correlated with AD phenotypes. Lipid modules comprising of phospholipids, triglycerides, sphingolipids and cholesterol esters, correlated with AD risk loci involved in immune response and lipid metabolism. Five protein modules involved in positive regulation of cytokine production, neutrophil mediated immunity, humoral immune responses were correlated with AD risk loci involved in immune and complement systems. DISCUSSIONWe have shown the first multi-omic study linking genes, proteins and lipids to study pathway dysregulation in AD. Results identified modules of tightly regulated lipids and proteins that were strongly associated with AD phenotypes and could be pathology drivers in lipid homeostasis and innate immunity. Research in ContextO_LILipid and protein modules were preserved amongst Alzheimers disease (AD) patients, participants with mild cognitive impairment (MCI) and controls. Protein modules were also externally validated. C_LIO_LIFive lipid and five protein modules out of a total of thirty-seven correlated with clinical AD diagnosis, brain atrophy measurements and the rate of cognitive decline in AD. C_LIO_LILipid and protein modules associated with AD phenotypes showed associations with established AD risk loci involved in lipid and immune pathways. C_LI

neuroscience↗
bioRxiv2020

Structures of a P4-ATPase lipid flippase in lipid bilayers

Type 4 P-type ATPases (P4-ATPases) are a group of key enzymes maintaining lipid asymmetry of eukaryotic membranes. Phospholipids are actively and selectively flipped by P4-ATPases from the exoplasmic leaflet to the cytoplasmic leaflet. How lipid flipping is coupled with ATP-hydrolysis by P4-ATPases is poorly understood. Here, we report the electron cryo-microscopy structures of a P4-ATPase, Dnf1-Cdc50 from Chaetomium thermophilum, which had been reconstituted into lipid nanodiscs and captured in two transport intermediate states. The structures reveal that transmembrane segment 1 of Dnf1 becomes highly flexible during lipid transport. The local lipid bilayers are distorted to facilitate the entry of the phospholipid substrates from the exoplasmic leaflet to a cross-membrane groove. During transport, the lipid substrates are relayed through four binding sites in the groove which constantly shields the lipid polar heads away from the hydrophobic environment of the membranes.

biophysics↗
bioRxiv2020

High sensitivity detection of SARS-CoV-2 using multiplex PCR and a multiplex-PCR-based metagenomic method

Many detection methods have been used or reported for the diagnosis and/or surveillance of COVID-19. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most commonly used because of its high sensitivity, typically claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by some RT-PCR methods, probably due to low viral load, timing of sampling, degradation of virus RNA in the sampling process, or possible mutations spanning the primer binding sites. Therefore, alternative and highly sensitive methods are imperative. With the goal of improving sensitivity and accommodating various application settings, we developed a multiplex-PCR-based method comprised of 343 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. We further amplified the entire SARS-CoV-2 genome from 8 to half a million viral copies purified from 13 COVID-19 positive specimens, and detected mutations through next generation sequencing. Finally, we developed a multiplex-PCR-based metagenomic method in parallel, that required modest sequencing depth for uncovering SARS-CoV-2 mutational diversity and potentially novel or emerging isolates.

genomics↗
bioRxiv2020

Quantification of Adeno-Associated Virus with Safe Nucleic Acid Dyes

Adeno-associated virus (AAV) is the most commonly used viral vector for both biological and gene therapeutic applications1. Although many methods have been developed to measure quantity attributes of AAV, they are often technically challenging and time consuming. Here we report a method to titer AAV with GelGreen(R) dye, a safe green fluorescence nucleic acid dye recently engineered by Biotium company (Fremont, CA). This method, hereinafter referred to as GelGreen method, provides a fast (~ 30 minutes) and reliable strategy for AAV titration. To validate GelGreen method, we measured genome titer of an AAV reference material AAV8RSM and compared our titration results with those determined by Reference Material Working Group (ARMWG). We showed that GelGreen results and capsid Elisa results are comparable to each other. We also showed that GelRed(R) dye, a red fluorescence dye from Biotium, can be used to directly "visualize" AAV genome titer on a conventional gel imager, presenting an especially direct approach to estimate viral quantity. In summary, we described a technique to titer AAV by using new generation of safe DNA dyes. This technique is simple, safe, reliable and cost-efficient. It has potential to be broadly applied for quantifying and normalizing AAV viral vectors.

molecular biology↗