Biology subjects

Fu, B.

Publications and source records attributed to Fu, B..

At least 55 records · Page 3Linked to original sources

bioRxiv2021

Nucleocapsid 203 mutations enhance SARS-CoV-2 immune evasion

Previous work indicated that the nucleocapsid 203 mutation increase the virulence and transmission of the SARS-CoV-2 Alpha variant. However, Delta later outcompeted Alpha and other lineages, promoting a new wave of infections. Delta also possesses a nucleocapsid 203 mutation, R203M. Large-scale epidemiological analyses suggest a synergistic effect of the 203 mutation and the spike L452R mutation, associated with Delta expansion. Viral competition experiments demonstrate the synergistic effect in fitness and infectivity. More importantly, we found that the combination of R203M and L452R brings in a 3.2-fold decrease in neutralizing titers to the neutralizing serum relative to L452R-only virus. R203M/L452R show an increased fitness after the initiation of global vaccination programmes, possibly associated with the enhanced immune evasion. Another rapidly emerging variant Omicron also bears the 203 mutation. Thus, we proposed that nucleocapsid mutations play an essential role for the rise and predominance of variants in concern.

microbiology↗

bioRxiv2021

Unconventional secretion of unglycosylated ORF8 is critical for the cytokine storm during SARS-CoV-2 infection

Coronavirus disease 2019 is a respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Evidence on the pathogenesis of SARS-CoV-2 is accumulating rapidly. In addition to structural proteins such as Spike and Envelope, the functional roles of non-structural and accessory proteins in regulating viral life cycle and host immune responses remain to be understood. Here, we show that open reading frame 8 (ORF8) acts as messenger for inter-cellular communication between alveolar epithelial cells and macrophages during SARS-CoV-2 infection. Mechanistically, ORF8 is a secretory protein that can be secreted by infected epithelial cells via both conventional and unconventional secretory pathways. The unconventionally secreted ORF8 recognizes the IL17RA receptor of macrophages and induces cytokine release. However, conventionally secreted ORF8 cannot bind to IL17RA due to N-linked glycosylation. Furthermore, we found that Yip1 interacting factor homolog B (YIF1B) is a channel protein that translocates unglycosylated ORF8 into vesicles for unconventional secretion. Blocking the unconventional secretion of ORF8 via a YIF1B knockout in hACE2 mice attenuates inflammation and yields delayed mortality following SARS-CoV-2 challenge.

microbiology↗

bioRxiv2021

Genome-wide analysis of DNA methylation reveals selection signatures of the grass carp during domestication

With the rapid development of aquaculture, more and more fish species from wild environments are artificially domesticated and cultured. In the process of domestication, the fish develop some adaptations and phenotypic traits, namely selection signatures. However, it is still unclear about the biological process underlying these selection signatures. Here, we used grass carp (Ctenopharyngodon idellus), an aquaculture fish with the largest production worldwide, to detect its selection signatures and investigate the roles of DNA methylation in the emergence of selection signatures during domestication based on whole-genome bisulfite sequencing technology. Our results showed that domesticated grass carp demonstrated four selection signatures, including growth and metabolism, immunity, foraging and learning behaviors, and 38 candidate genes were associated with these traits. 16 of candidate genes, such as IGF-1, GK, GYS1, etc., were found to play major roles in the growth and metabolism. Immunity signature was related to 11 of candidate genes, including MHCI, MHCII, C1QA, etc. The GRM1, TAS1R1 and TAS1R3 genes were essential for the adaptation of domesticated grass carp to commercial feed in artificial rearing condition. The C-FOS, POMC and CBP genes might be responsible for the acquisition of novel feeding habits and contribute to faster growth indirectly by enhancing food intake. These findings would provide new insights to expand our understanding on the role of DNA methylation in shaping physiological phenotypes in fish, and also contribute to efficient breeding of aquaculture stocks and restocking programs.

genetics↗

bioRxiv2021

Assembled chromosomes of the blood fluke Schistosoma mansoni provide insight into the evolution of its ZW sex-determination system

BackgroundSchistosoma mansoni is a flatworm that causes a neglected tropical disease affecting millions worldwide. Most flatworms are hermaphrodites but schistosomes have genotypically determined male (ZZ) and female (ZW) sexes. Sex is essential for pathology and transmission, however, the molecular determinants of sex remain unknown and is limited by poorly resolved sex chromosomes in previous genome assemblies. ResultsWe assembled the 391.4 Mb S. mansoni genome into individual, single-scaffold chromosomes, including Z and W. Manual curation resulted in a vastly improved gene annotation, resolved gene and repeat arrays, trans-splicing, and almost all UTRs. The sex chromosomes each comprise pseudoautosomal regions and single sex-specific regions. The Z-specific region contains 932 genes, but on W all but 29 of these genes have been lost and the presence of five pseudogenes indicates that degeneration of W is ongoing. Synteny analysis reveals an ancient chromosomal fusion corresponding to the oldest part of Z, where only a single gene--encoding the large subunit of pre-mRNA splicing factor U2AF--has retained an intact copy on W. The sex-specific copies of U2AF have divergent N-termini and show sex-biased gene expression. ConclusionOur assembly with fully resolved chromosomes provides evidence of an evolutionary path taken to create the Z and W sex chromosomes of schistosomes. Sex-linked divergence of the single U2AF gene, which has been present in the sex-specific regions longer than any other extant gene with distinct male and female specific copies and expression, may have been a pivotal step in the evolution of gonorchorism and genotypic sex determination of schistosomes.

genomics↗

bioRxiv2021

Nucleocapsid mutation R203K/G204R increases the infectivity, fitness and virulence of SARS-CoV-2

In addition to the mutations on the spike protein (S), co-occurring mutations on nucleocapsid (N) protein are also emerging in SARS-CoV-2 world widely. Mutations R203K/G204R on N, carried by high transmissibility SARS-CoV-2 lineages including B.1.1.7 and P.1, has a rapid spread in the pandemic during the past year. In this study, we performed comprehensive population genomic analyses and virology experiment concerning on the evolution, causation and virology consequence of R203K/G204R mutations. The global incidence frequency (IF) of 203K/204R has rose up from nearly zero to 76% to date with a shrinking from August to November in 2020 but bounced later. Our results show that the emergence of B.1.1.7 is associated with the second growth of R203K/G204R mutants. We identified positive selection evidences that support the adaptiveness of 203K/204R variants. The R203K/G204R mutant virus was created and compared with the native virus. The virus competition experiments show that 203K/204R variants possess a replication advantage over the preceding R203/G204 variants, possibly in relation to the ribonucleocapsid (RNP) assemble during the virus replication. Moreover, the 203K/204R virus increased the infectivity in a human lung cell line and induced an enhanced damage to blood vessel of infected hamsters lungs. In consistence, we observed a positive association between the increased severity of COVID-19 and the IF of 203K/204R from in silicon analysis of global clinical and epidemic data. In combination with the informatics and virology experiment, our work suggested the contribution of 203K/204R to the increased transmission and virulence of the SARS-CoV-2. In addition to mutations on the S protein, the mutations on the N protein are also important to virus spread during the pandemic.

evolutionary biology↗

bioRxiv2021

Integrated Genome and Transcriptome Analyses Reveal the Mechanism of Genome Instability in Ataxia with Oculomotor Apraxia 2

Mutations in the SETX gene, which encodes Senataxin, are associated with the progressive neurodegenerative diseases Ataxia with Oculomotor Apraxia 2 (AOA2) and Amyotrophic Lateral Sclerosis 4 (ALS4). To identify the causal defect in AOA2, patient-derived cells and SETX knockouts (human and mouse) were analyzed using integrated genomic and transcriptomic approaches. We observed a genome-wide increase in chromosome instability (gains and losses) within genes and at chromosome fragile sites, resulting in changes to gene expression profiles. Senataxin loss caused increased transcription stress near promoters that correlated with high GCskew and R-loop accumulation at promoter-proximal regions. Notably, the chromosomal regions with gains and losses overlapped with regions of elevated transcription stress. In the absence of Senataxin, we found that Cockayne Syndrome protein CSB was required for the recruitment of the transcription-coupled repair endonucleases (XPG and XPF) and recombination protein RAD52 to target and resolve transcription bubbles containing R-loops, leading to error prone repair and genomic instability. These results show that transcription stress is an important contributor to SETX mutation-associated chromosome fragility and AOA2.

molecular biology↗

bioRxiv2021

Elucidation of an anaerobic pathway for metabolism of L-carnitine-derived γ-butyrobetaine to trimethylamine in human gut bacteria

Trimethylamine (TMA) is an important gut microbial metabolite strongly associated with human disease. There are prominent gaps in our understanding of how TMA is produced from the essential dietary nutrient L-carnitine, particularly in the anoxic environment of the human gut where oxygen-dependent L-carnitine-metabolizing enzymes are likely inactive. Here, we elucidate the chemical and genetic basis for anaerobic TMA generation from the L-carnitine-derived metabolite {gamma}-butyrobetaine ({gamma}bb) by the human gut bacterium Emergencia timonensis. We identify a set of genes upregulated by {gamma}bb and demonstrate that the enzymes encoded by the induced {gamma}bb utilization (bbu) gene cluster convert {gamma}bb to TMA. The key TMA-generating step is catalyzed by a previously unknown type of TMA-lyase enzyme that utilizes a flavin cofactor to catalyze a redox neutral transformation. We identify additional cultured and uncultured host-associated bacteria that possess the bbu gene cluster, providing insights into the distribution of anaerobic {gamma}bb metabolism. Lastly, we present genetic, transcriptional, and metabolomic evidence that confirms the relevance of this metabolic pathway in the human gut microbiota. These analyses indicate that the anaerobic pathway is a more substantial contributor to TMA generation from L-carnitine in the human gut than the previously proposed aerobic pathway. The discovery and characterization of the bbu pathway provides the critical missing link in anaerobic metabolism of L-carnitine to TMA, enabling investigation into the connection between this microbial function and human disease. SIGNIFICANCETrimethylamine (TMA) is a disease-associated metabolite produced in the human body exclusively by microbes. Gut microbes generate TMA from essential nutrients consumed in the human diet, including L-carnitine. However, our understanding of the biochemical mechanisms involved in these transformations is incomplete. In this work, we define the biochemical pathway and genetic components in gut bacteria required for anaerobic production of TMA from {gamma}-butyrobetaine, a metabolite derived from L-carnitine. This discovery identifies a new type of TMA-producing enzyme and fills a critical gap in our knowledge of L-carnitine metabolism to TMA in the anaerobic environment of the human gut. This knowledge will enable evaluation of the link between L-carnitine metabolism and human disease, and the design of potential therapeutics.

biochemistry↗

bioRxiv2020

Virus-free and live-cell visualizing SARS-CoV-2 cell entry for studies of neutralizing antibodies and compound inhibitors

The ongoing COVID-19 pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and host ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system, we generated a recombinant fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process. In ACE2-expressing cells, we found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.

microbiology↗

bioRxiv2020

An ABA Synthesis Enzyme Allele OsNCED2 Promotes the Aerobic Adaption in Upland Rice

There are two ecotypes, upland rice and irrigate rice, during its evolution in rice. Upland rice exhibits aerobic adaptive phenotype via its stronger root system and more rapid drought responses compared with its counterpart, irrigated rice. We assessed the functional variation and applications of the aerobic adaptive allele OsNCED2T cloned from IRAT104 which is a cultivar of upland rice. OsNCED2-overexpressing transgenic rice and OsNCED2T-NILs exhibited significantly higher ABA contents at the seedling and reproductive stages, which can improve root development (RD) and drought tolerance (DT) to promote aerobic adaptation in upland rice. RNA-Seq-based expression profiling of transgenic versus wild-type rice identified OsNCED2-mediated pathways that regulate RD and DT. Meanwhile, OsNCED2-overexpressing rice exhibited significantly increased reactive oxygen species (ROS)-scavenging abilities and transcription levels of many stress- and development-related genes, which regulate RD and DT. A SNP mutation (C to T) from irrigated rice to upland rice, caused the functional variation of OsNCED2, and the enhanced RD and DT mediated by this site under aerobic conditions could promote higher yield of upland rice. These results show that OsNCED2T, through ABA synthesis, positively modulates RD and DT which confers aerobic adaptation in upland rice and might serve as a novel gene for breeding aerobic adaptive or water-saving rice.

plant biology↗

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

Tocilizumab treatment in severe COVID-19 patients attenuates the inflammatory storm incited by monocyte centric immune interactions revealed by single-cell analysis

ABSTRACTDespite the current devastation of the COVID-19 pandemic, several recent studies have suggested that the immunosuppressive drug Tocilizumab can powerfully treating inflammatory responses that occur in this disease. Here, by employing single-cell analysis of the immune cell composition of severe-stage COVID-19 patients and these same patients in post Tocilizumab-treatment remission, we have identified a monocyte subpopulation specific to severe disease that contributes to inflammatory storms in COVID-19 patients. Although Tocilizumab treatment attenuated the strong inflammatory immune response, we found that immune cells including plasma B cells and CD8+ T cells still exhibited an intense humoral and cell-mediated anti-virus immune response in COVID-19 patients after Tocilizumab treatment. Thus, in addition to providing a rich, very high-resolution data resource about the immune cell distribution at multiple stages of the COVID-19 disease, our work both helps explain Tocilizumab’s powerful therapeutic effects and defines a large number of potential new drug targets related to inflammatory storms.Competing Interest StatementJingwen Fang is the executive officer of HanGen BiotechView Full Text

genomics↗

bioRxiv2020

Aberrant pathogenic GM-CSF+ T cells and inflammatory CD14+CD16+ monocytes in severe pulmonary syndrome patients of a new coronavirus

Pathogenic human coronavirus infections, such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV), cause high morbidity and mortality 1,2. Recently, a severe pneumonia-associated respiratory syndrome caused by a new coronavirus was reported at December 2019 (2019-nCoV) in the city Wuhan, Hubei province, China3-5, which was also named as pneumonia-associated respiratory syndrome (PARS)6. Up to 9th of February 2020, at least 37, 251 cases have been reported with 812 fatal cases according to the report from China CDC. However, the immune mechanism that potential orchestrated acute mortality from patients of 2019-nCoV is still unknown. Here we show that after the 2019-nCoV infection, CD4+T lymphocytes are rapidly activated to become pathogenic T helper (Th) 1 cells and generate GM-CSF etc. The cytokines environment induces inflammatory CD14+CD16+ monocytes with high expression of IL-6 and accelerates the inflammation. These aberrant and excessive immune cells may enter the pulmonary circulation in huge numbers and play an immune damaging role to causing lung functional disability and quick mortality. Our results demonstrate that excessive non-effective host immune responses by pathogenic T cells and inflammatory monocytes may associate with severe lung pathology. Therefore, we suggest that monoclonal antibody that targets the GM-CSF or interleukin 6 receptor may potentially curb immunopathology caused by 2019-nCoV and consequently win more time for virus clearance.

immunology↗

bioRxiv2019

Mechanical stress compromises multicomponent efflux complexes in bacteria

Physical forces have long been recognized for their effects on the growth, morphology, locomotion, and survival of eukaryotic organisms1. Recently, mechanical forces have been shown to regulate processes in bacteria, including cell division2, motility3, virulence4, biofilm initiation5,6, and cell shape7,8, although it remains unclear how mechanical forces in the cell envelope lead to changes in molecular processes. In Gram-negative bacteria, multicomponent protein complexes that form rigid links across the cell envelope directly experience physical forces and mechanical stresses applied to the cell. Here we manipulate tensile and shear mechanical stress in the bacterial cell envelope and use single-molecule tracking to show that shear (but not tensile) stress within the cell envelope promotes disassembly of the tripartite efflux complex CusCBA, a system used by E. coli to resist copper and silver toxicity, thereby making bacteria more susceptible to metal toxicity. These findings provide the first demonstration that mechanical forces, such as those generated during colony overcrowding or bacterial motility through submicron pores, can inhibit the contact and function of multicomponent complexes in bacteria. As multicomponent, trans-envelope efflux complexes in bacteria are involved in many processes including antibiotic resistance9, cell division10, and translocation of outer membrane components11, our findings suggest that the mechanical environment may regulate multiple processes required for bacterial growth and survival.

biophysics↗

bioRxiv2019

Functional linkage of gene fusions to cancer cell fitness assessed by pharmacological and CRISPR/Cas9 screening

Many gene fusions have been reported in tumours and for most their role remains unknown. As fusions can be used clinically for diagnostic and prognostic purposes, and are targets for treatment, it is crucial to assess their functional implications in cancer. To investigate the role of fusions in tumor cell fitness, we developed a systematic analysis utilising RNA-sequencing data from 1,011 human cancer cell lines to functionally link 8,354 gene fusion events with genomic data, sensitivity to >350 anti-cancer drugs and CRISPR-Cas9 loss-of-fitness information. Established clinically-relevant fusions were readily identified. Overall, functional fusions were rare, including those involving cancer driver genes, suggesting that many fusions are dispensable for tumor cell fitness. Novel therapeutically actionable fusions involving RAF1, BRD4 and ROS1 were verified in new histologies. In addition, recurrent YAP1-MAML2 fusions were identified as activators of Hippo-pathway signaling in multiple cancer types, supporting therapeutic targeting of Hippo signalling. Our approach discriminates functional fusions, identifying new drivers of carcinogenesis and fusions that could have important clinical implications. SignificanceWe identify fusions as new potential candidates for drug repurposing and drivers of carcinogenesis. These results support histology agnostic marker-driven precision cancer medicine. Most fusions are not functional with implications for interpreting cancer fusions reported from clinical sequencing studies.

cancer biology↗

bioRxiv2018

Homogenization of capillary flow and oxygenation in deeper cortical layers correlates with increased oxygen extraction

Our understanding of how capillary blood flow and oxygen distribute across cortical layers to meet the local metabolic demand is incomplete. We addressed this question by using two-photon imaging of microvascular oxygen partial pressure (PO2) and flow in the whisker barrel cortex in awake mice at rest. Our measurements in layers I-V show that the capillary red-blood-cell flux and oxygenation heterogeneity, and the intracapillary resistance to oxygen delivery, all decrease with depth, reaching a minimum around layer IV, while the depth-dependent oxygen extraction fraction is increased in layer IV, where oxygen demand is presumably the highest. Our findings suggest that homogenization of physiological observables relevant to oxygen transport to tissue is an important part of the microvascular network adaptation to a local brain metabolism increase. These results will inform the biophysical models of layer-specific cerebral oxygen delivery and consumption and improve our understanding of diseases that affect the cerebral microcirculation.\n\nIMPACT STATEMENTHomogenization of cortical capillary blood flow and oxygenation underpins an important mechanism, by which the microvascular network adapts to an increase in the local brain oxidative metabolism.

neuroscience↗

bioRxiv2018

ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks

Mutations in the ATM tumor suppressor confer hypersensitivity to DNA-damaging agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase poison topotecan. Thus, we establish that loss of terminal components of the non-homologous end-joining (NHEJ) machinery or the BRCA1-A complex specifically confers topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase inhibitor olaparib is due to delayed homologous recombination repair at DNA-replication-fork-associated double-strand breaks (DSBs), resulting in toxic NHEJ-mediated chromosome fusions. Accordingly, restoring legitimate repair in ATM-deficient cells, either by preventing NHEJ DNA ligation or by enhancing DSB-resection by BRCA1-A complex inactivation, markedly suppresses this toxicity. Our work suggests opportunities for patient stratification in ATM-deficient cancers and when using ATM inhibitors in the clinic, and identifies additional therapeutic vulnerabilities that might be exploited when such cancers evolve drug resistance.\n\nOne Sentence SummaryATM counteracts toxic NHEJ at broken replication forks

cell biology↗

bioRxiv2018

Multiple laboratory mouse reference genomes define strain specific haplotypes and novel functional loci

The most commonly employed mammalian model organism is the laboratory mouse. A wide variety of genetically diverse inbred mouse strains, representing distinct physiological states, disease susceptibilities, and biological mechanisms have been developed over the last century. We report full length draft de novo genome assemblies for 16 of the most widely used inbred strains and reveal for the first time extensive strain-specific haplotype variation. We identify and characterise 2,567 regions on the current Genome Reference Consortium mouse reference genome exhibiting the greatest sequence diversity between strains. These regions are enriched for genes involved in defence and immunity, and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. Several immune related loci, some in previously identified QTLs for disease response have novel haplotypes not present in the reference that may explain the phenotype. We used these genomes to improve the mouse reference genome resulting in the completion of 10 new gene structures, and 62 new coding loci were added to the reference genome annotation. Notably this high quality collection of genomes revealed a previously unannotated gene (Efcab3-like) encoding 5,874 amino acids, one of the largest known in the rodent lineage. Interestingly, Efcab3-like-/- mice exhibit severe size anomalies in four regions of the brain suggesting a mechanism of Efcab3-like regulating brain development.

genomics↗

bioRxiv2017

Natural Variation of OsLG3 Controls Drought Stress Tolerance in Rice by Inducing ROS Scavenging

BackgroundImproving performance of rice under drought stress has potential to significant impact on rice productivity. Previously we reported that OsLG3 positively control rice grain length and yield.\n\nResultsIn this study, we found that OsLG3 was more strongly expressed in upland rice compared to lowland rice under drought stress condition. Candidate gene association analysis showed that the natural variation in OsLG3 was associated with tolerance to water deficit stress in germinating rice seeds. Transgenic rice with enhanced OsLG3 expression exhibited improved tolerance to drought and that is most likely due to enhanced ROS scavenging efficiency. Phylogenetic analysis and pedigree records indicated that the tolerant allele of OsLG3 has potential to improve drought tolerance of japonica rice.\n\nConclusionsCollectively, our work revealed that the natural variation of OsLG3 contributes to rice drought tolerance and the elite allele of OsLG3 is a promising genetic resource for the development of drought-tolerant and high-yield rice varieties.

plant biology↗