Biology subjects

Tong, Y.

Publications and source records attributed to Tong, Y..

106 records · Page 6Linked to original sources

bioRxiv2020

Simple cloning of large natural product biosynthetic gene cluster by CRISPR/Cas12a-mediated fast direct capturing strategy

Directly cloning of biosynthetic gene clusters (BGCs) from microbial genomes has been revolutionizing the natural product-based drug discovery. However, it is still very challenging to efficiently clone, for example, large (> 80kb) and GC-rich (> 70%), streptomycete originating BGCs. In this study, we developed a simple, fast yet efficient and low-cost in vitro platform for direct cloning large BGCs from streptomycete genomic DNA, named as CAT-FISHING (CRISPR/Cas12a- and Agarose plug-based sysTem for Fast bIoSyntHetIc geNe cluster cloninG), by combining the advantages of CRISPR/Cas12a cleavage and bacterial artificial chromosome (BAC) library construction. CAT-FISHING was demonstrated by directly cloning large DNA fragments ranging from 47 to 139 kb with GC content of > 70% from the S. albus J1074 genome in a relatively efficient manner. Moreover, surugamides, encoded by a captured 87-kb BGC with GC content of 76%, was heterologously expressed in a Streptomyces chassis. These results indicate that CAT-FISHING is a powerful platform for BGCs batch cloning, which would be greatly beneficial to the natural products-based drug discovery. We believe that this system will lead a renaissance of interest in microorganisms as a source for drug development.

synthetic biology↗

bioRxiv2020

The Genetic Architecture of DNA Replication Timing in Human Pluripotent Stem Cells

DNA replication follows a strict spatiotemporal program that intersects with chromatin structure and gene regulation. However, the genetic basis of the mammalian DNA replication timing program is poorly understood1-3. To systematically identify genetic regulators of DNA replication timing, we exploited inter-individual variation in 457 human pluripotent stem cell lines from 349 individuals. We show that the human genomes replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs4) - base-pair-resolution sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, to affect replication timing. Analysis of rtQTL locations reveals a histone code for replication initiation, composed of bivalent histone H3 trimethylation marks on a background of histone hyperacetylation. The H3 trimethylation marks are individually repressive yet synergize to promote early replication. We further identify novel positive and negative regulators of DNA replication timing, the former comprised of pluripotency-related transcription factors while the latter involve boundary elements. Human replication timing is controlled by a multi-layered mechanism that operates on target DNA sequences, is composed of dozens of effectors working combinatorially, and follows principles analogous to transcription regulation: a histone code, activators and repressors, and a promoter-enhancer logic.

genomics↗

bioRxiv2020

The draft genome sequence of herbaceous diploid bamboo Raddia distichophylla

Bamboos are important non-timber forest plants widely distributed in the tropical and subtropical regions of Asia, Africa, America, and Pacific islands. They comprise the Bambusoideae in the grass family (Poaceae), including approximately 1,700 described species in 127 genera. In spite of the widespread uses of bamboo for food, construction and bioenergy, the gene repertoire of bamboo still remains largely unexplored. Raddia distichophylla (Schrad. ex Nees) Chase, belonging to the tribe Olyreae (Bambusoideae, Poaceae), is diploid herbaceous bamboo with only slightly lignified stems. In this study, we report a draft genome assembly of the approximately [~]589 Mb whole-genome sequence of R. distichophylla with a contig N50 length of 86.36 Kb. Repeated sequences account for [~]49.08% of the genome, of which LTR retrotransposons occupy [~]35.99% of whole genome. A total of 30,763 protein-coding genes were annotated in the R. distichophylla genome with an average transcript size of 2,887 bp. Access to this herbaceous bamboo genome sequence will provide novel insights into biochemistry, molecular-assisted breeding programs and germplasm conservation for bamboo species world-wide.

genomics↗

bioRxiv2020

The Coxiella burnetii QpH1 plasmid is a virulence factor for colonizing bone marrow-derived murine macrophages

Coxiella burnetii carries a large conserved plasmid or plasmid-like chromosomally integrated sequence of unknown function. Here we report the curing of QpH1 plasmid from C. burnetii Nine Mile phase II, the characterization of QpH1-deficient C. burnetii in in vitro and in vivo infection models, and the characterization of plasmid biology. A shuttle vector pQGK, which is composed of the CBUA0036-0039a region (predicted for QpH1 maintenance), an E. coli plasmid ori, eGFP and kanamycin resistance genes was constructed. The pQGK vector can be stably transformed into Nine Mile II and maintained at a similar low copy like QpH1. Importantly, transformation with pQGK cured the endogenous QpH1 due to plasmid incompatibility. Compared to a Nine Mile II transformant of a RSF1010-based vector, the pQGK transformant shows an identical one-step growth curve in axenic media, a similar growth curve in Buffalo green monkey kidney cells, an evident growth defect in macrophage-like THP-1 cells, and dramatically reduced ability of colonizing bone marrow-derived murine macrophages. In the SCID mouse infection model, the pQGK transformants caused a lesser extent of splenomegaly. Moreover, the plasmid biology was investigated by mutagenesis. We found CBUA0037-0039 are essential for plasmid maintenance, and CBUA0037-0038 account for plasmid compatibility. Taken together, our data suggest that QpH1 encodes factor(s) essential for colonizing murine macrophages, and to a lesser extent for colonizing human macrophages. This study highlights a critical role of QpH1 for C. burnetii persistence in rodents, and expands the toolkit for genetic studies in C. burnetii. Author summaryIt is postulated that C. burnetii recently evolved from an inherited symbiont of ticks by the acquisition of novel virulence factors. All C. burnetii isolates carry a large plasmid or have a chromosomally integrated plasmid-like sequence. The plasmid is a candidate virulence factor that contributes to C. burnetii evolution. Here we describe the construction of novel shuttle vectors that allow to make plasmid-deficient C. burnetii mutants. With this plasmid-curing approach, we characterized the role of the QpH1 plasmid in in vitro and in vivo C. burnetii infection models. We found that the plasmid plays a critical role for C. burnetii growth in macrophages, especially in murine macrophages, but not in axenic media and BGMK cells. Our work highlights an essential role of the plasmid for the acquisition of colonizing capability in rodents by C. burnetii. This study represents a major step toward unravelling the mystery of the C. burnetii cryptic plasmids.

microbiology↗

bioRxiv2020

Novel and potent inhibitors targeting DHODH, a rate-limiting enzyme in de novo pyrimidine biosynthesis, are broad-spectrum antiviral against RNA viruses including newly emerged coronavirus SARS-CoV-2

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of coronavirus SARS-CoV-2. Existing direct-acting antiviral (DAA) drugs cannot be applied immediately to new viruses because of virus-specificity, and the development of new DAA drugs from the beginning is not timely for outbreaks. Thus, host-targeting antiviral (HTA) drugs have many advantages to fight against a broad spectrum of viruses, by blocking the viral replication and overcoming the potential viral mutagenesis simultaneously. Herein, we identified two potent inhibitors of DHODH, S312 and S416, with favorable drug-like and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus (H1N1, H3N2, H9N2), Zika virus, Ebola virus, and particularly against the recent novel coronavirus SARS-CoV-2. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knocking-out cells. We also proposed the drug combination of DAA and HTA was a promising strategy for anti-virus treatment and proved that S312 showed more advantageous than Oseltamivir to treat advanced influenza diseases in severely infected animals. Notably, S416 is reported to be the most potent inhibitor with an EC50 of 17nM and SI value >5882 in SARS-CoV-2-infected cells so far. This work demonstrates that both our self-designed candidates and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-repression may have clinical potentials not only to influenza but also to COVID-19 circulating worldwide, no matter such viruses mutate or not.

microbiology↗

bioRxiv2020

A single nonsynonymous mutation on gene encoding E protein of Zika virus leads to increased neurovirulence in vivo

Zika virus can infect a wide range of tissues including the developmental brain of human fetuses, causing from mild to severe clinical diseases. Whether its genetic characteristics impacts on viral pathogenesis is incompletely understood. We have obtained viral variants through serially passage of a clinical Zika virus isolate (SW01) in neonatal mice in vivo and found some of which exhibited markedly increased virulence and neurotropism. By deep sequencing analysis, the more pathogenic viral variants were found to contain four dominant nonsynonymous nucleotide mutations on genes encoding E and NS2A proteins. Further investigation using molecularly cloned viruses revealed that a single 67D (Aspatic acid) to N (Asparagine) substitution on E protein is sufficient to confer the increased virulence and neurotropism. These findings provide new insight into Zika virus pathogenesis and suggest novel targets for the development of therapeutics. Author SummaryRecent large outbreaks of Zika virus infection worldwide have revealed an association between the viral infection and increased cases of specific neurological problems including Congenital Zika Syndrome (including microcephaly) and adult Guillain-Barre Syndrome. However, the determinants of the increased neurovirulence of Zika virus remain uncertain. One hypothesis is that some unique changes across the Zika viral genome have led to the occurrence of these neurological diseases. To test this hypothesis, we continuously propagated a clinical isolate of contemporary Zika virus (SW01) in neonatal mice brain for 11 times to obtain an mouse central nervous system (CNS) adapted Zika virus (MA-SW01) that showed significantly increased neurovirulence in vivo. We then discovered that a single G to A nucleotide substitution at the 1069 site of Zika virus open reading frame leading to a D (aspartic acid) to N (asparagine) in viral Envelope protein is responsible for the increased neurovirulence. These findings improve our understanding of the neurological pathogenesis of Zika virus and provide clues for the development of antiviral strategy.

microbiology↗

bioRxiv2020

SMRT sequencing of the Oryza rufipogon genome reveals the genomic basis of rice adaptation

Asian cultivated rice is believed to have been domesticated from an immediate ancestral progenitor, Oryza rufipogon, which provides promising sources of novel alleles for world rice improvement. Here we first present a high-quality de novo assembly of the typical O. rufipogon genome through the integration of single-molecule sequencing (SMRT), 10x and Hi-C technologies. This chromosome-based reference genome allows a multi-species comparative analysis of the annual selfing O. sativa and its two wild progenitors, the annual selfing O. nivara and perennial outcrossing O. rufipogon, identifying massive numbers of dispensable genes that are functionally enriched in reproductive process. Comparative genomic analyses identified millions of genomic variants, of which large-effect mutations (e.g., SVs, CNV and PAVs) may affect the variation of agronomically significant traits. We demonstrate how lineage-specific expansion of rice gene families may have contributed to the formation of reproduction isolation (e.g., the recognition of pollen and male sterility), thus brightening the role in driving mating system evolution during the evolutionary process of recent speciation. We document thousands of positively selected genes that are mainly involved in flower development, ripening, pollination, reproduction and response to biotic- and abiotic stresses. We show that selection pressures may serve as crucial forces to govern substantial genomic alterations among the three rice species that form the genetic basis of rapid evolution of mating and reproductive systems under diverse habitats. This first chromosome-based wild rice genome in the genus Oryza will become powerful to accelerate the exploration of untapped genomic diversity from wild rice for the enhancement of elite rice cultivars.

genomics↗

bioRxiv2020

SMRT sequencing yields the chromosome-scale reference genome of tea tree, Camellia sinensis var. sinensis

Tea is the oldest and most popular nonalcoholic beverage consumed in the world. It provides abundant secondary metabolites that account for its diverse flavors and health benefits. Here we present the first high-quality chromosome-length reference genome of C. sinensis var. sinensis using long read single-molecule real time (SMRT) sequencing and Hi-C technologies to anchor the [~]2.85-Gb genome assembly into 15 pseudo-chromosomes with a scaffold N50 length of [~]195.68 Mb. We annotated at least 2.17 Gb ([~]74.13%) of repetitive sequences and high-confidence prediction of 40,812 protein-coding genes in the [~]2.92-Gb genome assembly. This accurately assembled genome allows us to comprehensively annotate functionally important gene families such as those involved in the biosynthesis of catechins, theanine and caffeine. The contiguous genome assembly provides the first view of the repetitive landscape allowing us to accurately characterize retrotransposon diversity. The large tea tree genome is dominated by a handful of Ty3-gypsy long terminal repeat (LTR) retrotransposon families that recently expanded to high copy numbers. We uncover the latest bursts of numerous non-autonomous LTR retrotransposons that may interfere with the propagation of autonomous retroelements. This reference genome sequence will largely facilitate the improvement of agronomically important traits relevant to the tea quality and production.

genomics↗

bioRxiv2019

Engineering oleaginous yeast Yarrowia lipolytica for violacein production: extraction, quantitative measurement and culture optimization

Violacein is a naturally occurring anticancer therapeutic with deep purple color. Yeast fermentation represents an alternative approach to efficiently manufacturing violacein from inexpensive feedstocks. In this work, we optimized the extraction protocol to improve violacein recovery ratio and purity from yeast culture, including the variations of organic solvents, the choice of mechanical shear stress, incubation time and the use of cell wall-degrading enzymes. We also established the quantitative correlation between HPLC and microplate reader method. We demonstrated that both HPLC and microplate reader are technically equivalent to measure violacein from yeast culture. Furthermore, we optimized the yeast cultivation conditions, including carbon/nitrogen ratio and pH conditions. Our results indicated that ethyl acetate is the best extraction solvent with glass beads grinding the cell pellets, the maximum violacein and deoxyviolacein production was 70.04 mg/L and 5.28 mg/L in shake flasks, respectively. Violacein purity reaches 86.92% at C/N ratio of 60, with addition of 10 g/L CaCO3 to control the media pH. Taken together, the development of efficient extraction protocol, quantitative correlation between HPLC and microplate reader, and the optimization of culture conditions set a new stage for engineering violacein production in Y. lipolytica. This information should be valuable for us to build a renewable and scalable violacein production platform from the novel host oleaginous yeast species.

microbiology↗

bioRxiv2019

Versatile polyketide biosynthesis platform for production of aromatic compounds in yeast

To accelerate the shift to bio-based production and overcome complicated functional implementation of natural and artificial biosynthetic pathways to industry relevant organisms, development of new, versatile, bio-based production platforms is required. Here we present a novel yeast platform for biosynthesis of bacterial aromatic polyketides. The platform is based on a synthetic polyketide synthase system enabling a first demonstration of bacterial aromatic polyketide biosynthesis in a eukaryotic host.

synthetic biology↗

bioRxiv2019

CRISPR-BEST: a highly efficient DSB-free base editor for filamentous actinomycetes

Filamentous actinomycetes serve as major producers of various natural products including antimicrobial compounds. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSB) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem). Specifically targeted by an sgRNA, the cytidine deaminase component of CRISPR-BEST efficiently converts C:G to T:A within a window of approximately seven-nucleotides. The system was validated and successfully used in different Streptomyces species.

synthetic biology↗

bioRxiv2018

Transection of the ventral hippocampal commissure impairs spatial but not contextual memory in split-brain mice

Plasticity is a neural phenomenon in which experience induces long-lasting changes to neuronal circuits and is at the center of most neurobiological theories of learning and memory. However, too much plasticity is maladaptive and must be balanced with substrate stability. Area CA3 of the hippocampus is lateralized with the left hemisphere dominant in plasticity and the right specialized for stability. Left and right CA3 project bilaterally to CA1; however, it is not known whether this downstream merging of lateralized plasticity and stability is functional. We hypothesized that interhemispheric integration of input from these pathways is essential for integrating spatial memory stored in the left CA3 with spatial working memory facilitated by the right CA3. To test this, we severed interhemispheric connections between the left and right hippocampi in mice and assessed learning and memory. Despite damage to this major hippocampal fiber tract, hippocampus-dependent spatial working memory and short- and long-term memory were both spared. However, tasks that required the integration of information retrieved from memory with ongoing spatial working memory and navigation were impaired. We propose that one function of interhemispheric communication in the mouse hippocampus is to integrate lateralized processing of plastic and stable circuits to facilitate memory-guided spatial navigation.

neuroscience↗

bioRxiv2018

Drug target identification and virtual screening in pursuit of phytochemical intervention of Mycobacterium chelonae

Mycobacterium chelonae is a rapidly growing mycobacterium present in the environment. It is associated with skin and soft tissue infections including abscess, cellulitis and osteomyelitis. Other infections by this bacterium are post-operative/transplant-associated, catheter, prostheses and even concomitant to haemodialytic procedures. In this study, we employ a subtractive genomics approach to predict the potential therapeutic candidates, intended for experimental research against this bacterium. A computational workflow was devised and executed to procure core proteome targets essential to the pathogen but with no similarity to the human host. Initially, essential Mycobacterium chelonae proteins were predicted through homology searching of core proteome content from 19 different bacteria. Druggable proteins were then identified and N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) was chosen as a case study from identified therapeutic targets, based on its important bifunctional role. Structure modeling was followed by virtual screening of phytochemicals (N > 10,000) against it. 4,4-[(1E)-5-hydroxy-4-(methoxymethyl)-1-pentene-1,5-diyl]diphenol, apigenin-7-O-beta-gluconopyranoside and methyl rosmarinate were screened as compounds having best potential for binding GlmU. Phytotherapy helps curb the menace of antibiotic resistance so treatment of Mycobacterium chelonae infection through this method is recommended.

bioinformatics↗

bioRxiv2017

Epidemic Potential for Human Infection with Influenza A (H7N9) Virus in China through Web Search Behaviors: A Data-Driven Study

Since the beginning of September 2016, a steep upsurge of the human cases of avian influenza A (H7N9) virus has been reported in China, which are alarming public concern for the pandemic potential of the H7N9 virus. In this study, we collected the data from H7N9 epidemics and H7N9-related Baidu Search Index (BSI) in China between September 2013 and June 2017. And we observed a strong correlation between the numbers of Influenza A (H7N9) cases and H7N9-related BSI in Guangdong province and Shanghai municipality (p<0.001). Autoregressive integrated moving average (ARIMA) models were constructed for the dynamic estimation of seasonal H7N9 outbreaks in 2016-2017 and the online search data acted as an external regressor with the historical H7N9 epidemic data in the forecasting model to improve the quality of predictions. Predictions by the models closely matched the actual numbers of reported cases during current H7N9 epidemic season. Especially, the estimated numbers of reported cases sharply increased to reach 49.88 (95% CI: 0-194.05) in Guangdong and 9.05 (95% CI: 0-37.43) in Shanghai from December 2016 to June 2017. Moreover, this accessible and flexible dynamic forecast model could be used in the monitoring of H7N9 virus to provide advanced warning of future emerging infection diseases.\n\nAuthor summaryAs the availability and popularity of the internet has greatly increased in recent years, an increasing number of cyber users, including patients and their family members, search online for health information on personal computers (PCs) and mobile phones (MPs) before seeking medical attention, making it possible to investigate the influenza prevalence by monitoring changes in frequencies of uses of particular search terms. In this study, we collected the data from H7N9 epidemics and H7N9-related Baidu Search Index (BSI) in China between September 2013 and June 2017. And then, we showed a strong correlation between the numbers of Influenza A (H7N9) cases and H7N9-related BSI in Guangdong province and Shanghai municipality (p<0.001). Furthermore, we reconstructed an improved dynamic forecasting method for outbreaks of H7N9 influenza using Autoregressive integrated moving average (ARIMA) models to predict future patterns of H7N9 transmission and the online search data acted as an external regressor with the historical H7N9 epidemic data in the forecasting model to improve the quality of predictions. Our results suggest that data from the Baidu search engine, combed with data from a traditional disease surveillance system, may be considered for early detection of H7N9 influenza outbreaks in mainland China.

epidemiology↗

bioRxiv2017

Intestinal cell kinase regulates chondrocyte proliferation and maturation during skeletal development

An autosomal recessive loss-of-function mutation R272Q in human ICK (intestinal cell kinase) gene causes profound multiplex developmental defects in human ECO (endocrine-cerebro-osteodysplasia) syndrome. ECO patients exhibit a wide variety of skeletal abnormalities, yet the underlying cellular and molecular mechanisms by which ICK regulates skeletal development remain largely unknown. The goal of this study is to understand the structural and mechanistic basis underlying skeletal anomalies caused by ICK dysfunction. Ick R272Q knock in transgenic mouse model not only recapitulated major ECO skeletal defects such as short limbs and polydactyly but also revealed a deformed spine with deficient intervertebral disc. Loss of ICK functions markedly reduces mineralization in the spinal column, ribs, and long bones. Ick mutants show a significant decrease in the number of proliferating chondrocytes and type X collagen-expressing hypertrophic chondrocytes in the spinal column and the growth plate of long bones. Our results demonstrate that ICK plays an important role in bone and intervertebral disc development by promoting chondrocyte proliferation and maturation, and thus provide novel mechanistic insights into the skeletal phenotypes of human ECO syndrome.

developmental biology↗

bioRxiv2017

Gαq Mediates Clozapine Effects in Caenorhabditis elegans

Clozapine binds and has significant effects on multiple neurotransmitter receptors, notably including some dopamine receptors. Downstream of these receptors, clozapine affects the balance of Gi- and Gq-dependent second-messenger signaling. We used Caenorhabiditis elegans as a genetic model to study further how clozapine affects both dopamine receptors and downstream Gq mediated signaling. Four of six worm dopamine receptor orthologs, dop-1, dop-2, dop-4, and dop-5 produced resistance to clozapine induced developmental delay when mutated, suggesting that both type I and type II dopamine receptors mediate the behavioral effects of clozapine in C. elegans. Beyond these receptors, reduction of function of one of the G proteins, egl-30 (Gq), produced greatly increased susceptibility to clozapine. Gq has multiple known downstream effects. Among these is the control of acetylcholine release, which is in balance with monoamines in the human brain and is another target of clozapine and other antipsychotic drugs. We tested for downstream effects on acetylcholine at the neuromuscular junction upon clozapine treatment but found no evidence for effects of clozapine. In contrast, modulation of Gq upstream leads to worms that are either more resistant or more susceptible to clozapine, emphasizing the importance of Gq proteins in mediating effects of clozapine. A genetic screen for suppressors of egl-30 recovered eight mutants. By characterizing the behavioral effects of these mutants, we found that clozapine exerts its function on development by affecting Gq signaling through control of the pharyngeal pumping rate. A whole-genome sequencing technique was utilized and identified a list of candidate genes for these suppressor mutations. Further characterization of these mutants promises the discovery of novel components participating in Gq signaling and a better understanding of the mechanisms of action of clozapine.

genetics↗