Biology subjects

Wan, Y.

Publications and source records attributed to Wan, Y..

98 records · Page 6Linked to original sources

bioRxiv2019

Quantitative measurements of chromatin modification dynamics during zygotic genome activation

Histone posttranslational modifications (PTMs) are key gene expression regulators, but their rapid dynamics during development remain difficult to capture. We describe an approach that images fluorescent antibody fragments to quantify PTM enrichment and active transcription at defined loci during zygotic genome activation in living zebrafish embryos. The technique reveals a drastic increase in histone H3 Lys27 acetylation (H3K27ac) before genome activation both at a specific locus and globally.

cell biology↗

bioRxiv2019

Tomato spotted wilt virus manipulates the reproduction of its insect vector, western flower thrips (Frankliniella occidentalis), to facilitate transmission

BACKGROUNDTomato spotted wilt virus (TSWV), one of the most devastating viruses of ornamental plants and vegetable crops worldwide, is transmitted by the western flower thrips, Frankliniella occidentalis (Pergande), in a persistent-propagative manner. How TSWV manipulates the reproduction of its vector to enhance transmission and whether infection with TSWV changes the mating behaviour of this thrips vector are not fully understood.\n\nRESULTSIn this study, we found that TSWV-exposed thrips, in general, had a significantly longer developmental time than did non-exposed individuals. Such an increase was predominantly seen in adults, a stage associated with dispersal and virus transmission. TSWV-exposed F. occidentalis produced substantially more progeny than did non-exposed thrips. Interestingly, most of the increase in progeny came from an increase in males, a sex with a greater dispersal and virus transmission capability. Specifically, the sex ratio of progeny shifted from female biased (2-7:1) to evenly split or male biased. Regarding mating behaviour, compared to virus-free controls, TSWV-exposed F. occidentalis had significantly longer copulation duration, were more active in males, and remated less often in females.\n\nCONCLUSIONThese combined results suggest that TSWV alters the reproductive behaviour of its insect vector, F. occidentalis, to promote virus transmission. Consequently, a monitoring program capable of earlier detection of the virus and a reduced economic threshold for vector (thrips) control should be in consideration for the long-term, sustainable management of TSWV.

ecology↗

bioRxiv2019

Interkinetic nuclear migration in the zebrafish retina as a diffusive process

A major hallmark of neural development is the oscillatory movement of nuclei between the apical and basal surfaces of the neuroepithelium during the process of interkinetic nuclear migration (IKNM). Here, we employ long-term, rapid lightsheet and two-photon imaging of Zebrafish retinas in vivo during early development to uncover the physical processes that govern the behavior of nuclei during IKNM. These images allow the capture of reliable tracks of nuclear movements and division during early retinogenesis for many tightly packed nuclei. These tracks are then used to create and test a theory of retinal IKNM as a diffusive process across a nuclear concentration gradient generated by the addition of new nuclei at the apical surface. The analysis reveals the role of nuclear packing at the apical surface on the migration dynamics of nuclei, provides a robust quantitative explanation for the distribution of nuclei across the retina, and may have implications for stochastic fate choice in this system.

developmental biology↗

bioRxiv2019

Streamlined low-input transcriptomics through EASY-RNAseq

High-throughput sequencing for transcriptome profiling is an increasingly accessible and important tool for biological research. However, accurate profiling of small cell populations remains challenging due to issues with gene detection sensitivity and experimental complexity. Here we describe a streamlined RNAseq protocol (EASY RNAseq) for sensitive transcriptome assessment starting from low amount of input materials. EASY RNAseq is technically robust enough for sequencing small pools of homogenous and heterogeneous cells, recovering higher numbers of genes and with a more even expression distribution pattern than other commonly used methods. Application of EASY RNAseq to single human embryos at the 8-cell stage was able to achieve detection of 70% protein-coding genes. This workflow may thus serve as a useful tool for sensitive interrogation of rare cell populations.

genomics↗

bioRxiv2018

Microbial Cells Harboring a Mitochondrial Gene Are Capable of CO2 Capture

Global warming is escalating with increased temperatures reported worldwide. Given the enormous land mass on the planet, biological capture of CO2 remains a viable approach to mitigate the crisis as it is economical and easy to implement. In this study, a gene capable of CO2 capture was identified via selection in minimal media. This mitochondrial gene named as OG1 encodes the OK/SW-CL.16 protein and shares homology with cytochrome oxidase subunit III of various species and PII uridylyl-transferase from Loktanella vestfoldensis SKA53. CO2 capture experiments indicate that {delta}13C was substantially higher in the cells harboring the gene OG1 than the control in the nutrition-poor media. This study suggests that CO2 capture using engineered microorganisms in barren land can be exploited to address the soaring CO2 level in the atmosphere, opening up vast land resources to cope with global warming.\n\nIMPORTANCEGlobal warming crisis is deteriorating with increased CO2 levels in the atmosphere each year. Action must be taken before catastrophic consequences occur in the not-so-distant future. Biological capture of CO2 is a feasible approach to alleviate the current crisis. We have identified a mitochondrial gene which demonstrated CO2 utilization capability. Data presented in this study suggest that CO2 capture using engineered microorganisms can be harnessed to address the ever-rising CO2 level in the atmosphere.

bioengineering↗

bioRxiv2018

A Novel Method for the Capture-based Purification of Whole Viral Native RNA Genomes

Current technologies for targeted characterization and manipulation of viral RNA either involve amplification or ultracentrifugation with isopycnic gradients of viral particles to decrease host RNA background. The former strategy is non-compatible for characterizing properties innate to RNA strands such as secondary structure, RNA-RNA interactions, and also for nanopore direct RNA sequencing involving the sequencing of native RNA strands. The latter strategy, ultracentrifugation, causes loss in genomic information due to its inability to retrieve unassembled viral RNA. We developed a novel nucleic acid manipulation technique involving the capture of whole viral native RNA genomes for downstream RNA assays using hybridization baits in solution to circumvent these problems. This technique involves hybridization of biotinylated baits at 500 nucleotides (nt) intervals, stringent washes and release of free native RNA strands using DNase I treatment, with a turnaround time of about 6 h 15 min. Proof of concept was primarily done using RT-qPCR with dengue virus infected Huh-7 cells. We report that this protocol was able to purify viral RNA (561-791 fold). We also describe a successful application of our capture-based purification method to direct RNA sequencing, with a 77.47% of reads mapping to the target viral genome. We observed a reduction in human host RNA background by 1580 fold, a 99.91% recovery of viral genome with at least 15x coverage, and a mean coverage across the genome of 120x. This report is, to the best of our knowledge, the first description of a capture-based purification method for whole viral RNA genomes. The fundamental advantages of using our capture-based purification method makes it a superior alternative to conventional viral purification methods and would potentially pave a new path for the direct characterization and sequencing of native RNA molecules.

genomics↗

bioRxiv2018

Structure mapping of dengue and Zika viruses reveals new functional long-range interactions

Dengue and Zika are clinically important members of the Flaviviridae family that utilizes an 11kb positive strand RNA for genome regulation. While structures have been mapped primarily in the UTRs, much remains to be learnt about how the rest of the genome folds to enable function. Here, we performed secondary structure and pair-wise interaction mapping on four dengue serotypes and four Zika strains in their native virus particles and infected cells. Comparative analysis of SHAPE reactivities across serotypes nominated potentially functional regions that are highly structured, show structure conservation, and low synonymous mutation rates, including a structure associated with ribosome pausing. Pair-wise interaction mapping by SPLASH further reveals new pair-wise interactions, in addition to the known circularization sequence. 40% of pair-wise interactions form alternative structures, suggesting extensive structural heterogeneity. Analysis of shared pair-wise interactions between serotypes revealed macro-organization whereby interactions are preserved at their physical locations, beyond their sequence identities. In addition, structure mapping of virus genomes released in solution-as well as inside host cells-showed that other helicases, in addition to the ribosome, play a role in unwinding viral structures inside cells. Mutational experiments that disrupt in cell and in virion pair-wise interactions result in virus attenuation, demonstrating their importance during the virus life-cycle.

genomics↗

bioRxiv2018

The identification of an anti-thrombin molecule via the screening of semi-random DNA libraries

Thrombosis remains one of the leading causes of mortality and morbidity in the world. Thrombin is a key enzyme involved in the blood clotting processes, which can be intervened by low concentrations of Hirudin. The C-terminal dodecapeptide of Hirudin was capable of inhibiting thrombosis. This peptide has been partially randomized in this report, and the coding sequences have been expressed in yeast as chimerical peptides for secretion into the culture media. Two other semi-random modules have been processed likewise. The supernatant was subsequently tested for anti-thrombin activities. DNA sequencing indicated that the putative positive clone encoded a single serine residue followed by a stop codon. The Ninhydrin assay of the culture supernatant of the positive clone indicated a high content of amino acid. Electrospray Mass Spectrometry showed a distinct peak at 430.5 when the expression products from Pichia pastoris were examined, suggesting that the compound may be a dimannosylated serine, as yeast possesses glycosylation at serine residues. The observed effects of -Mannosidase treatments on the function of yeast induction products are consistent with this assumption. Partial randomization of peptides and proteins may accelerate directed evolution, yielding unprecedented number of variants for functional interrogation and drug development.

molecular biology↗