Biology subjects

Wu, Y.

Publications and source records attributed to Wu, Y..

At least 1,099 records · Page 61Linked to original sources

bioRxiv2017

A high-throughput analysis method of microdroplet PCR coupled with fluorescence spectrophotometry

Here we report a novel microdroplet PCR method combined with fluorescence spectrophotometry (MPFS), which allows for qualitative, quantitative and high -throughput detection of multiple DNA targets. In this study, each pair of primers was labeled with a specific fluorophore. Through microdroplet PCR, a target DNA was amplified and labeled with the same fluorophore. After products purification, the DNA products tagged with different fluorophores could be analyzed qualitatively by the fluorescent intensity determination. The relative fluorensence unit was also measured to construct the standard curve and to achieve quantitative analysis. In a reaction, the co -amplified products with different fluorophores could be simultaneously analyzed to achieve high -throughput detection. We used four kinds of GM maize as a model to confirm this theory. The qualitative results revealed high specificity and sensitivity of 0.5% (w / w). The quantitative results revealed that the limit of detection was 103copies and with good repeatability. Moreover, reproducibility assay were further performed using four foodborne pathogenic bacteria. Consequently, the same qualitative, quantitative and high-throughput results were confirmed as the four GM maize.

biochemistry↗

bioRxiv2017

Single-shot super-resolution total internal reflection fluorescence microscopy

We demonstrate a simple method for combining instant structured illumination microscopy (SIM) with total internal reflection fluorescence microscopy (TIRF), doubling the spatial resolution of TIRF (down to 115 +/-13 nm) and enabling imaging frame rates up to 100 Hz over hundreds of time points. We apply instant TIRF-SIM to multiple live samples, achieving rapid, high contrast super-resolution imaging in close proximity to the coverslip surface.

bioengineering↗

bioRxiv2017

A scheme for 3-dimensional morphological reconstruction and force inference in the early C. elegans embryo

In this study, we present novel schemes for the reconstruction of cellular morphology and the inference of forces in the early C. elegans embryo. We have developed and bench-marked a morphological reconstruction scheme that transforms live-imaging of cellular membranes into a point cloud of smoothed surface patches, which facilitates accurate estimation of membrane curvatures and the angles between membranes.\n\nAssuming an isotropic and homogeneous distribution of tensions along a membrane, we infer a pattern of forces that are 7% deviated from force balance at edges, and 10% deviated from the Young-Laplace relation at membrane faces. We have also demonstrated the stability of our scheme by sensitivity analysis of the coefficient matrices involved and the reproducibility of our image-analysis and force inference pipeline.

biophysics↗

bioRxiv2017

Immunologic Effect of Polysaccharides Extracted from Sipunculus nudus (SNP) on Hepatoma HepG2-bearing Mice

Since many studies have clarified the biological activity of polysaccharides, we investigated the effect of SNP which was the water-soluble polysaccharides extracted from Sipunculus nudus on Hepatoma HepG2-bearing Mice to verify the potential of SNP as an effective clinical agent for liver cancer therapy. SNP were administered at the doses of 50,100, and 200 mg/kg to HepG2-bearing mice to determine their antitumor effects. SNP had an inhibitory effect on the growth of HepG2 cells and enhanced the immunological effect on HepG2 tumor-bearing mice. SNP increased the expression of IL-2, IFN-{gamma}, and TNF- cytokines in serum, suggesting that SNP can strengthen the antitumor immune response. In addition, SNP increased ATF4, DDIT3, and IkB expression and decreased CYR61, HSP90, and VEGF expression, all of which are proteins involved in antitumor activity and cell death/survival. our results suggested that SNP may be a novel antitumor agent.\n\nSummary statementSNP(polysaccharides extracted from Sipunculus nudus) mediates anti-tumor activity through influencing immunoregulation, and SNP can be explored as a promising candidate for future anticancer drug.

cancer biology↗

bioRxiv2017

Expanding the Atlas of Functional Missense Variation for Human Genes

Although we now routinely sequence human genomes, we can confidently identify only a fraction of the sequence variants that have a functional impact. Here we developed a deep mutational scanning framework that produces exhaustive maps for human missense variants by combining random codon-mutagenesis and multiplexed functional variation assays with computational imputation and refinement. We applied this framework to four proteins corresponding to six human genes: UBE2I (encoding SUMO E2 conjugase), SUMO1 (small ubiquitin-like modifier), TPK1 (thiamin pyrophosphokinase), and CALM1/2/3 (three genes encoding the protein calmodulin). The resulting maps recapitulate known protein features, and confidently identify pathogenic variation. Assays potentially amenable to deep mutational scanning are already available for 57% of human disease genes, suggesting that DMS could ultimately map functional variation for all human disease genes.

molecular biology↗

bioRxiv2017

Causal associations between risk factors and common diseases inferred from GWAS summary data

Health risk factors such as body mass index (BMI), serum cholesterol and blood pressure are associated with many common diseases. It often remains unclear whether the risk factors are cause or consequence of disease, or whether the associations are the result of confounding. Genetic methods are useful to infer causality because genetic variants are present from birth and therefore unlikely to be confounded with environmental factors. We develop and apply a method (GSMR) that performs a multi-SNP Mendelian Randomization analysis using summary-level data from large genome-wide association studies (sample sizes of up to 405,072) to test the causal associations of BMI, waist-to-hip ratio, serum cholesterols, blood pressures, height and years of schooling (EduYears) with a range of common diseases. We identify a number of causal associations including a protective effect of LDL-cholesterol against type-2 diabetes (T2D) that might explain the side effects of statins on T2D, a protective effect of EduYears against Alzheimers disease, and bidirectional associations with opposite effects (e.g. higher BMI increases the risk of T2D but the effect T2D of BMI is negative). HDL-cholesterol has a significant risk effect on age-related macular degeneration, and the effect size remains significant accounting for the other risk factors. Our study develops powerful tools to integrate summary data from large studies to infer causality, and provides important candidates to be prioritized for further studies in medical research and for drug discovery.

genetics↗

bioRxiv2017

confFuse: high-confidence fusion gene detection across tumor entities

BackgroundFusion genes play an important role in the tumorigenesis of many cancers. Next-generation sequencing (NGS) technologies have been successfully applied in fusion gene detection for the last several years, and a number of NGS-based tools have been developed for identifying fusion genes during this period. Most fusion gene detection tools based on RNA-seq data report a large number of candidates (mostly false positives), making it hard to prioritize candidates for experimental validation and further analysis. Selection of reliable fusion genes for downstream analysis becomes very important in cancer research. We therefore developed confFuse, a scoring algorithm to reliably select high-confidence fusion genes which are likely to be biologically relevant.\n\nResultsConfFuse takes multiple parameters into account in order to assign each fusion candidate a confidence score, of which score [≥]8 indicates high-confidence fusion gene predictions. These parameters were manually curated based on our experience and on certain structural motifs of fusion genes. Compared with alternative tools, based on 96 published RNA-seq samples from different tumor entities, our method can significantly reduce the number of fusion candidates (301 high-confidence from 8,083 total predicted fusion genes) and keep high detection accuracy (recovery rate 85.7%). Validation of 18 novel, high-confidence fusions detected in three breast tumor samples resulted in a 100% validation rate.\n\nConclusionsConfFuse is a novel downstream filtering method that allows selection of highly reliable fusion gene candidates for further downstream analysis and experimental validations. confFuse is available at https://github.com/Zhiqin-HUANG/confFuse.

bioinformatics↗

bioRxiv2017

In vivo Imaging β-cell Function Reveals Two Waves of β-cell Maturation

The insulin-secreting cells generated from stem cells in vitro are less glucose responsive than primary {beta}-cells. To search for the missing ingredients that are needed for {beta}-cell maturation, we have longitudinally monitored function of every {beta}-cell in Tg (ins:Rcamp1.07) zebrafish embryos with a newly-invented two-photon light-sheet microscope. We have shown that {beta}-cell maturation begins from the islet mantle and propagates to the islet core during the hatching period, coordinated by the islet vascularization. Lower concentration of glucose is optimal to initiate {beta}-cell maturation, while increased glucose delivery to every cell through microcirculation is required for functional boosting of the {beta}-cells. Both the initiation and the boosting of {beta}-cell maturation demands activation of calcineurin/NFAT by glucose. Calcineurin activator combined with glucose promotes mouse neonatal {beta}-cells cultured in vitro to mature to a functional state similar to adult {beta}-cells, suggesting a new strategy for improving stem cell-derived {beta}-like cell function in vitro.

developmental biology↗

bioRxiv2017

BPG: Seamless, Automated and Interactive Visualization of Scientific Data

We introduce BPG, an easy-to-use framework for generating publication-quality, highly-customizable plots in the R statistical environment. This open-source package includes novel methods of displaying high-dimensional datasets and facilitates generation of complex multi-panel figures, making it ideal for complex datasets. A web-based interactive tool allows online figure customization, from which R code can be downloaded for seamless integration with computational pipelines. BPG is available at http://labs.oicr.on.ca/boutros-lab/software/bpg

bioinformatics↗

bioRxiv2017

Reflective imaging improves resolution, speed, andcollection efficiency in light sheet microscopy

Light-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, gentle imaging of live biological specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of multiple views, obtaining 4 complementary views in 250 ms, half the period it would otherwise take to collect only two views in symmetric dual-view selective plane illumination microscopy (diSPIM). We also report a modified deconvolution algorithm that removes the associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to < 300 nm in all three dimensions) by applying our method to a new asymmetric diSPIM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture (NA). We demonstrate the broad applicability of our method in a variety of samples of moderate (< 50 m) thickness, studying mitochondrial, membrane, Golgi, and microtubule dynamics in single cells and calcium activity in nematode embryos.

bioengineering↗

bioRxiv2017

Widespread signatures of negative selection in the genetic architecture of human complex traits

Estimation of the joint distribution of effect size and minor allele frequency (MAF) for genetic variants is important for understanding the genetic basis of complex trait variation and can be used to detect signature of natural selection. We develop a Bayesian mixed linear model that simultaneously estimates SNP-based heritability, polygenicity (i.e. the proportion of SNPs with nonzero effects) and the relationship between effect size and MAF for complex traits in conventionally unrelated individuals using genome-wide SNP data. We apply the method to 28 complex traits in the UK Biobank data (N = 126,752), and show that on average across 28 traits, 6% of SNPs have nonzero effects, which in total explain 22% of phenotypic variance. We detect significant (p < 0.05/28 =1.8x10-3) signatures of natural selection for 23 out of 28 traits including reproductive, cardiovascular, and anthropometric traits, as well as educational attainment. We further apply the method to 27,869 gene expression traits (N = 1,748), and identify 30 genes that show significant (p < 2.3x10-6) evidence of natural selection. All the significant estimates of the relationship between effect size and MAF in either complex traits or gene expression traits are consistent with a model of negative selection, as confirmed by forward simulation. We conclude that natural selection acts pervasively on human complex traits shaping genetic variation in the form of negative selection.

genetics↗

bioRxiv2017

The Cell Polarity Proteins Boi1p And Boi2p Stimulate Vesicle Fusion At The Plasma Membrane Of Yeast Cells

Eukaryotic cells can direct secretion to defined regions of their plasma membrane. These regions are distinguished by an elaborate architecture of proteins and lipids that are specialized to capture and fuse post-Golgi vesicles. Here we show that the proteins Boi1p and Boi2p are important elements of this area of active exocytosis at the tip of growing yeast cells. Cells lacking Boi1p and Boi2p accumulate secretory vesicles in their bud. The essential PH domains of Boi1p and Boi2p interact with Sec1p, a protein required for SNARE complex formation and vesicle fusion. Sec1p loses its tip localization in cells depleted of Boi1p and Boi2p but can partially compensate for their loss upon overexpression. The capacity to simultaneously bind phospholipids, Sec1p, multiple subunits of the exocyst, Cdc42p, and the module for generating active Cdc42p identify Boi1p and Boi2p as essential mediators between exocytosis and polar growth.\n\nSummary statementA novel protein complex connects vesicle fusion with Cdc42p activation. Genetic and protein interaction data suggest that its central members Boi1p and Boi2p chaperone the formation of the docking complex.

cell biology↗

bioRxiv2017

Simple And Multiplexed Enrichment Of Rare DNA Variants Via Sequence-Selective And Temperature-Robust Amplification

Rare DNA sequence variants hold important clinical and biological information, but are chal-lenging for existing methods (e.g. PCR, NGS) to profile in an inexpensive, multiplexed, simple-to-implement, and sequence-general way. Here, we present Blocker Displacement Amplification (BDA), a temperature-robust PCR method that selectively amplifies all sequence variants within a roughly 20 nt window by 1000-fold over wildtype sequences, allowing easy detection and quantitation of hundreds of potentials variants originally at [≤]0.1% allele frequency. BDA employs a rationally designed competitive hybridization reaction to achieve similar enrichment performance across anneal temperatures ranging from 56{degrees}C to 64{degrees}C. This temperature robustness facilitates multiplexed enrichment of many different variants across the genome, and furthermore enables the use of in-expensive and portable thermocycling instruments for rare DNA variant detection. To show the sequence generality of BDA, we demonstrated enrichment on 156 single-nucleotide variants (SNVs). BDA has been validated on multiple different PCR platforms, DNA polymerases, and sample types including clinical cell-free DNA samples collected from the blood plasma of lung cancer patients. BDA quantitation of mutation allele fraction is generally consistent with deep sequencing results.

genomics↗

bioRxiv2017

Deep-Learning Based, Automated Segmentation Of Macular Edema In Optical Coherence Tomography

Evaluation of clinical images is essential for diagnosis in many specialties and the development of computer vision algorithms to analyze biomedical images will be important. In ophthalmology, optical coherence tomography (OCT) is critical for managing retinal conditions. We developed a convolutional neural network (CNN) that detects intraretinal fluid (IRF) on OCT in a manner indistinguishable from clinicians. Using 1,289 OCT images, the CNN segmented images with a 0.911 cross-validated Dice coefficient, compared with segmentations by experts. Additionally, the agreement between experts and between experts and CNN were similar. Our results reveal that CNN can be trained to perform automated segmentations.

bioinformatics↗

bioRxiv2017

CircMarker: A Fast and Accurate Algorithm for Circular RNA Detection

While RNA is often created from linear splicing during transcription, recent studies have found that non-canonical splicing sometimes occurs. Non-canonical splicing joins 3 and 5 and forms the socalled circular RNA. It is now believed that circular RNA plays important biological roles such as affecting susceptibility in some diseases. within these few years, several experimental methods have been developed to enrich circular RNA while degrade linear RNA. Although several useful software tools for circRNA detection have been developed as well, these tools may miss many circular RNA. Also, existing tools are slow for large data because those tools often depend on reads mapping. In this paper, we present a new computational approach, named CircMarker, based on k-mers rather than reads mapping for circular RNA detection. CircMarker takes advantage of transcriptome annotation files to create k-mer table for circular RNA detection. Empirical results show that CircMarker outperforms existing tools in circular RNA detection on accuracy and efficiency in many simulated and real datasets. CircMarker can be downloaded from https://github.com/lxwgcool/CircMarker.

bioinformatics↗

bioRxiv2017

Role of Membrane-tension Gated Ca Flux in Cell Mechanosensation

Eukaryotic cells are sensitive to mechanical forces that they experience from the environment. The process of mechanosensation is complex, and involves elements such as the cytoskeleton and active contraction from myosin motors. Ultimately, mechanosensation is connected to changes in gene expression in the cell, or mechanotransduction. While the involvement of the cytoskeleton in mechanosensation is known, processes upstream to cytoskeletal changes is unclear. In this paper, using a microfluidic device that mechanically compresses live cells, we demonstrate that calcium currents and membrane tension-sensitive ion channels directly signals to the Rho GTPase and myosin contraction. In response to membrane tension changes, cell actively regulates cortical myosin contraction to balance external forces. The process is captured by a mechanochemical model where membrane tension, myosin contraction and the osmotic pressure difference between the cytoplasm and extracellular environment are connected by mechanical force-balance. Finally, to complete the picture of mechanotransduction, we find that the tension-sensitive transcription factor YAP translocates from the nucleus to the cytoplasm in response to mechanical compression.

cell biology↗

bioRxiv2017

Pooled CRISPR interference screens enable high-throughput functional genomics study and elucidate new rules for guide RNA library design in Escherichia coli

Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology provides potential advantages in high-throughput functional genomics analysis in prokaryotes over previously established platforms based on recombineering or transposon mutagenesis. In this work, as a proof-of-concept to adopt CRISPR/Cas9 method as a pooled functional genomics analysis platform in prokaryotes, we developed a CRISPR interference (CRISPRi) library consisting of 3,148 single guide RNAs (sgRNAs) targeting the open reading frame (ORF) of 67 genes with known knockout phenotypes and performed pooled screens under two stressed conditions (minimal and acidic medium) in Escherichia coli. Our approach confirmed most of previously described gene-phenotype associations while maintaining < 5% false positive rate, suggesting that CRISPRi screen is both sensitive and specific. Our data also supported the ability of this method to narrow down the candidate gene pool when studying operons, a unique structure in prokaryotic genome. Meanwhile, assessment of multiple loci across treatments enables us to extract several guidelines for sgRNA design for such pooled functional genomics screen. For instance, sgRNAs locating at the first 5% upstream region within ORF exhibit enhanced activity and 10 sgRNAs per gene is suggested to be enough for robust identification of gene-phenotype associations. We also optimized the hit-gene calling algorithm to identify target genes more robustly with even fewer sgRNAs. This work showed that CRISPRi could be adopted as a powerful functional genomics analysis tool in prokaryotes and provided the first guideline for the construction of sgRNA libraries in such applications.\n\nImportanceTo fully exploit the valuable resource of explosive sequenced microbial genomes, high-throughput experimental platform is needed to associate genes and phenotypes at the genome level, giving microbiologists the insight about the genetic structure and physiology of a microorganism. In this work, we adopted CRISPR interference method as a pooled high-throughput functional genomics platform in prokaryotes with Escherichia coli as the model organism. Our data suggested that this method was highly sensitive and specific to map genes with previously known phenotypes, potent to act as a new strategy for high-throughput microbial genetics study with advantages over previously established methods. We also provided the first guideline for the sgRNA library design by comprehensive analysis of the screen data. The concept, gRNA library design rules and open-source scripts of this work should benefit prokaryotic genetics community to apply high-throughput mapping of defined gene set with phenotypes in a broad spectrum of microorganisms.

microbiology↗

bioRxiv2017

Modeling Zika Virus Congenital Eye Disease: Differential Susceptibility of Fetal Retinal Progenitor Cells and iPSC-Derived Retinal Stem Cells to Zika Virus Infection

Zika virus (ZIKV) causes microcephaly and congenital eye disease that is characterized by macular pigment mottling, macular atrophy, and loss of foveal reflex. The cell and molecular basis of congenital ZIKV infection are not well understood. Here, we utilized a biologically relevant cell-based system on human fetal retinal pigment epithelial cells (FRPE) and iPSC-derived retinal stem cells (iRSCs) to model ZIKV-ocular cell injury processes. FRPEs were highly susceptible to ZIKV, resulting in apoptosis and decreased viability, whereas iRSCs showed reduced susceptibility. Transcriptomics and proteomics analyses of infected FRPE cells revealed the activation of innate immune and inflammatory response genes, and dysregulation of cell survival pathways, mitochondrial transmembrane potential, phagocytosis, and particle internalization. Nucleoside analogue drug treatment inhibited ZIKV replication and prevented apoptosis. In conclusion, ZIKV affects ocular cells of different developmental stages resulting in cellular injury and death, further providing molecular insight into the pathogenesis of congenital eye disease.

microbiology↗