Biology subjects

Ning, W.

Publications and source records attributed to Ning, W..

23 records · Page 2Linked to original sources

bioRxiv2020

New targets acquired: improving locus recovery from the Angiosperms353 probe set

Universal target enrichment kits maximise utility across wide evolutionary breadth while minimising the number of baits required to create a cost-efficient kit. Locus assembly requires a target reference, but the taxonomic breadth of the kit means that target references files can be phylogenetically sparse. The Angiosperms353 kit has been successfully used to capture loci throughout angiosperms but includes sequence information from 6-18 taxa per locus. Consequently, reads sequenced from on-target DNA molecules may fail to map to references, resulting in fewer on-target reads for assembly, reducing locus recovery. We expanded the Angiosperms353 target file, incorporating sequences from 566 transcriptomes to produce a mega353 target file, with each gene represented by 17-373 taxa. This mega353 file is a drop-in replacement for the original Angiosperms353 file in HybPiper analyses. We provide tools to subsample the file based on user-selected taxon groups, and to incorporate other transcriptome or protein-coding gene datasets. Compared to the default Angiosperms353 file, the mega353 file increased the percentage of on-target reads by an average of 31%, increased loci recovery at 75% length by 61.9%, and increased the total length of the concatenated loci by 30%. The mega353 file and associated scripts are available at: https://github.com/chrisjackson-pellicle/NewTargets

bioinformatics↗

bioRxiv2020

Mechanical Regulation of Stem Cell Proliferation and Fate Decisions by their Differentiated Daughters

Basal stem cells fuel development, homeostasis, and regeneration of the epidermis. The proliferation and fate decisions of these cells are highly regulated by their microenvironment, including the basement membrane and underlying mesenchymal cells. Basal progenitors give rise to differentiated progeny that serve an essential role in generating the epidermal barrier. Here, we present data that differentiated progeny also regulate the proliferation, differentiation, and migration of basal progenitor cells. Using two distinct mouse lines, we found that increasing contractility of differentiated cells resulted in non-cell autonomous hyperproliferation of stem cells and prevented their commitment to a hair follicle lineage. These phenotypes were rescued by pharmacological inhibitors of contractility. Live-imaging revealed that increasing the contractility of differentiated cells resulted in stabilization of adherens junctions and impaired movement of basal progenitors during hair placode morphogenesis, as well as a defect in migration of melanoblasts. These data suggest that intra-tissue tension regulates stem cell proliferation, fate decisions and migration, similar to the known roles of extracellular matrix rigidity. Additionally, this work demonstrates that differentiated epidermal keratinocytes are a component of the stem cell niche that regulates development and homeostasis of the skin.

cell biology↗

bioRxiv2020

Constructing an ethanol utilization pathway in Escherichia coli to produce acetyl-CoA derived compounds

Engineering microbes to utilize non-conventional substrates could create short and efficient pathways to convert substrate into product. In this study, we designed and constructed a two-step heterologous ethanol utilization pathway (EUP) in Escherichia coli by using acetaldehyde dehydrogenase (encoded by ada) from Dickeya zeae and alcohol dehydrogenase (encoded by adh2) from Saccharomyces cerevisiae. This EUP can convert ethanol into acetyl-CoA without ATP consumption, and generate two molecules of NADH per molecule of ethanol. We optimized the expression of these two genes and found that ethanol consumption could be improved by expressing them in a specific order (ada-adh2) with a constitutive promoter (PgyrA). The engineered E. coli strain with EUP consumed approximately 8 g/L of ethanol in 96 hours when it was used as sole carbon source. Subsequently, we combined EUP with the biosynthesis of polyhydroxybutyrate (PHB), a biodegradable polymer derived from acetyl-CoA. The engineered E. coli strain carrying EUP and PHB biosynthetic pathway produced 1.1 g/L of PHB from 10 g/L of ethanol and 1 g/L of aspartate family amino acids in 96 hours. We also engineered E. coli strain to produced 24 mg/L of prenol from 10 g/L of ethanol in 48 hours, supporting the feasibility of converting ethanol into different classes of acetyl-CoA derived compounds. HighlightsO_LIEngineered Escherichia coli strains to grow on ethanol as sole carbon source C_LIO_LIDemonstrated that ethanol was converted into acetyl-CoA (AcCoA) through two pathways (acetaldehyde-acetate-AcCoA and acetaldehyde-AcCoA) C_LIO_LIConverted ethanol into two acetyl-CoA derived products with low structural similarity (polyhydroxybutyrate and prenol) C_LIO_LIDiscovered that supplementation of the aspartate family amino acids can substantially improve cell growth on ethanol C_LI

bioengineering↗

bioRxiv2019

Improve Protein Solubility and Activity based on Machine Learning Models

Improving catalytic ability of protein biocatalysts leads to reduction in the production cost of biocatalytic manufacturing process, but the search space of possible proteins/mutants is too large to explore exhaustively through experiments. To some extent, highly soluble recombinant proteins tend to exhibit high activity. Here, we demonstrate that an optimization methodology based on machine learning prediction model can effectively predict which peptide tags can improve protein solubility quantitatively. Based on the protein sequence information, a support vector machine model we recently developed was used to evaluate protein solubility after randomly mutated tags were added to a target protein. The optimization algorithm guided the tags to evolve towards variants that can result in higher solubility. Moreover, the optimization results were validated successfully by adding the tags designed by our optimization algorithm to a model protein, expressing it in vivo and experimentally quantifying its solubility and activity. For example, solubility of a tyrosine ammonium lyase was more than doubled by adding two tags to its N- and C-terminus. Its protein activity was also increased nearly 3.5 fold by adding the tags. Additional experiments also supported that the designed tags were effective for improving activity of multiple proteins and are better than previously reported tags. The presented optimization methodology thus provides a valuable tool for understanding the correlation between amino acid sequence and protein solubility and for engineering protein biocatalysts.\n\nContactkang.zhou@nus.edu.sg, chewxia@nus.edu.sg

bioinformatics↗

bioRxiv2019

Circular RNA profiling in the oocyte and cumulus cells reveals that circARMC4 is essential for porcine oocyte maturation

Thousands of circular RNAs (circRNAs) have been recently discovered in cumulus cells and oocytes from several species. However, the expression and function of circRNA during porcine oocyte meiotic maturation have been never examined. Here, we separately identified 7,067 and 637 circRNAs in both the cumulus cell and the oocyte via deep sequencing and bioinformatic analysis. Further analysis revealed that a faction of circRNAs is differentially expressed (DE) in a developmental stage-specific manner. The host genes of DE circRNAs are markedly enriched to multiple signaling pathways associated with cumulus cell function and oocyte maturation. Additionally, most DE circRNAs harbor several miRNA targets, suggesting that these DE circRNAs potentially act as miRNA sponge. Importantly, we found that maternal circARMC4 knockdown by siRNA microinjection caused a severely impaired chromosome alignment, and significantly inhibited first polar body extrusion and early embryo development. Taken together, these results demonstrate for the first time that circRNAs are abundantly and dynamically expressed in a developmental stage-specific manner in cumulus cells and oocytes, and maternally expressed circARMC4 is essential for porcine oocyte meiotic maturation and early embryo development.

developmental biology↗