Biology subjects

Yong, J.

Publications and source records attributed to Yong, J..

23 records · Page 2Linked to original sources

bioRxiv2020

AS-Quant: Detection and Visualization of Alternative Splicing Events with RNA-seq Data

A simplistic understanding of the central dogma falls short in correlating the number of genes in the genome to the number of proteins in the proteome. Post-transcriptional alternative splicing contributes to the complexity of proteome and are critical in understanding gene expression. mRNA-sequencing (RNA-seq) has been widely used to study the transcriptome and provides opportunity to detect alternative splicing events among different biological conditions. Despite the popularity of studying transcriptome variants with RNA-seq, few efficient and user-friendly bioinformatics tools have been developed for the genome-wide detection and visualization of alternative splicing events. We have developed AS-Quant (Alternative Splicing Quantitation), a robust program to identify alternative splicing events and visualize the short-read coverage with gene annotations. AS-Quant works in three steps: (i) calculate the read coverage of the potential splicing exons and the corresponding gene; (ii) categorize the splicing events into five different types based on annotation, and assess the significance of the events between two biological conditions; (iii) generate the short reads coverage plot with a complete gene annotation for user specified splicing events. To evaluate the performance, two significant alternative splicing events identified by AS-Quant between two biological contexts were validated by RT-PCR. ImplementationAS-Quant is implemented in Python. Source code and a comprehensive users manual are freely available at https://github.com/CompbioLabUCF/AS-Quant

bioinformatics↗

bioRxiv2020

Factor VIII exhibits chaperone-dependent and glucose-regulated reversible amyloid formation in the endoplasmic reticulum

Factor VIII (FVIII) is the coagulation factor deficient in hemophilia A, which is treated by protein replacement. Unfortunately, this regimen is costly due to the expense of producing recombinant FVIII as a consequence of its low level secretion. FVIII expression activates the endoplasmic reticulum (ER) stress response, causes oxidative stress and induces apoptosis. Importantly, little is known about the factors that cause protein misfolding and aggregation in metazoans. Here we identified intrinsic and extrinsic factors that cause FVIII to form aggregates in the ER. We show that FVIII forms amyloid-like fibrils within the ER upon increased FVIII synthesis or inhibition of glucose metabolism. Significantly, FVIII amyloids can be dissolved upon restoration of glucose metabolism to produce functional secreted FVIII. Two ER chaperones and their co-chaperones, BiP and CANX/CRT, promote FVIII solubility in the ER, where the former is also required for disaggregation. A short aggregation motif in the FVIII A1 domain (termed Aggron) is necessary and sufficient to seed {beta}-sheet polymerization and BiP binding to this Aggron prevents amyloidogenesis. Our findings provide novel insight into mechanisms that limit FVIII secretion and ER protein folding in general and have implication for ongoing hemophilia A gene therapy clinical trials. Key Points- FVIII forms amyloid aggregates in the ER that are dissolved in a chaperone- and glucose-dependent manner to produce secreted active FVIII. - A short amino acid sequence in the A1 domain causes {beta} sheet polymerization and ER chaperone BiP binding to this site prevents aggregation.

molecular biology↗

bioRxiv2020

Chop/Ddit3 depletion in β-cells alleviates ER stress and corrects hepatic steatosis

Type 2 diabetes (T2D) is a metabolic disorder characterized by hyperglycemia, hyperinsulinemia and insulin resistance (IR). During the early phase of T2D, insulin synthesis and secretion by pancreatic {beta} cells is enhanced, which can lead to proinsulin (ProIns) misfolding that aggravates endoplasmic reticulum (ER) homeostasis in {beta} cells. Moreover, increased insulin in the circulation may contribute to fatty liver disease. Medical interventions aimed at alleviating ER stress in {beta} cells while maintaining optimal insulin secretion are therefore an attractive therapeutic strategy for T2D. Previously, we demonstrated that germline Chop gene deletion preserved {beta} cells in high fat diet (HFD) fed mice and in leptin receptor-deficient db/db mice. In the current study, we further investigated whether targeting Chop/Ddit3 specifically in murine {beta} cells confers therapeutic benefits. First, we show that Chop deletion in {beta} cells alleviates {beta} cell ER stress and delays glucose-stimulated insulin secretion (GSIS) in HFD fed mice. Second, importantly, {beta} cell-specific Chop deletion prevented liver steatosis and hepatomegaly in aged HFD fed mice without affecting basal glucose homeostasis. Third, we provide the first mechanistic evidence that ER remodeling secondary to Chop deletion modulates glucose-induced islet Ca2+ oscillations. Finally, using state-of-the-art GLP1-conjugated Chop AntiSense Oligonucleotides (GLP1-Chop ASO), we demonstrated that the Chop deletion induced GSIS change is a long term complex event in {beta} cells. In summary, our results demonstrate that Chop depletion in {beta} cells is a new therapeutic strategy to alleviate dysregulated insulin secretion and the consequently fatty liver disease in T2D.

physiology↗

bioRxiv2019

Quantitative analysis and medium components optimizing for culturing a fastidious bacterium Christensenella minuta

Christensenella minuta is a heritable bacterium with controversial physiologies associating with both obesity and potential pathogenicity. Since this bacterium is fastidious to culture, it is hardly to well understand its biological feature. We develop a strategy for statistical analysis of this low abundant strain and optimize culture condition to make a significant improvement on its biomass and facilitate the researches about the metabolism and function of this bacterium. Basing on the fluorogenic quantitative technology, a quantitative approach was successfully constructed for Christensenella minuta by plotting Ct value from fluorescence quantitative PCR against the logarithm of concentration gradient of plasmids containing 16S rDNA of the strain. This method exhibited to have specificity on analyzing the strain biomass statistically. For improving the strain biomass, \"komodo\" predicted to optimize medium components and metabonomics analysis explored the catabolites addition effects on culture improvement. With the aid of Plackett-Burman and Box-Behnken in Design-Expert 8.0.6, the PB and response surface experiment were designed and analyzed from the single factor results. On the modified GAM medium, the strain concentration was found increasing markedly by 10 times. The addition of some amino acids, vitamins and inorganic salts has contributions for the strain multiplication, especially L-cysteine, VB6 and NaCl. The addition of 55 mg/L of L-cysteine, 20.5 mg/L VB6 and 55 g/L NaCl into the modified GAM medium increased the biomass by 3.59 times compared to the biomass on only modified GAM medium according to the response surface experiment. Through the newly constructed method, we successfully analyzed the amount of Christensenella minuta and obtained a novel medium to increase biomass significantly.\n\nImportanceChristensenella minuta is a heritable bacterium with controversial physiologies associating with both obesity and potential pathogenicity. Since this bacterium is fastidious to culture, it is hard to well understand its biological feature. We develop a strategy for statistical analysis of this low abundant strain and optimize culture condition to make a significant improvement on its biomass and facilitate the researches about the metabolism and function of this bacterium. This work combined the prediction tools and experiments to improve the medium components of C. munita and successfully enhance the culturing and increase biomass by more than 10-fold. From this perspective, the project throws some new ideas and also enables access to new knowledge and information in uncultured microbial resources.

microbiology↗

bioRxiv2019

Mitochondria supply ATP to the ER through a mechanism antagonized by cytosolic Ca2+

The endoplasmic reticulum (ER) imports ATP and uses energy from ATP hydrolysis for protein folding and trafficking. However, little is known about this vital ATP transport process across the ER membrane. Here, using three commonly used cell lines (CHO, INS1 and HeLa), we report that ATP enters the ER lumen through a cytosolic Ca2+-antagonized mechanism, or CaATiER (Ca2+-Antagonized Transport into ER) mechanism for brevity. Significantly, we observed that a Ca2+ gradient across the ER membrane is necessary for ATP transport into the ER. Therefore Ca2+ signaling in the cytosol is inevitably coupled with ATP supply to the ER. We propose that under physiological conditions, cytosolic Ca2+ inhibits ATP import into the ER lumen to limit ER ATP consumption. Furthermore, the ATP level in the ER is readily depleted by oxidative phosphorylation (OxPhos) inhibitors, and that ER protein misfolding increases ATP trafficking from mitochondria into the ER. These findings suggest that ATP usage in the ER may increase mitochondrial OxPhos while decreasing glycolysis, i.e., an \"anti-Warburg\" effect.\n\nSignificance StatementWe report that ATP enters the ER lumen through an AXER-dependent, cytosolic Ca2+-antagonized mechanism, or CaATiER (Ca2+-Antagonized Transport into ER) mechanism. In addition, our findings suggest that ATP usage in the ER may render an \"anti-Warburg\" effect by increasing ATP regeneration from mitochondrial OxPhos while decreasing the portion of ATP regeneration from glycolysis.

cell biology↗