dissection-histological-processing-gene-expression-analysis-murine

Dissection, Histological Processing, and Gene Expression Analysis of Murine Supraclavicular Brown Adipose Tissue

Brown adipose tissue (BAT)-mediated thermogenesis plays an important role in the regulation of metabolism, and its morphology and function can be greatly ...

Baylor College of Medicine

In order to investigate if deletion patterns of the preS region can predict liver disease advancement, the preS region of the hepatitis B virus (HBV) genome in 45 chronic hepatitis B (CHB) and 94 HBV-related hepatocellular carcinoma (HCC) patients was sequenced by next-generation sequencing (NGS) and the percentages of nucleotide deletion in the preS region were analysed. Hierarchical clustering and heatmaps based on deletion percentages of preS revealed different deletion patterns between CHB and HCC patients. Intergenotype comparison also indicated divergence in preS deletions between HBV genotype B and C. No significant difference was found in preS deletion patterns between sera and matched adjacent non-tumour tissues. Based on hierarchical clustering, HCC patients were classed into two groups with different preS deletion patterns and different clinical features. Finally, the support vector machine (SVM) model was trained on preS nucleotide deletion percentages and used to predict HCC versus CHB patients. The prediction performance was assessed with fivefold cross-validation and independent cohort validation. The median area under the curve (AUC) was 0.729 after repeating SVM 500 times with fivefold cross-validations. After parameter optimization, the SVM model was used to predict an independent cohort with 51 CHB patients and 72 HCC patients and the AUC was 0.727. In conclusion, the use of the NGS method revealed a prominent divergence in preS deletion patterns between disease groups and virus genotypes, but not between different tissue types. Quantitative NGS data combined with a machine learning method could be a powerful approach for prediction of the status of different diseases.

Next-generation sequencing revealed divergence in deletions of the preS region in the HBV genome between different HBV-related liver diseases.

Glycogen, a randomly branched glucose polymer, provides energy storage in organisms. It forms small β particles which in animals bind to form composite α particles, which give better glucose release. Simulations imply β particle size is controlled only by activities and sizes of glycogen biosynthetic enzymes and sizes of polymer chains. Thus, storing more glucose requires forming more β particles, which are expected to sometimes form α particles. No α particles have been reported in bacteria, but the extraction techniques might have caused degradation. Using milder glycogen extraction techniques on Escherichia coli, transmission electron microscopy and size-exclusion chromatography showed α particles, consistent with this hypothesis for α-particle formation. Molecular density and size distributions show similarities with animal glycogen, despite very different metabolic processes. These general polymer constraints are such that any organism which needs to store and then release glucose will have similar α and β particle structures: a type of convergent evolution.

Molecular Structure of Glycogen in Escherichia coli.

Glycogen, a highly-branched glucose polymer, functions as a sugar reservoir in many organs and tissues. Liver glycogen comprises small β particles which can bind to form into large agglomerates (α particles) which readily degrade to β particles in diabetic livers. Muscle glycogen has only β particles, optimal for quick energy release. Healthy kidney contains negligible glycogen, but there is an abnormally high accumulation in diabetic kidneys. We here compare the molecular structure of glycogen in diabetic kidneys with that in liver and muscle, using a diabetic rat model. This involved exploring extraction techniques to minimize glycogen degradation. Using size exclusion chromatography and transmission electron microscopy, it was found that there were only β particles in diabetic kidneys. These are postulated to form during periods of abnormally high blood sugar, the driving force being the need to reduce blood sugar under such circumstances.

Some molecular structural features of glycogen in the kidneys of diabetic rats.

Diurnal alteration of glycogen molecular structure has been identified in healthy mice. Recently, both fragile (disintegration in dimethyl sulfoxide) and stable (not disintegrating in DMSO) glycogen particles were found in Escherichia coli. However, how glycogen structure changes dynamically in E. coli is not clear. The question examined here is whether fragile, stable glycogen α particles occur in bacteria, following a similar pattern as in mice. In this study, we examine the dynamic changes of glycogen molecular structure over 24-h in E. coli BL21(DE3), using transmission electron microscopy, size exclusion chromatography and fluorophore-assisted carbohydrate electrophoresis at representative time points. It was found that glycogen structure was mainly fragile at the synthesis stage and largely stable during the degradation stage. qRT-PCR results indicated that balance of anabolic and catabolic gene expression levels in glycogen metabolism could be a key factor affecting the fragility of glycogen α particles in bacteria.

The dynamic changes of glycogen molecular structure in Escherichia coli BL21(DE3).

Liver glycogen is a branched glucose polymer that functions as a blood-sugar buffer in animals. Previous studies have shown that glycogen's molecular structure affects its properties. This makes it important to develop a technique that extracts and purifies a representative sample of glycogen. Here we aim to optimize the sucrose density gradient centrifugation method for preserving glycogen's molecular structure by varying the density of the sucrose solution. The preservation of glycogen's structure involves: 1) minimizing molecular damage and 2) obtaining a structurally representative sample of glycogen. The addition of a 10-minute boiling step was also tested as a means for denaturing any glycogen degrading enzymes. Lower sucrose concentrations and the introduction of the boiling step were shown to be beneficial in obtaining a more structurally representative sample, with the preservation of smaller glycogen particles and decreased glycogen chain degradation.

Optimization of liver glycogen extraction when considering the fine molecular structure.

Glycogen is a complex branched glucose polymer found in many tissues and acts as a blood-glucose buffer. In the liver, smaller β glycogen particles can bind into larger composite α particles. In mouse models of diabetes, these liver glycogen particles are molecularly fragile, breaking up into smaller particles in the presence of solvents such as dimethyl sulfoxide (DMSO). If this occurs in vivo, such a rapid enzymatic degradation of these smaller particles into glucose could exacerbate the poor blood-glucose control that is characteristic of the disease. High-amylose resistant starch (RS) can escape digestion in the small intestine and ferment in the large intestine, which elicits positive effects on glycemic response and type 2 diabetes. Here we postulate that RS would help attenuate diabetes-related liver glycogen fragility. Normal maize starch and two types of high-amylose starch were fed to diabetic and non-diabetic mice. Molecular size distributions and chain-length distributions of liver glycogen from both groups were characterized to test glycogen fragility before and after DMSO treatment. Consistent with the hypothesis that high blood glucose is associated with glycogen fragility, a high-amylose RS diet prevented the fragility of liver-glycogen α particles. The diets had no significant effect on the glycogen chain-length distributions.

Article	Year
Next-generation sequencing revealed divergence in deletions of the preS region in the HBV genome between different HBV-related liver diseases. The Journal of general virology\| PubMed ID: 29022863	2017
Molecular Structure of Glycogen in Escherichia coli. Biomacromolecules\| PubMed ID: 31244022	2019
Some molecular structural features of glycogen in the kidneys of diabetic rats. Carbohydrate polymers\| PubMed ID: 31826402	2020
The dynamic changes of glycogen molecular structure in Escherichia coli BL21(DE3). Carbohydrate polymers\| PubMed ID: 33674016	2021
Optimization of liver glycogen extraction when considering the fine molecular structure. Carbohydrate polymers\| PubMed ID: 33766374	2021
The effect of high-amylose resistant starch on the glycogen structure of diabetic mice. International journal of biological macromolecules\| PubMed ID: 34968551	2021

ZiYi Wang

Biography

JoVE Journal Publications

Other Publications