Adipose triacylglycerol lipase (ATGL) is a dynamic lipid droplet-associated protein involved in cellular lipolysis, which is conserved from bacteria to humans. Recent methods that measure the enzymatic activity of ATGL in vitro are established using lipid emulsions. However, the lipid emulsion platforms contain various membranous structures which reduce the accuracy of enzymatic activity determination. Therefore, a new platform and corresponding method are required for accurate measurement of ATGL enzymatic activity that represents cellular lipid and energy homeostasis. Adiposomes are artificial lipid nanostructures mimicking lipid droplets. Employing adiposome as a platform, we have developed an assay to measure the enzymatic activity of ATGLin vitro. Here, a detailed protocol is described to explain how to measure the activity of ATGL using adiposomes. This method successfully proves the concept of lipid droplet-mimetic lipase activity determining platform and provides a tool to identify the active sites of lipases.
3D genomics mainly focuses on the 3D position of single genes at the cell level, while spatial genomics focuses more on the tissue level. In this exciting new era of 3D/spatial genomics, half-century old FISH and its derivative methods, including Tn5-FISH, play important roles. In this review, we introduce the Tn5-FISH we developed recently, and present six different applications published by our collaborators and us, based on (Tn5-)FISH, which can be either general BAC clone-based FISH or Tn5-FISH. In these interesting cases, (Tn5-)FISH demonstrated its vigorous ability of targeting sub-chromosomal structures across different diseases and cell lines (leukemia, mESCs (mouse embryonic stem cells), and differentiation cell lines). Serving as an effective tool to image genomic structures at the kilobase level, Tn5-FISH holds great potential to detect chromosomal structures in a high-throughput manner, thus bringing the dawn for new discoveries in the great era of 3D/spatial genomics.
The thioredoxin system is composed of thioredoxin (Trx), thioredoxin reductase (TR) and reduced nicotinamide adenine dinucleotide phosphate. Trx is an important antioxidant molecule that can resist cell death caused by various stresses and plays a prominent role in redox reactions. TR is a protein that contains selenium (selenocysteine), in three main forms, namely, TR1, TR2 and TR3. TR1, TR2 and TR3 are mainly distributed in the cytoplasm, mitochondria, and testes, respectively. TR can regulate cell growth and apoptosis. After a cell becomes cancerous, the expression of TR is increased to promote cell growth and metastasis. The Trx system is closely related to neurodegenerative diseases, parasitic infections, acquired immunodeficiency syndrome, rheumatoid arthritis, hypertension, myocarditis, and so on. In addition, the Trx system can remove the reactive oxygen species in the body and keep the inside and outside of the cell in a balanced state. In summary, the Trx system is an important target for the drug treatment of many diseases.
Gna12 has been identified as one of the reported inflammatory bowel disease (IBD) susceptibility genes in genome-wide association studies (GWAS). However, the function of GNA12 in intestinal homeostasis remains unknown. Here we report that GNA12, a G-protein α subunit, regulates C5a-induced migration in macrophages. Deficiency of GNA12 results in enhanced migration induced by C5a in macrophages. Mechanistically, GNA12 suppresses C5a-induced migration by downregulating the C5aR1-PLCβ2-PI3K-AKT-ERK1/2 signaling. Therefore, our study reveals that GNA12 is an anti-inflammatory factor, which might alleviate the development of inflammation by inhibiting the excessive chemotactic migration of macrophages.
Abnormal histone modifications (HMs) can promote the occurrence of breast cancer. To elucidate the relationship between HMs and gene expression, we analyzed HM binding patterns and calculated their signal changes between breast tumor cells and normal cells. On this basis, the influences of HM signal changes on the expression changes of breast cancer-related genes were estimated by three different methods. The results showed that H3K79me2 and H3K36me3 may contribute more to gene expression changes. Subsequently, 2109 genes with differential H3K79me2 or H3K36me3 levels during cancerogenesis were identified by the Shannon entropy and submitted to perform functional enrichment analyses. Enrichment analyses displayed that these genes were involved in pathways in cancer, human papillomavirus infection, and viral carcinogenesis. Univariate Cox, LASSO, and multivariate Cox regression analyses were then adopted, and nine potential breast cancer-related driver genes were extracted from the genes with differential H3K79me2/H3K36me3 levels in the TCGA cohort. To facilitate the application, the expression levels of nine driver genes were transformed into a risk score model, and its robustness was tested via time-dependent receiver operating characteristic curves in the TCGA dataset and an independent GEO dataset. At last, the distribution levels of H3K79me2 and H3K36me3 in the nine driver genes were reanalyzed in the two cell lines and the regions with significant signal changes were located.
