Pdxl and its role in activating Ngn3 and Pax6 to induce differentiation of iPSCs into islet ß cells.

The pancreatic and duodenal homeobox factor 1 (Pdx1) protein is the most pivotal transcription factor in the development of islet ß cells. This study investigated the role of Pdx1 and its mechanism in differentiating induced pluripotent stem cells (iPSCs) into islet ß cells. iPSCs derived from human skin fibroblasts were cultured in vitro and directionally induced to differentiate for 20 days. The expression of insulin-related genes was then detected by RT-PCR, and the expression of several differentiation-related transcription factors was assessed both before and after the differentiation process. Lastly, the specific promoter regions where Pdx1 binds were detected by ChIP. The insulin-related genes, MafA, insulin, Glut2, Nkx6.1, GCK, and Tcf1, showed increased expression during differentiation, and nearly peaked on the 20th day. Similarly, the expression of transcription factors, Pdx1, Ngn3, and Pax6 showed enhanced expression during differentiation as compared with that of the control group. ChIP experiments confirmed that Pdx1 activates the expression of the downstream transcription factors, Ngn3 and Pax6, by combined with the promoter regions of insulin (Insulin-P), Ngn3 (Ngn3-P), and Pax6 (Pax6-P). In conclusion, Pdx1 activates downstream transcription factors Ngn3 and Pax6, and may be one of the mechanisms that promote differentiation of iPSCs into islet ß cells.

Role of adiponectin in adipose tissue wound healing.

The purpose of this study was to investigate the mechanism behind adipose tissue wound healing (ATWH). The preadipocyte cell line 3T3-L1 was cultured and expression of adiponectin receptors (AdipoR1/2) was detected by immunohistochemistry and reverse transcription polymerase chain reaction. The concentration of adiponectin secreted at different cell densities was measured by enzyme-linked immunosorbent assay, while preadipocyte proliferation and migration were determined in vitro by MTT and wound closure assays. AdipoR1/2 were found to be expressed in 3T3-L1 preadipocytes. There were no statistically significant differences in the concentrations of adiponectin secreted by cell solutions of different densities (P > 0.05). In addition, adiponectin was seen to promote the growth and migration of preadipocytes. In conclusion, adiponectin may regulate ATWH by promoting preadipocyte proliferation and migration, and its systemic and/or local application is proposed as a promising therapeutic approach for the treatment of wounds incurred as a result of surgery.

SYNTHESIS AND CHARACTERIZATION OF POLYSIALIC ACID/CARBOXYMETHYL CHITOSAN HYDROGEL WITH POTENTIAL FOR DRUG DELIVERY.

A novel hydrogel was prepared from polysialic acid (PSA) and carboxymethyl chitosan (CMCS) using glutaraldehyde as the cross-linking agent. The resulting PSA-CMCS hydrogel exhibited pH sensitivity, in which the swelling ratio under acidic conditions was higher than those under neutral or alkaline conditions. The swelling ratio of PSA-CMCS hydrogel at equilibrium depended on the medium pH, the cross-linking agent concentration, and the ratio of PSA to CMCS (w/w). Bovine serum albumin (BSA) and 5-fluorouracil (5-FU) were used as model drugs to prepare hydrogel delivery systems. The loading efficiencies of the hydrogel for BSA and 5-FU were 26.25 and 36.74%, respectively. Release behaviors of BSA and 5-FU were influenced by the pH. MTT assays confirmed that PSA-CMCS hydrogel has no cytotoxicity toward the NIH-3T3 cell line; in fact, the 100% aqueous extract of the PSA-CMCS hydrogel enhanced cell growth. These results suggest that PSA-CMCS hydrogel may be a promising pH-sensitive delivery system, especially for hydrophobic chemicals.

An increase of curdlan productivity by integration of carbon/nitrogen sources control and sequencing dual fed-batch fermentors operation.

Curdlan is produced by Agrobacterium sp. ATCC 31749 under nitrogen-limited conditions not associated with cell growth. A novel curdlan production process was developed based on the different nutrient requirements for microbial cell growth and its efficiency was increased by integrating carbon/nitrogen sources control and sequencing dual fed-batch fermentors operation. By feeding ammonium solution to supply abundant nitrogen source and controlling pH in Fermentor I, cell growth was accelerated. High cell density of 29 g/L was attained. The culture broth in Fermentor I was then inoculated into sequencing Fermentor II which alleviated the high requirement for dissolved oxygen and accumulation of inhibitory metabolic by-products during curdlan production. Fermentor I promoted cell growth. Curdlan production started instantaneously in Fermentor II. By feeding nutrient solution with high carbon/nitrogen ratio and NaOH solution for pH adjustment, a feasible and optimal curdlan production process was formulated. The productivity, conversion efficiency and curdlan yield were achieved of 0.98 g/(L h), 57% (w) and 67 g/L, respectively. Such novel process can be scaled up for significant cost reduction at the industrial level.

Changes in gene transcription and protein expression involved in the response of Agrobacterium sp. ATCC 31749 to nitrogen availability during curdlan production.

The changes in transcription of genes involved in nitrogen metabolism and curdlan biosynthesis, and total protein expression were firstly analyzed to define the responses of Agrobacterium sp. ATCC 31749 to nitrogen source availability during curdlan fermentation. The transcription of all nitrogen metabolism and regulation genes increased significantly under nitrogen limitation. The genes of carbon (exoC) and nitrogen (ntrB, ntrC, and nifR) metabolism showed distinctive transcriptional responses to nitrogen limitation. Their relative expression level was increased by 14, 9, 7 and 7-fold, respectively. Two-dimentional electrophoresis (2-DE) revealed that the expression of 14 proteins were elevated and 6 proteins were down-regulated significantly under nitrogen starvation. Furthermore, 4 proteins (GroEL, ABC transporter, Atu1730 and enoyl-acyl carrier protein reductase) in which the expression level changed significantly were identified. The results showed that Agrobacterium sp. regulates its carbon flux and nitrogen assimilation effectively for better survival.