Design of a Dual Agonist of Exendin-4 and FGF21 as a Potential Treatment for Type 2 Diabetes Mellitus and Obesity.

Background: Fibroblast growth factor 21 (FGF21) is a metabolic, endocrine hormone regulating insulin sensitivity, energy expenditure, and lipid metabolism. It has significant potential as a therapeutic drug for treating type 2 diabetes and obesity. However, the clinical efficacy of FGF21 analogs is limited due to their instability and short half-life. Glucagon-like peptide 1 (GLP-1) receptor agonists have been recognized as effective medications for type 2 diabetes mellitus and obesity over the past two decades. Methods: This study designed a new long-acting dual-agonist, exendin-4/FGF21, utilizing albumin-binding-designed ankyrin repeat proteins (DARPins) as carriers. The purified fusion proteins were subcutaneously injected into mice for pharmacokinetic and biological activity studies. Results: Ex-DARP-FGF21 had a high binding affinity for human serum albumin (HSA) in vitro and a prolonged half-life of 27.6 hours in vivo. Bioactivity results reveal that Ex-DARP-FGF21 significantly reduced blood glucose levels in healthy mice. Moreover, compared to Ex-DARP alone, the Ex-DARP-FGF21 dual agonist displayed enhanced blood glucose lowering bioactivity and superior body weight management in the diet-induced obesity (DIO) mouse model. Conclusions: These results indicate that the long-acting dual agonist of exendin-4 and FGF21 holds considerable potential as a treatment for type 2 diabetes mellitus (T2DM) and obesity in the future.

Improving pharmacological activities of thrombopoietin mimetic peptide by genetic fusion to albumin-binding domain.

OBJECTIVE: Thrombopoietin mimetic peptide (TMP), an analog of natural thrombopoietin, can be used to treat primary immune thrombocytopenia. However, the short half-life of TMP limits its application in clinics. The present study aimed to improve the stability and biological activity of TMP in vivo via genetic fusion to the albumin-binding protein domain (ABD). RESULTS: TMP dimer was genetically fused to the N-terminal or C-terminal of ABD, denoted as TMP-TMP-ABD and ABD-TMP-TMP. A Trx-tag was used to improve the fusion proteins' expression levels effectively. ABD-fusion TMP proteins were produced in Escherichia coli and purified by Ni2+-NTA and SP ion exchange column. Albumin binding studies in vitro showed that the fusion proteins could effectively bind to serum albumin to extend their half-lives. The fusion proteins effectively induced platelet proliferation in healthy mice, and the platelet count was increased by more than 2.3-fold compared with the control group. The increased platelet count induced by the fusion proteins lasted 12 days compared with the control group. The increasing trend was maintained for 6 days before a decline occurred after the last injection in the fusion-protein-treated mice group. CONCLUSIONS: ABD can effectively improve the stability and pharmacological activity of TMP by binding to serum albumin, and the ABD-fusion TMP protein can promote platelet formation in vivo.

Albumin-binding DARPins as scaffold improve the hypoglycemic and anti-obesity effects of exendin-4 in vivo.

Type 2 diabetes mellitus (T2DM) and obesity have been considered epidemics and threats to public health worldwide. Exendin-4 (Ex), a GLP-1R agonist, has potential for treating T2DM and obesity. However, Ex has a half-life of only 2.4 h in humans and needs to be administered twice daily, which hampers its clinical application. In this study, we synthesized four new GLP-1R agonists by genetically fusing Ex to the N-terminus of HSA-binding ankyrin repeat proteins (DARPins) via linkers of different lengths, denoted as Ex-DARPin-GSx fusion proteins (x = 0, 1, 2, and 3). The Ex-DARPin fusion proteins were substantially stable, resulting in incomplete denaturation even at 80 °C. The in vitro bioactivity results demonstrated that Ex-DARPin fusion proteins could bind to HSA and activate GLP-1R. The Ex-DARPin fusion proteins had a comparable half-life (29-32 h), which is much longer than that of native Ex (0.5 h in rats). Subcutaneous injection of 25 nmol/kg Ex-DARPin fusion protein normalized blood glucose (BG) levels for at least 72 h in mice. The Ex-DARPin fusion proteins, injected at 25 nmol/kg every three days, significantly lowered BG, inhibited food consumption, and reduced body weight (BW) for 30 days in STZ-induced diabetic mice. Histological analysis of pancreatic tissues using H&E staining revealed that Ex-DARPin fusion proteins significantly improved the survival of pancreatic islets in diabetic mice. The differences in in vivo bioactivity of fusion proteins with different linker lengths were not significant. According to the findings in this study, long-acting Ex-DARPin fusion proteins designed by us hold promise for further development as antidiabetic and antiobesity therapeutic agents. Our findings also indicate that DARPins are a universal platform for generating long-acting therapeutic proteins via genetic fusion, thus broadening the application scope of DARPins.

The Effects of Helicobacter pylori Infection on Gastric Microbiota in Children With Duodenal Ulcer.

Background: Helicobacter pylori (H. pylori) infection is the main cause of chronic gastritis and duodenal ulcer in children. Little is known about the effect of H. pylori on gastric microbiota in children with duodenal ulcer. This study is aimed at the characteristics of gastric microbiota in children with duodenal ulcer on H. pylori infection. Methods: We studied 23 children diagnosed with duodenal ulcer by gastric endoscopy because of the gastrointestinal symptoms, 15 children were diagnosed with H. pylori infection, while 8 children were without H. pylori infection. Endoscopic mucosal biopsy samples were obtained for DNA extraction. Microbiomes were analyzed by 16S rRNA profiling and microbial functions were predicted using the software Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Results: Bacterial richness and diversity of gastric microbiota in duodenal ulcer with H. pylori-positive were lower than those negative. The gastric microbiota in H. pylori-positive group significantly reduced proportions of six phyla and fifteen genera; only Helicobacter taxa were more abundant in H. pylori-positive group. Co-expression network analysis showed a more complex network of interactions in the H. pylori-positive group than that in the H. pylori-negative group. For the predicted functions, lower abundance in the pathways of carbohydrate metabolism, signal transduction, amino acid metabolism, and lipid metabolism were found in H. pylori-positive group than the H. pylori-negative group. H. pylori colonization reduces a microbial community with genotoxic potential in the gastric mucosa of children with duodenal ulcer. Conclusions: The presence of H. pylori significantly influences gastric microbiota and results in a lower abundance of multiple taxonomic levels in children with duodenal ulcer. Children with duodenal ulcer exhibit a dysbiotic microbial community with genotoxic potential, which is distinct from that of children with H. pylori infection. Clinical Trial Registration: [http://www.chictr.org.cn], identifier [ChiCTR1800015190].

Effect of ursodeoxycholic acid on gallstone formation after bariatric surgery: An updated meta-analysis.

OBJECTIVE: Bariatric surgery increases the risk of postoperative gallstone formation. Many studies have proposed ursodeoxycholic acid (UDCA) as a preventive agent for postoperative gallstone formation. This study aimed to investigate the effect of UDCA on gallstone formation after bariatric surgery in patients without preoperative gallstones. METHODS: PubMed, the Web of Science, the Cochrane Library, and EBSCO were searched for articles assessing the effect of UDCA on gallstone formation after bariatric surgery. The outcome was the incidence of postoperative gallstones. Odds ratios were used to assess dichotomous variables, and random-effects models were used for statistical analyses. RESULTS: A total of 18 studies including 4,827 participants met the inclusion criteria. The statistical results showed that the incidence of gallstones in the UDCA group was significantly lower than in the control group. Furthermore, the occurrence of symptomatic gallstones and cholecystectomy was significantly reduced. CONCLUSIONS: In patients without preoperative gallstones, UDCA can effectively prevent the formation of gallstones after bariatric surgery. In addition, UDCA can significantly reduce the occurrence of symptomatic gallstones and the risk of postoperative cholecystectomy. Doses of 500 to 600 mg/d can be used as a measure to prevent postoperative gallstone formation.

Knockdown of lncRNA ZFAS1-suppressed non-small cell lung cancer progression via targeting the miR-150-5p/HMGA2 signaling.

Non-small cell lung cancer (NSCLC) is the main type of lung malignancy. Early diagnosis and treatments for NSCLC are far from satisfactory due to the limited knowledge of the molecular mechanisms regarding NSCLC progression. Long noncoding RNA (lncRNA) ZNFX1 antisense RNA1 (ZFAS1) has been implicated for its functional role in the progression of malignant tumors. This study aimed to determine the ZFAS1 expression from lung cancer clinical samples and to explore the molecular mechanisms underlying ZFAS1-modulated NSCLC progression. Experimental assays revealed that clinical samples and cell lines of lung malignant tumors showed an upregulation of ZFSA1. ZFAS1 expression was markedly upregulated in the lung tissues from patients with advanced stage of this malignancy. The loss-of-function assays showed that knockdown of ZFAS1-suppressed NSCLC cell proliferative, as well as invasive potentials, increased NSCLC cell apoptotic rates in vitro and also attenuated tumor growth of NSCLC cells in the nude mice. Further experimental evidence showed that ZFAS1 inversely affected miR-150-5p expression and positively affected high-mobility group AT-hook 2 (HMGA2) expression in NSCLC cell lines. MiR-150-5p inhibition or HMGA2 overexpression counteracted the effects of ZFAS1 knockdown on NSCLC cell proliferative, invasive potentials and apoptotic rates. In light of examining the clinical lung cancer samples, miR-150-5p expression was downregulated and the HMGA2 expression was highly expressed in the lung cancer tissues compared with normal ones; the ZFAS1 expression showed a negative correlation with miR-150-5p expression but a positive correlation with HMGA2 expression in lung cancer tissues. To summarize, we, for the first time, demonstrated the inhibitory effects of ZFAS1 knockdown on NSCLC cell progression, and the results from mechanistic studies indicated that ZFAS1-mediated NSCLC progression cells via targeting miR-150-5p/HMGA2 signaling.

Coexistence of NDM-1-producing Escherichia coli and Citrobacter freundii in the same patient.

OBJECTIVES: Although blaNDM-1 has been widely detected in various Enterobacteriaceae clinical isolates, multiple blaNDM-1 colonisations within the same patient remain rare. The aim of the study was to describe a patient with respiratory tract colonisation with NDM-1-producing Escherichia coli and Citrobacter freundii during hospitalisation in China. METHODS: Two carbapenem-resistant isolates were analysed. Antimicrobial susceptibility testing, metallo-ß-lactamase production, conjugation assay, plasmid analysis and molecular typing were performed. RESULTS: The two clinical isolates carried the blaNDM-1 gene and showed resistance to carbapenems. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) revealed that E. coli NB753 harboured a plasmid of ca. 80kb, and C. freundii NB865 harboured three plasmids (ca. 70, 80 and 135kb). Southern blot and Inc replicon typing further demonstrated that both isolates carried the blaNDM-1 gene on a self-transferrable IncX3 plasmid. Moreover, the two NDM-producing plasmids were conjugative and the transconjugants showed increased resistance to carbapenems. CONCLUSION: An NDM-1-encoding plasmid harboured by various clinical isolates in a single patient is worrying considering that this plasmid may be widespread in our hospital. Furthermore, the threat of carbapenemase-producing bacterial epidemics should be closely monitored. However, a limitation of this study was the extremely small sample size.

Peak expiratory flow, breath rate and blood pressure in adults with changes in particulate matter air pollution during the Beijing Olympics: a panel study.

OBJECTIVES: This study aims to examine whether changes in short-term exposures to particulate matter are associated with changes in lung function, breath rate, and blood pressure among healthy adults and whether smoking status modifies the association. METHODS: We took advantage of the artificially controlled changes in air pollution levels that occurred during the 2008 Olympic Games in Beijing, China and conducted a panel study of 201 Beijing residents. Data were collected before, during, and after the Olympics, respectively. Linear mixed-effect models and generalized estimating equation models were used to compare measurements of peak expiratory flow, breath rate and blood pressure across three time points. RESULTS: The mean values of peak expiratory flow were 346.0 L/min, 399.3 L/min, and 364.1L/min over the three study periods. Peak expiratory flow levels increased in 78% of the participants when comparing the during- with pre- Olympics time points, while peak expiratory flow levels decreased in 80% of participants for the post- and during-Olympic periods comparison. In subgroup analyses comparing the during-Olympic to pre-Olympic time points, we found a larger percentage change in peak expiratory flow (+17%) among female, younger and non-smoking participants than among male, elderly and smoking participants (+12%). The percentage of participants with a fast breath rate (>20/min) changed from 9.7% to 4.9% to 30.1% among females, and from 7.9% to 2.6% to 27.3% among males over the three time points. The changes in blood pressure over the three study periods were not very clear, although there is an increase in diastolic pressure and a decrease in pulse pressure among males during the games. CONCLUSIONS: The results suggest that exposure to different air pollution levels has significant effects on respiratory function. Smoking, age and gender appear to modify participants' biological response to changes in air quality.