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关键词： Gastric glands   Glycoconjugates   Lectin histochemistry   Mucins   Zymogens   O-LINKED OLIGOSACCHARIDES   HISTOCHEMICAL-DEMONSTRATION   GLYCOPROTEIN   PEPSINOGENS   STOMACH   LIPASE   MUCUS   SITE  

摘要： Many studies have been conducted to determine the composition of the glycoconjugates of the mucus-secreting cells of the fundic glands of the stomach. However, the chief cells of these glands have been largely ignored because they secrete mainly zymogens with a lower glycosylation. The aim of this work was to analyze the glycoconjugates of the gastric chief cells by a battery of 17 different lectins, recognizing Fucose,N-acetylgalactosamine, Galactose,N-acetylneuraminic acid,N-acetylglucosamine and Mannose containing oligosaccharides. Histochemical techniques were performed with several lectins and also combined with two pre-treatments; beta-elimination, which removes O-linked oligosaccharides, and incubation with Peptide-N-Gycosidase F, which removes N-linked oligosaccharides. In addition, acid hydrolysis was performed before WGA histochemistry, and incubation with glucose oxidase before Con A labeling. Many lectins did not stain the chief cells. In addition, the presence ofO-glycans in the apical cell membrane was demonstrated with the lectins AAL, HPA, MPA/MPL, PNA, RCA-I, and WGA. Some of theseO-glycans were resistant to short-term beta-elimination pre-treatments. Mannose-binding lectins stained the basal cytoplasm of the chief cells. The level of glycosylation of the chief cells was lower than that of the mucous cells. The presence ofO-glycans in the apical cell membrane is consistent with the presence of mucins such as MUC1 in the apical membrane of chief cells. Moreover, Mannose-binding lectins revealedN-glycosylation in the basal cytoplasm. The knowledge of gastric chief cell glycoconjugates is relevant because of their potential involvement not only in in physiological but also in pathological processes, such as cancer.

关键词： Basalinsulin + GLP-1RA combination therapies   Basal oral therapy   Delphi method   Expert consensus   Insulin intensification   Type 2 diabetes   FIXED-RATIO COMBINATION   GLYCEMIC CONTROL   HEART-FAILURE   BOLUS INSULIN   DUAL VII   GLARGINE   DEGLUDEC   LIXISENATIDE   LIRAGLUTIDE   IDEGLIRA  

摘要： Introduction The aim of this study was to elaborate a consensus on treatment intensification strategies in patients with type 2 diabetes failing basal insulin supported oral therapy (BOT). The panel focused on glucagon-like peptide-1 receptor agonists (GLP-1RA) and basal insulin (BI) combinations. Methods The authors developed a Delphi questionnaire organized into ten statements and 77 items that focused on: the definition of BOT and BOT failure, intensification strategies, fixed-dose combinations in general and the BI/GLP-1RA fixed combination. The survey was administered in two rounds to a panel of 80 Italian diabetes specialists, who rated their level of agreement with each item on a 5-point Likert scale. Consensus was predefined as > 66% of the panel agreeing/disagreeing on any given item. Results Consensus was achieved for 71 of the 77 items. The panel agreed that the use of sulfonylureas in the BOT regimen is inappropriate. BOT failure was defined as individualized targets not being met for glycated hemoglobin, fasting plasma glucose and/or postprandial plasma glucose. There was agreement that postprandial hyperglycaemia and/or presence of nocturnal hypoglycaemia or weight gain define BOT failure. Addition of a GLP-1RA to BI therapy was considered to be the best option for BOT intensification. There was consensus for the use of BI/GLP-1RA fixed combinations as valuable options to increase compliance and safely improve glycaemic control. The panel agreed in considering the fixed-ratio combination insulin degludec/liraglutide (IDegLira) to be preferable to the fixed-ratio combination insulin glargine/lixisenatide (iGlarLixi) in the control of glycaemia, body weight and cardiovascular risk. Conclusion According to this Delphi consensus, the addition of a GLP-1RA may be the best option to intensify BOT. The BI/GLP-1RA fixed combinations may increase compliance and optimize the advantages of each of these molecules.

关键词： Soluble ESAT6-CFP10   Fusion protein   Synthetic gene   Codon optimization   Antigenicity  

摘要： Translation engineering and bioinformatics have accelerated the rate at which gene sequences can be improved to generate multi-epitope proteins. Strong antigenic proteins for tuberculosis diagnosis include individual ESAT6 and CFP10 proteins or derived peptides. Obtention of heterologous multi-component antigens in E. coli without forming inclusion bodies remain a biotechnological challenge. The gene sequence for ESAT6-CFP10 fusion antigen was optimized by codon bias adjust for high-level expression as a soluble protein. The obtained fusion protein of 23.7 kDa was observed by SDS-PAGE and Western blot analysis after Ni-affinity chromatography and the yield of expressed soluble protein reached a concentration of approximately 67 mg/L in shake flask culture after IPTG induction. Antigenicity was evaluated at 4 mu g/mL in whole blood cultures from bovines, and protein stimuli were assessed using a specific in vitro IFN-gamma release assay. The hybrid protein was able to stimulate T-cell specific responses of bovine TB suspects. The results indicate that improved E. coli codon usage is a good and cost-effective strategy to potentialize large scale production of multi-epitope proteins with sustained antigenic properties for diagnostic purposes. (C) 2020 The Author(s). Published by Elsevier B.V.

关键词： HNSCC   Second primary tumor   Lung   Diagnostic biomarker   Methylation   CpG  

摘要： Background Patients with head and neck squamous cell carcinoma (HNSCC) can develop lung squamous cell carcinoma (LuSCC), which could be the second primary tumor or HNSCC metastasis. Morphologically it is difficult to distinguish metastatic HNSCC from a second primary tumor which presents a significant diagnostic challenge. Differentiation of those two malignancies is important because the recommended treatments for metastatic HNSCC and primary LuSCC differ significantly. We investigated if the quantification of the promotor methylation status in HNSCC and LuSCC differs. Methods Primary HNSCC (N = 36) and LuSCC (N = 17) were included in this study. Methylation status in the ASC/TMS1/PYCARD (apoptosis-associated speck-like protein containing a caspase recruitment domain; 8 CpG sites) and MyD88 (Myeloid differentiation primary response protein 88; 10 CpG sites) promoters was analyzed. Bisulfite converted DNA, isolated from tumor tissue was quantified using pyrosequencing. Results of pyrosequencing analysis were expressed as a percentage for each tested CpG site. Receiver-operating characteristic (ROC) curve analysis was used for the evaluation of the diagnostic properties of selected biomarkers. Results CpG sites located in the promoters of ASC/TMS1/PYCARD_CpG8 (- 65 upstream) and MyD88_CpG4 (- 278 upstream) are significantly hypermethylated in the HNSCC when compared with LuSCC (p <= 0.0001). By performing ROC curve analysis we showed that corresponding areas under the curve (AUC) were 85-95%, indicating that selected CpG sites are useful for a distinction between primary LuSCC and primary HNSCC. Conclusions Results of the present study indicate that there is a significant difference in the methylation status of tested genes between primary HNSCC and LuSCC. However, to prove this approach as a useful tool for distinguishing second primary LuSCC from HNSCC metastasis, it would be necessary to include a larger number of samples, and most importantly, metastatic samples.

关键词： Locoweed   Swainsonine   Apoptosis   Autophagy   Lysosomal  

摘要： Swainsonine (SW), an indolizidine alkaloid, is the primary toxin in locoweeds that causes toxicity syndrome in livestock. Current research shows that SW can induce both apoptosis and autophagy. However, the relationship between, and regulatory mechanism of, autophagy and apoptosis in SW-mediated cytotoxicity remain unclear. In this study, we investigated the role of autophagy and apoptosis in SW-induced cytotoxicity in rat primary renal tubular epithelial cells (RTECs). We examined the effect of SW on lysosomal function using western blotting, transmission electron microscopy, fluorescent microscopy, and flow cytometry. The results showed that SW induced both autophagy and apoptosis, and autophagy protected RTECs from cellular damage. Activating autophagy using rapamycin (Rapa) inhibited apoptosis, while suppressing autophagy using bafilomycin A1 (Baf A1) greatly enhanced SW-induced SW and aloxistatin (E64d) further promoted apoptosis and LC3-II accumulation in RTECs. These results suggest that SW causes toxicity by disrupting lysosomal dysfunction, inhibiting autophagic degradation, and promoting apoptosis.