Immunohistochemical assessment of Pax8 expression during pancreatic islet development and in human neuroendocrine tumors

The paired box transcription factor Pax8 is critical for development of the eye, thyroid gland as well as the urinary and reproductive organs. In adult, Pax8 overexpression is associated with kidney, ovarian and thyroid tumors and has emerged as a specific marker for these cancers. Recently, Pax8 expression was also reported in human pancreatic islets and in neuroendocrine tumors, identifying Pax8 as a novel member of the Pax family expressed in the pancreas. Herein, we sought to provide a comprehensive analysis of Pax8 expression during pancreogenesis and in adult islets. Immunohistochemical analysis using the most employed Pax8 polyclonal antibody revealed strong nuclear staining in the developing mouse pancreas and in mature human and mouse islets. Astonishingly, Pax8 mRNA in mouse islets was undetectable while human islets exhibited low levels. These discrepancies raised the possibility of antibody cross-reactivity. This premise was confirmed by demonstrating that the polyclonal Pax8 antibody also recognized the islet-enriched Pax6 protein both by Western blotting and immunohistochemistry. Thus, in islets polyclonal Pax8 staining corresponds mainly to Pax6. In order to circumvent this caveat, a novel Pax8 monoclonal antibody was used to re-evaluate whether Pax8 was indeed expressed in islets. Surprisingly, Pax8 was not detected in neither the developing pancreas or in mature islets. Reappraisal of pancreatic neuroendocrine tumors using this Pax8 monoclonal antibody exhibited no immunostaining as compared to the Pax8 polyclonal antibody. In conclusion, Pax8 is not expressed in the pancreas and cast doubts on the value of Pax8 as a pancreatic neuroendocrine tumor marker

The CAPN10 Gene Is Associated with Insulin Resistance Phenotypes in the Spanish Population

Cardiovascular disease is the leading cause of morbidity and mortality in the industrialized world. Familial aggregation of cardiovascular risk factors is a frequent finding, but genetic factors affecting its presentation are still poorly understood. The calpain 10 gene (CAPN10) has been associated with type 2 diabetes (T2DM), a complex metabolic disorder with increased risk of cardiovascular disease. Moreover, the CAPN10 gene has been associated with the presence of metabolic syndrome (MS) in T2DM and in polycystic ovary syndrome (PCOS). In this work, we have analysed whether the polymorphisms UCSNP44, -43, -19 and -63 are related to several cardiovascular risk factors in the context of MS. Molecular analysis of CAPN10 gene was performed in 899 individuals randomly chosen from a cross-sectional population-based epidemiological survey. We have found that CAPN10 gene in our population is mainly associated with two indicators of the presence of insulin resistance: glucose levels two hours after a 75-g oral glucose tolerance test (OGTT) and HOMA values, although cholesterol levels and blood pressure values are also influenced by CAPN10 variants. In addition, the 1221/1121 haplogenotype is under-represented in individuals that fulfil the International Diabetes Federation (IDF) diagnostic criteria for MS. Our results suggest that CAPN10 gene is associated with insulin resistance phenotypes in the Spanish population

Comparative Therapeutic Effects of Velaglucerase Alfa and Imiglucerase in a Gaucher Disease Mouse Model

Gaucher disease type 1 is caused by the defective activity of the lysosomal enzyme, acid β-glucosidase (GCase). Regular infusions of purified recombinant GCase are the standard of care for reversing hematologic, hepatic, splenic, and bony manifestations. Here, similar in vitro enzymatic properties, and in vivo pharmacokinetics and pharmacodynamics (PK/PD) and therapeutic efficacy of GCase were found with two human GCases, recombinant GCase (CHO cell, imiglucerase, Imig) and gene-activated GCase (human fibrosarcoma cells, velaglucerase alfa, Vela), in a Gaucher mouse, D409V/null. About 80+% of either enzyme localized to the liver interstitial cells and <5% was recovered in spleens and lungs after bolus i.v. injections. Glucosylceramide (GC) levels and storage cell numbers were reduced in a dose (5, 15 or 60 U/kg/wk) dependent manner in livers (60–95%) and in spleens (∼10–30%). Compared to Vela, Imig (60 U/kg/wk) had lesser effects at reducing hepatic GC (p = 0.0199) by 4 wks; this difference disappeared by 8 wks when nearly WT levels were achieved by Imig. Anti-GCase IgG was detected in GCase treated mice at 60 U/kg/wk, and IgE mediated acute hypersensitivity and death occurred after several injections of 60 U/kg/wk (21% with Vela and 34% with Imig). The responses of GC levels and storage cell numbers in Vela- and Imig-treated Gaucher mice at various doses provide a backdrop for clinical applications and decisions

Plant-Type Trehalose Synthetic Pathway in Cryptosporidium and Some Other Apicomplexans

The trehalose synthetic pathway is present in bacteria, fungi, plants and invertebrate animals, but is absent in vertebrates. This disaccharide mainly functions as a stress protectant against desiccation, heat, cold and oxidation. Genes involved in trehalose synthesis have been observed in apicomplexan parasites, but little was known about these enzymes. Study on trehalose synthesis in apicomplexans would not only shed new light into the evolution of this pathway, but also provide data for exploring this pathway as novel drug target.We have observed the presence of the trehalose synthetic pathway in Cryptosporidium and other apicomplexans and alveolates. Two key enzymes (trehalose 6-phosphate synthase [T6PS; EC 2.4.1.15] and trehalose phosphatase [TPase; EC 3.1.3.12] are present as Class II bifunctional proteins (T6PS-TPase) in the majority of apicomplexans with the exception of Plasmodium species. The enzyme for synthesizing the precursor (UDP-glucose) is homologous to dual-substrate UDP-galactose/glucose pyrophosphorylases (UGGPases), rather than the "classic" UDP-glucose pyrophosphorylase (UGPase). Phylogenetic recontructions indicate that both T6PS-TPases and UGGPases in apicomplexans and other alveolates are evolutionarily affiliated with stramenopiles and plants. The expression level of T6PS-TPase in C. parvum is highly elevated in the late intracellular developmental stage prior to or during the production of oocysts, implying that trehalose may be important in oocysts as a protectant against environmental stresses. Finally, trehalose has been detected in C. parvum oocysts, thus confirming the trehalose synthetic activity in this parasite.A trehalose synthetic pathway is described in the majority of apicomplexan parasites including Cryptosporidium and the presence of trehalose was confirmed in the C. parvum oocyst. Key enzymes in the pathway (i.e., T6PS-TPase and UGGPase) are plant-type and absent in humans and animals, and may potentially serve as novel drug targets in the apicomplexans

Whole Grain Products, Fish and Bilberries Alter Glucose and Lipid Metabolism in a Randomized, Controlled Trial: The Sysdimet Study

Due to the growing prevalence of type 2 diabetes, new dietary solutions are needed to help improve glucose and lipid metabolism in persons at high risk of developing the disease. Herein we investigated the effects of low-insulin-response grain products, fatty fish, and berries on glucose metabolism and plasma lipidomic profiles in persons with impaired glucose metabolism.Altogether 106 men and women with impaired glucose metabolism and with at least two other features of the metabolic syndrome were included in a 12-week parallel dietary intervention. The participants were randomized into three diet intervention groups: (1) whole grain and low postprandial insulin response grain products, fatty fish three times a week, and bilberries three portions per day (HealthyDiet group), (2) Whole grain enriched diet (WGED) group, which includes principally the same grain products as group (1), but with no change in fish or berry consumption, and (3) refined wheat breads (Control). Oral glucose tolerance, plasma fatty acids and lipidomic profiles were measured before and after the intervention. Self-reported compliance with the diets was good and the body weight remained constant. Within the HealthyDiet group two hour glucose concentration and area-under-the-curve for glucose decreased and plasma proportion of (n-3) long-chain PUFAs increased (False Discovery Rate p-values <0.05). Increases in eicosapentaenoic acid and docosahexaenoic acid associated curvilinearly with the improved insulin secretion and glucose disposal. Among the 364 characterized lipids, 25 changed significantly in the HealthyDiet group, including multiple triglycerides incorporating the long chain (n-3) PUFA.The results suggest that the diet rich in whole grain and low insulin response grain products, bilberries, and fatty fish improve glucose metabolism and alter the lipidomic profile. Therefore, such a diet may have a beneficial effect in the efforts to prevent type 2 diabetes in high risk persons.ClinicalTrials.gov NCT00573781

Association between TCF7L2 gene polymorphisms and susceptibility to Type 2 Diabetes Mellitus: a large Human Genome Epidemiology (HuGE) review and meta-analysis

<p>Abstract</p> <p>Background</p> <p>Transcription factor 7-like 2 (<it>TCF7L2</it>) has been shown to be associated with type 2 diabetes mellitus (T2MD) in multiple ethnic groups in the past two years, but, contradictory results were reported for Chinese and Pima Indian populations. The authors then performed a large meta-analysis of 36 studies examining the association of type 2 diabetes mellitus (T2DM) with polymorphisms in the <it>TCF7L2 </it>gene in various ethnicities, containing rs7903146 C-to-T (IVS3C>T), rs7901695 T-to-C (IVS3T>C), a rs12255372 G-to-T (IVS4G>T), and rs11196205 G-to-C (IVS4G>C) polymorphisms and to evaluate the size of gene effect and the possible genetic mode of action.</p> <p>Methods</p> <p>Literature-based searching was conducted to collect data and three methods, that is, fixed-effects, random-effects and Bayesian multivariate mete-analysis, were performed to pool the odds ratio (<it>OR</it>). Publication bias and study-between heterogeneity were also examined.</p> <p>Results</p> <p>The studies included 35,843 cases of T2DM and 39,123 controls, using mainly primary data. For T2DM and IVS3C>T polymorphism, the Bayesian <it>OR </it>for TT homozygotes and TC heterozygotes versus CC homozygote was 1.968 (95% credible interval (<it>CrI</it>): 1.790, 2.157), 1.406 (95% <it>CrI</it>: 1.341, 1.476), respectively, and the population attributable risk (PAR) for the TT/TC genotypes of this variant is 16.9% for overall. For T2DM and IVS4G>T polymorphism, TT homozygotes and TG heterozygotes versus GG homozygote was 1.885 (95%<it>CrI</it>: 1.698, 2.088), 1.360 (95% <it>CrI</it>: 1.291, 1.433), respectively. Four <it>OR</it>s among these two polymorphisms all yielded significant between-study heterogeneity (P < 0.05) and the main source of heterogeneity was ethnic differences. Data also showed significant associations between T2DM and the other two polymorphisms, but with low heterogeneity (<it>P </it>> 0.10). Pooled <it>OR</it>s fit a codominant, multiplicative genetic model for all the four polymorphisms of <it>TCF7L2 </it>gene, and this model was also confirmed in different ethnic populations when stratification of IVS3C>T and IVS4G>T polymorphisms except for Africans, where a dominant, additive genetic mode is suggested for IVS3C>T polymorphism.</p> <p>Conclusion</p> <p>This meta-analysis demonstrates that four variants of <it>TCF7L2 </it>gene are all associated with T2DM, and indicates a multiplicative genetic model for all the four polymorphisms, as well as suggests the <it>TCF7L2 </it>gene involved in near 1/5 of all T2MD. Potential gene-gene and gene-environmental interactions by which common variants in the <it>TCF7L2 </it>gene influence the risk of T2MD need further exploration.</p

Production of mannosylglycerate in Saccharomyces cerevisiae by metabolic engineering and bioprocess optimization

Mannosylglycerate (MG) is one of the most widespread compatible solutes among marine microorganisms adapted to hot environments. This ionic solute holds excellent ability to protect proteins against thermal denaturation, hence a large number of biotechnological and clinical applications have been put forward. However, the current prohibitive production costs impose severe constraints towards large-scale applications. All known microbial producers synthesize MG from GDP-mannose and 3-phosphoglycerate via a two-step pathway in which mannosyl-3-phosphoglycerate is the intermediate metabolite. In an early work, this pathway was expressed in Saccharomyces cerevisiae with the goal to confirm gene function (Empadinhas et al. in J Bacteriol 186:4075--4084, 2004), but the level of MG accumulation was low. Therefore, in view of the potential biotechnological value of this compound, we decided to invest further effort to convert S. cerevisiae into an efficient cell factory for MG production.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte and also by project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI). Cristiana Faria was supported by a Ph.D. Grant from FCT (Ref. SFRH/ BD/79552/2011).info:eu-repo/semantics/publishedVersio