Selection of Reference Genes for RT-qPCR Studies in Different Organs of Rice Cultivar BRS AG Submitted to Recurrent Saline Stress.

Quantitative real-time polymerase chain reactions (RT-qPCR) have become one of the most widely used methods for analyzing gene expression, provided suitable reference genes are available to normalize the data. RNA was isolated from leaves, grain, rachises and sheaths of rice (Oryza sativa L. cv. BRS AG) submitted to different saline stress events for seven days, and expression analysis was carried out by RT-qPCR. Expression levels of ten candidate reference genes were assessed, actin11 (ACT11), ubiquitin conjugating enzyme E2 (UBC-E2), eukaryotic elongation factor1-a (Eef-1a), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), B-tubulin (B-Tub), eukaryotic initiation factor 4a (Eif-4-a), ubiquitin10 (UBQ10), ubiquitin5 (UBQ5), aquaporin TIP41 (TIP41-like). Gene expression stability was calculated using the common statistical algorithms geNorm, BestKeeper and ?Ct method, NormFinder and RefFinder. The most stably expressed genes were UBC2E and GAPDH for leaves, UBQ5 and UBQ10 for sheaths, TIP41 and UBQ10 for rachises, and TIP41 and cyclophilin for grain. Gene expression of triose phosphate translocator (TPT1), ADP-glucose transporter (BT1-1), choline monooxygenase (CMO) was used to validate the selected reference genes. The results highlighted the importance of using suitable reference gene to normalize gene expression data in rice plants

Prospective evaluation of glutamine and phospholipids levels in first degree relatives of patients with Type 1 Diabetes from a multiethnic population

A dysregulation in the metabolism of lipids may be an early marker of autoimmunity in Type 1 Diabetes (T1D). It would be of general importance to identify metabolic patterns that would predict the risk for T1D later in life. The aim of this study was to perform a prospective evaluation of glutamine and phospholipids levels in Brazilian first degree relatives (FDR) of patients with T1D in a mean interval of 5 years

Relationship between the prevalence of anti-glutamic acid decarboxylase autoantibodies and duration of type 1 diabetes mellitus in Brazilian patients

The objective of the present study was to determine whether the duration of disease has any influence on the prevalence of glutamic acid decarboxylase autoantibodies (GADA) in Brazilian patients with type 1 diabetes (T1D) and variable disease duration. We evaluated 83 patients with T1D. All participants were interviewed and blood was obtained for GADA measurement by a commercial radioimmunoassay (RSR Limited, Cardiff, UK). Four groups of patients were established according to disease duration: A) 1-5 years of disease (N = 24), B) 6-10 years of disease (N = 19), C) 11-15 years of disease (N = 25), and D) >15 years of disease (N = 15). GADA prevalence and its titers were determined in each group. GADA was positive in 38 patients (45.8%) and its frequency did not differ between the groups. The prevalence was 11/24 (45.8%), 8/19 (42.1%), 13/25 (52%), and 6/15 (40%) in groups A, B, C, and D, respectively (P = 0.874). Mean GADA titer was 12.54 ± 11.33 U/ml for the sample as a whole and 11.95 ± 11.8, 12.85 ± 12.07, 10.57 ± 8.35, and 17.45 ± 16.1 U/ml for groups A, B, C, and D, respectively (P = 0.686). Sex, age at diagnosis or ethnic background had no significant effect on GADA (+) frequency. In conclusion, in this transversal study, duration of disease did not affect significantly the prevalence of GADA or its titers in patients with T1D after one year of diagnosis. This was the first study to report this finding in the Brazilian population

MULTICENTER STUDY of the PREVALENCE of DIABETES-MELLITUS and IMPAIRED GLUCOSE-TOLERANCE in the URBAN BRAZILIAN POPULATION AGED 30-69 YR

OBJECTIVE - To assess the prevalence of diabetes and IGT in the urban adult Brazilian population.RESEARCH DESIGN and METHODS- We used a two-stage, multicenter, cross-sectional survey in a random sample of 21,847 individuals aged 30-9 yr from nine large cities. Subjects were first screened by FCG. All positive screenees (FCG greater-than-or-equal-to 5.6 mM/L) and every sixth consecutive negative screenee were administered a 75 g OGTT and classified as diabetic, IGT, or normal (nondiabetic) according to WHO recommendations. OGTT findings from the negative screenees were extrapolated to all negative screenees after adjustments for potential biases.RESULTS - the overall rates were 7.6 and 7.8% for diabetes and IGT, respectively Men (7.5%) and women (7.6%) had similar rates of diabetes. Similar rates resulted with whites (7.8%) and nonwhites (7.3%). Diabetes prevalence increased from 2.7% in the 30-39-yr age-group to 17.4% in the 60-69-yr age-group. Diabetes was more prevalent among less educated people, but this difference disappeared after adjusting for age. Family history of diabetes was associated with a twofold increase in diabetes prevalence (I 2.5 vs. 5.8%); the same increase occurred with obesity (I 1.6 vs. 5.2%). Undiagnosed diabetes accounted for 46% of the total prevalence. Among previously diagnosed cases, 22.3% were not under treatment, 7.9% were on insulin, 40.7% were on oral agents, and 29.1% were on dietary treatment only. Self-reported diabetes prevalence was 0.1, 3.2, and 11.6% in the age groups 70 yr, respectively.CONCLUSIONS - the prevalence of diabetes in Brazil is comparable with that of more developed countries, where it is considered a major health problem.UNIV São Paulo,SCH MED,DIV ENDOCRINOL,HUMAN NUTR & METAB DIS LAB LIM25,São Paulo,BRAZILESCOLA PAULISTA MED,DEPT PREVENT MED,BR-04023 São Paulo,BRAZILESCOLA PAULISTA MED,DEPT PREVENT MED,BR-04023 São Paulo,BRAZILWeb of Scienc

The prevalence of diabetes in Rio de Janeiro, Brazil

OBJECTIVE - To assess the prevalence of diabetes and impaired glucose tolerance (IGT) in the adult population of Rio de Janeiro, a two-stage cross-sectional survey was carried out in a random sample of 2,051 individuals aged 30-69 years from Rio de Janeiro city in Brazil.RESEARCH DESIGN and METHODS - Subjects were first screened by fasting capillary glycemia (FCG). All individuals who screened positive (FCG > 5.6 mmol/l) and every sixth consecutive person who screened negative (FCG 70 years of age.CONCLUSIONS - the numbers found for Rio de Janeiro are similar to those for more developed countries and lead us to conclude that the impact of diabetes on public health is the same as in those countries where this disease is considered an important health problem.UNIV HOSP CLEMENTINO FRAGA FILHO,DIV NUTROL,RIO JANEIRO,BRAZILESCOLA PAULISTA MED,DEPT PREVENT MED,São Paulo,BRAZILUNIV FED RIO de JANEIRO,DEPT INTERNAL MED,BR-21945 RIO JANEIRO,BRAZILESCOLA PAULISTA MED,DEPT PREVENT MED,São Paulo,BRAZILWeb of Scienc

Target validation of the inosine monophosphate dehydrogenase (IMPDH) gene in Cryptosporidium using Phylomer® peptides

Cryptosporidiosis, a gastroenteric disease characterised mainly by diarrheal illnesses in humans and mammals is caused by infection with the protozoan parasite Cryptosporidium. Treatment options for cryptosporidiosis are limited, with the current therapeutic nitazoxanide, only partly efficacious in immunocompetent individuals. The parasite lacks de novo purine synthesis, and is exclusively dependant on purine salvage from its host. Inhibition of the inosine 5' monophosphate dehydrogenase (IMPDH), a purine salvage enzyme that is essential for DNA synthesis, thereby offers a potential drug target against this parasite. In the present study, a yeast-two-hybrid system was used to identify Phylomer peptides within a library constructed from the genomes of 25 phylogenetically diverse bacteria that targeted the IMPDH of Cryptosporidium parvum (IMPcp) and Cryptosporidium hominis (IMPch). We identified 38 unique interacting Phylomers, of which, 12 were synthesised and screened against C. parvum in vitro. Two Phylomers exhibited significant growth inhibition (81.2-83.8% inhibition; P < 0.05), one of which consistently exhibited positive interactions with IMPcp and IMPch during primary and recapitulation yeast two-hybrid screening and did not interact with either of the human IMPDH proteins. The present study highlightsthe potential of Phylomer peptides as target validation tools for Cryptosporidium and other organisms and diseases because of their ability to bind with high affinity to target proteins and disrupt function

A platform for discovery of functional cell-penetrating peptides for efficient multi-cargo intracellular delivery

Cell penetrating peptides (CPPs) offer great potential to deliver therapeutic molecules to previously inaccessible intracellular targets. However, many CPPs are inefficient and often leave their attached cargo stranded in the cell’s endosome. We report a versatile platform for the isolation of peptides delivering a wide range of cargos into the cytoplasm of cells. We used this screening platform to identify multiple “Phylomer” CPPs, derived from bacterial and viral genomes. These peptides are amenable to conventional sequence optimization and engineering approaches for cell targeting and half-life extension. We demonstrate potent, functional delivery of protein, peptide, and nucleic acid analog cargos into cells using Phylomer CPPs. We validate in vivo activity in the cytoplasm, through successful transport of an oligonucleotide therapeutic fused to a Phylomer CPP in a disease model for Duchenne’s muscular dystrophy. This report thus establishes a discovery platform for identifying novel, functional CPPs to expand the delivery landscape of druggable intracellular targets for biological therapeutics