Fabiane Gomes de Moraes Rego

Genome Structure of the Genus Azospirillum

Azospirillum species are plant-associated diazotrophs of the alpha subclass of Proteobacteria. The genomes of five of the six Azospirillum species were analyzed by pulsed-field gel electrophoresis. All strains possessed several megareplicons, some probably linear, and 16S ribosomal DNA hybridization indicated multiple chromosomes in genomes ranging in size from 4.8 to 9.7 Mbp. The nifHDK operon was identified in the largest replicon.

Characterization of the orf1glnKamtB operon of Herbaspirillum seropedicae

Herbaspirillum seropedicae is an endophytic nitrogen-fixing bacterium that colonizes economically important grasses. In this organism, the amtB gene is co-transcribed with two other genes: glnK that codes for a PII-like protein and orf1 that codes for a probable periplasmatic protein of unknown function. The expression of the orf1glnKamtB operon is increased under nitrogen-limiting conditions and is dependent on NtrC. An amtB mutant failed to transport methylammonium. Post-translational control of nitrogenase was also partially impaired in this mutant, since a complete switch-off of nitrogenase after ammonium addition was not observed. This result suggests that the AmtB protein is involved in the signaling pathway for the reversible inactivation of nitrogenase in H. seropedicae.

The plasma logarithm of the triglyceride/HDL-cholesterol ratio is a predictor of low risk gestational diabetes in early pregnancy.

BACKGROUND The plasma lipid profile changes atherogenically during normal pregnancy. Gestational diabetes mellitus (GDM) can exacerbate the changes in metabolism. The logarithm of the ratio triglycerides/HDL-cholesterol is an atherogenic index of the plasma (AIP) and can be used as a marker for plasma atherogenicity. METHODS Serum of 576 unrelated Euro-Brazilian pregnant women was collected and the subjects were classified as healthy pregnant women (control, n=288) and gestational diabetic patients (GDM, n=288) according to the ADA 2010 criteria. Both studied groups were sub classified in 4 gestational periods: (i) 12-23, (ii) 24-28, (iii) 29-32 and (iv) >32 weeks of gestation. RESULTS Except for the AIP, the other parameters showed low discrimination between control and GDM groups (ROC curves). When analyzed by ROC curves the AIP of subjects in the early period of gestation showed sensitivity and specificity of 82.6% and 83.4%, respectively, with a cut-off point of 0.099 (AUC 0.886, P<0.0001). CONCLUSIONS The AIP is a valuable index to identify pregnant women with low risk of gestational diabetes before 24 weeks of gestation.

Is phlebotomy part of the dark side in the clinical laboratory struggle for quality

Objective: Blood collection is a critical part of the preanalytical phase of laboratory testing. Only a few procedures are evaluable for detecting errors in this non-automatic activity. Information about potential sources of error is frequently absent from quality-control procedures and training materials. Objective: To evaluate the performance of phlebotomists and to identify the major sources of errors during diagnostic blood collection. Methods: We evaluated the performance of 3 phlebotomists each from 10 laboratories regarding tourniquet time, request for fist clenching, excessive friction during skin cleaning, sequence of vacuum-tube usage, and mixing of tube contents after specimen collection. The total number of these laboratories represented an equal number of private (ie, owned by private parties) and public (ie, administration by government organizations) settings. Results: An error rate of greater than 60% was observed in the performance of procedures we examined, and the error rate was not significantly different between public and private settings other than more adequate mixing of the contents of primary collection tubes in private facilities. Conclusions: Most facilities did not achieve adequate quality standards in phlebotomy. In procedural manuals, training materials and practices for phlebotomists, greater emphasis should be placed on presenting information on possible sources of error and on the correct implementation of procedures that ensure the quality of diagnostic blood specimens collected for laboratory analysis.

Processing of Diagnostic Blood Specimens: Is It Really Necessary to Mix Primary Blood Tubes after Collection with Evacuated Tube System?

BACKGROUND The preanalytical phase is considered the most vulnerable phase in biopreservation, biobanking, and laboratory diagnostics. Accurate mixing after blood collection is claimed to be important and recommended by the manufacturers. OBJECTIVE To evaluate whether it is really necessary to mix the primary blood tubes immediately after blood collection by means of evacuated tube systems. MATERIAL AND METHODS Blood from 300 outpatients was equally and randomly divided into three groups: G1, sodium citrate vacuum tubes; G2, lithium heparin vacuum tubes; and G3, K2EDTA vacuum tubes. All vacuum tubes were processed using three different procedures. Procedure 1: Gold Standard (P1): All specimens mixed gently and carefully by inverting five times as recommended; Procedure 2: Rest time (P2): All specimens remained 5 min in the upright position, followed by gentle careful mixing by inverting five times; Procedure 3: No mix (P3): All specimens were left in upright position without mixing afterwards. The influence of the primary mixing tube procedure was evaluated for clinical chemistry, hematology, and coagulation parameters by paired t-test. The bias from the mixing procedure was also compared with quality specifications derived from biological variation. RESULTS Significant differences (p<0.017) were found for: i) red blood cell count and hematocrit when P1 was compared with P2; ii) alanine aminotransferase and erythrocyte sedimentation rate when P1 was compared with P3; iii) red blood cell count, hematocrit, and hemolysis index when P2 was compared with P3. Surprisingly, clinically significant differences were found only for sodium when P1 was compared with P2, and P1 was compared with P3. No fibrin filaments or microclots were observed in any samples. CONCLUSION Primary blood tubes mixing after collection with evacuated tube system appears to be unnecessary.

Polymorphisms in FTO and TCF7L2 genes of Euro-Brazilian women with gestational diabetes

OBJECTIVE To investigate the association between fat mass and obesity-associated (FTO) gene polymorphisms rs8050136C>A and rs9939609T>A, and transcription factor 7-like 2 (TCF7L2) gene polymorphisms rs12255372G>T and rs7903146C>T, in a sample group of pregnant Euro-Brazilian women with or without gestational diabetes mellitus (GDM). METHODS Subjects were classified as either healthy pregnant control (n=200) or GDM (n=200) according to the 2010 criteria of the American Diabetes Association. The polymorphisms were genotyped using fluorescent probes (TaqMan®). RESULTS All groups were in the Hardy-Weinberg equilibrium. The genotype and allele frequencies of the examined polymorphisms did not exhibit significant difference (P>0.05) between the groups. In the healthy and GDM pregnant women groups, the A-allele frequencies (95% CI) of FTO polymorphisms rs8050136 and rs9939609 were 39% (34-44%); 38% (33-43%) and 40% (35-45%); 41% (36-46%), respectively; and the T-allele frequencies of TCF7L2 polymorphisms rs12255372 and rs7903146 were 30% (26-35%), 32% (27-37%) and 29% (25-34%), 36% (31-41%), respectively. CONCLUSION The examined polymorphisms were not associated with GDM in the Euro-Brazilian population studied.

ATP-dependent potassium channels and type 2 diabetes mellitus

Diabetes mellitus is a public health problem, which affects a millions worldwide. Most diabetes cases are classified as type 2 diabetes mellitus, which is highly associated with obesity. Type 2 diabetes is considered a multifactorial disorder, with both environmental and genetic factors contributing to its development. An important issue linked with diabetes development is the failure of the insulin releasing mechanism involving abnormal activity of the ATP-dependent potassium channel, KATP. This channel is a transmembrane protein encoded by the KCNJ11 and ABCC8 genes. Furthermore, polymorphisms in these genes have been linked to type 2 diabetes because of the role of KATP in insulin release. While several genetic variations have been reported to be associated with this disease, the E23K polymorphism is most commonly associated with this pathology, as well as to obesity. Here, we review the molecular genetics of the potassium channel and discusses its most described polymorphisms and their associations with type 2 diabetes mellitus.

The Streptococcus mutans GlnR protein exhibits an increased affinity for the glnRA operon promoter when bound to GlnK

The control of nitrogen metabolism in pathogenic Gram-positive bacteria has been studied in a variety of species and is involved with the expression of virulence factors. To date, no data have been reported regarding nitrogen metabolism in the odontopathogenic species Streptococcus mutans. GlnR, which controls nitrogen assimilation in the related bacterial species, Bacillus subtilis, was assessed in S. mutans for its DNA and protein binding activity. Electrophoretic mobility shift assay of the S. mutans GlnR protein indicated that GlnR binds to promoter regions of the glnRA and amtB-glnK operons. Cross-linking and pull-down assays demonstrated that GlnR interacts with GlnK, a signal transduction protein that coordinates the regulation of nitrogen metabolism. Upon formation of this stable complex, GlnK enhances the affinity of GlnR for the glnRA operon promoter. These results support an involvement of GlnR in transcriptional regulation of nitrogen metabolism-related genes and indicate that GlnK relays information regarding ammonium availability to GlnR.

The GCKR Gene Polymorphism rs780094 is a Risk Factor for Gestational Diabetes in a Brazilian Population.

The glucokinase regulatory protein (GCKR) regulates the activity of the glucokinase (GCK), which plays a key role in glucose homeostasis. Genetic variants in GCK have been associated with diabetes and gestational diabetes (GDM). Due to the relationship between GCKRP and GCK, polymorphisms in GCKR are also candidates for genetic association with GDM. The aim of this study was to evaluate the association between the GCKR rs780094 polymorphism and GDM in a Brazilian population.

Type 2 diabetes-associated genetic variants of FTO, LEPR, PPARg, and TCF7L2 in gestational diabetes in a Brazilian population

Objective Gestational diabetes mellitus (GDM) is a metabolic disorder that shares pathophysiologic features with type 2 diabetes mellitus. The aim of this study was to investigate the association of the polymorphisms fat mass and obesity-associated (FTO) rs1421085, leptin receptor (LEPR) rs1137100, rs1137101, peroxisome proliferator-activated receptor gamma (PPARg) rs1801282, and transcription factor 7-like 2 (TCF7L2) rs7901695 with GDM. Subjects and methods 252 unrelated Euro-Brazilian pregnant women were classified into two groups according to the 2015 criteria of the American and Brazilian Diabetes Association: healthy pregnant women (n = 125) and pregnant women with GDM (n = 127), matched by age. The polymorphisms were genotyped using fluorescent probes (TaqMan®). Results All groups were in Hardy-Weinberg equilibrium. The genotype and allele frequencies of the studied polymorphisms did not show significant differences between the groups (P > 0.05). In the healthy and GDM groups, the C allele frequencies (95% CI) of the FTO rs1421085 polymorphism were 36.8% [31-43%] and 35.0% [29-41%]; the G allele frequencies (95% CI) of the LEPR rs1137100 polymorphism were 24.8% [19-30%] and 22.8% [18-28%]; the G allele frequencies (95% CI) of the LEPR rs1137101 polymorphism were 43.6% [37-50%] and 42.9% [37-49%]; the G allele frequencies (95% CI) of the PPARg rs1801282 polymorphism were 7.6% [4-11%] and 8.3% [5-12%]; and the C allele frequencies (95% CI) of the TCF7L2 rs7901695 polymorphism were 33.6% [28-39%] and 39.0% [33-45%], respectively. Conclusion The studied polymorphisms were not associated with GDM in a Brazilian population.