A cost-effective melting temperature assay for the detection of single-nucleotide polymorphism in the MBL2 gene of HIV-1-infected children

We report a fast (less than 3 h) and cost-effective melting temperature assay method for the detection of single-nucleotide polymorphisms in the MBL2 gene. The protocol, which is based on the Corbett Rotor Gene real time PCR platform and SYBR Green I chemistry, yielded, in the cohorts studied, sensitive (100%) and specific (100%) PCR amplification without the use of costly fluorophore-labeled probes or post-PCR manipulation. At the end of the PCR, the dissociation protocol included a slow heating from 60º to 95ºC in 0.2ºC steps, with an 8-s interval between steps. Melting curve profiles were obtained using the dissociation software of the Rotor Gene-3000 apparatus. Samples were analyzed in duplicate and in different PCR runs to test the reproducibility of this technique. No supplementary data handling is required to determine the MBL2 genotype. MBL2 genotyping performed on a cohort of 164 HIV-1-positive Brazilian children and 150 healthy controls, matched for age and sex and ethnic origin, yielded reproducible results confirmed by direct sequencing of the amplicon performed in blind. The three MBL2 variants (Arg52Cys, Gly54Asp, Gly57Glu) were grouped together and called allele 0, while the combination of three wild-type alleles was called allele A. The frequency of the A/A homozygotes was significantly higher among healthy controls (0.68) than in HIV-infected children (0.55; P = 0.0234) and the frequency of MBL2 0/0 homozygotes was higher among HIV-1-infected children than healthy controls (P = 0.0296). The 0 allele was significantly more frequent among the 164 HIV-1-infected children (0.29) than among the 150 healthy controls (0.18; P = 0.0032). Our data confirm the association between the presence of the mutated MBL2 allele (allele 0) and HIV-1 infection in perinatally exposed children. Our results are in agreement with the literature data which indicate that the presence of the allele 0 confers a relative risk of 1.37 for HIV-1 infection through vertical transmission.

In vivo antithrombotic properties of a heparin from the oocyte test cells of the sea squirt Styela plicata(Chordata-Tunicata)

In the ascidian Styela plicata, the oocytes are surrounded by two types of accessory cells named follicle cells and test cells. A heparin-like substance with an anticoagulant activity equivalent to 10% of mammalian heparin and about 5% as potent as the mammalian counterpart for the inhibition of thrombin by antithrombin was isolated from the oocyte test cells. In the present study, we compared the antithrombotic and hemorrhagic effects of sea squirt oocyte test cell heparin with those of porcine heparin in rat models of venous thrombosis and blood loss. Intravenous administration of the oocyte test cell heparin to Wistar rats (both sexes, weighing ~300 g, N = 4 in each group) at a dose of 5.0 mg/kg body weight, which produced a 1.8-fold increase in plasma activated partial thromboplastin time, inhibited thrombosis by 45 ± 13.5% (mean ± SD) without any bleeding effect. The same dose of porcine heparin inhibited thrombosis by 100 ± 1.4%, but produced a blood loss three times greater than that of the saline-treated control. However, 10-fold reduction of the dose of porcine heparin to 0.5 mg/kg body weight, which produced a 5-fold increase in plasma-activated partial thromboplastin time, inhibited thrombosis by 70 ± 13% without any bleeding effect. The antithrombotic properties of a new heparin isolated from test cells of the sea squirt S. plicata, reported here for the first time, indicate that, although sea squirt oocyte test cell heparin was a poor anticoagulant compared to porcine heparin, it had a significant antithrombotic effect without causing bleeding.

Rapid test for the evaluation of the activity of the prodrug hydroxymethylnitrofurazone in the processing of Trypanosoma cruzi messenger RNAs

No fully effective treatment has been developed since the discovery of Chagas' disease by Carlos Chagas in 1909. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effectiveness in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Many natural and/or synthetic substances showing trypanocidal activity have been used, even though they are not likely to be turned into clinically approved drugs. Originally, drug screening was performed using natural products, with only limited knowledge of the molecular mechanism involved in the development of diseases. Trans-splicing, which is unusual RNA processing reaction and occurs in nematodes and trypanosomes, implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. In the present study, permeable cells of T. cruzi epimastigote forms (Y, BOL and NCS strains) were treated to evaluate the interference of two drugs (hydroxymethylnitrofurazone - NFOH-121 and nitrofurazone) in the trans-splicing reaction using silver-stained PAGE analysis. Both drugs induced a significant reduction in RNA processing at concentrations from 5 to 12.5 µM. These data agreed with the biological findings, since the number of parasites decreased, especially with NFOH-121. This proposed methodology allows a rapid and cost-effective screening strategy for detecting drug interference in the trans-splicing mechanism of T. cruzi.

Efficacy of the FEV1/FEV6 ratio compared to the FEV1/FVC ratio for the diagnosis of airway obstruction in subjects aged 40 years or over

The present cross-sectional, population-based study was designed to evaluate the performance of the FEV1/FEV6 ratio for the detection of airway-obstructed subjects compared to the FEV1/FVC <0.70 fixed ratio test, as well as the lower limit of normality (LLN) for 1000 subjects ³40 years of age in the metropolitan area of São Paulo, SP, Brazil. After the exclusion of 37 (3.7%) spirometries, a total of 963 pre-bronchodilator (BD) and 918 post-BD curves were constructed. The majority of the post-BD curves (93.1%) were of very good quality and achieved grade A (762 curves) or B (93 curves). The FEV1/FEV6 and FEV1/FVC ratios were highly correlated (r² = 0.92, P < 0.000). Two receiver operator characteristic curves were constructed in order to express the imbalance between the sensitivity and specificity of the FEV1/FEV6 ratio compared to two FEV1/FVC cut-off points for airway obstruction: equal to 70 (area under the curve = 0.98, P < 0.0001) and the LLN (area under the curve = 0.97, P < 0.0001), in the post-BD curves. According to an FEV1/FVC <0.70, the cut-off point for the FEV1/FEV6 ratio with the highest sum for sensitivity and specificity was 0.75. The FEV1/FEV6 ratio can be considered to be a good alternative to the FEV1/FVC ratio for the diagnosis of airway obstruction, both using a fixed cut-off point or below the LLN as reference. The FEV1/FEV6 ratio has the additional advantage of being an easier maneuver for the subjects and for the lung function technicians, providing a higher reproducibility than traditional spirometry maneuvers.

Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test

A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r² = 0.95, P < 0.0001) and for diphtheria (r² = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

Moderate-term reproducibility of heart rate variability during rest and light to moderate exercise in children

Previous studies have demonstrated the high reproducibility of heart rate variability (HRV) measures in adults while little information exists concerning HRV reproducibility in children. Subsequently, the aim of the current study was to examine the moderate-term reproducibility of heart rate and frequency domain measures of HRV during rest and light to moderate exercise in children. Ten healthy children (6 males, 4 females) aged between 7 and 12 years of age volunteered for this study with HRV recordings obtained during supine rest and three treadmill walking exercise work rates (≤60% maximum heart rate), initially and then 8 weeks later. Differences (P < 0.05) between variables were examined using paired t-tests or Wilcoxon signed rank tests while reliability and reproducibility were examined by intraclass correlation coefficients (ICC), coefficients of variation (CV), and mean bias ratio and ratio limits of agreement (LOA). Heart rate and all measures of HRV at rest and exercise were unchanged after 8 weeks. Significant ICC were documented primarily during rest (0.72-0.85) while weaker relationships (-0.02-0.87) were evident during exercise. A large range of CV was identified during rest (6-33%) and exercise (3-128%) while the ratio LOA were variable and substantial (1.04-2.73). Despite similar HRV over an 8-week period, variable ICC and sizable CV and ratio LOA indicate moderate to poor reproducibility of HRV in children, particularly during light to moderate exercise. Studies examining HRV in children should include age- or maturation stage-matched control participants to address the age-related change in HRV and inadequate HRV reliability.

Evaluation of a 1-h 75-g oral glucose tolerance test in the diagnosis of gestational diabetes

In order to evaluate the performance of a 1-h 75-g oral glucose tolerance test (OGTT) for the diagnosis of gestational diabetes mellitus (GDM), a cohort of 4998 women, 20 years or older, without previous diabetes being treated in prenatal care clinics in Brazil answered a questionnaire and performed a 75-g OGTT including fasting, 1-h and 2-h glucose measurements between their 24th and 28th gestational weeks. Pregnancy outcomes were transcribed from medical registries. GDM was defined according to WHO criteria (fasting: ≥126 mg/dL; 2-h value: ≥140 mg/dL) and macrosomia as a birth weight equal to or higher than 4000 g. Areas under the receiver operator characteristic curve (AUC) were compared and diagnostic properties of various cut-off points were evaluated. The AUCs for the prediction of macrosomia were 0.606 (0.572-0.637) for the 1-h and 0.589 (0.557-0.622) for the 2-h plasma glucose test. Similar predictability was demonstrable regarding combined adverse outcomes: 0.582 (0.559-0.604) for the 1-h test and 0.572 (0.549-0.595) for the 2-h test. When the 1-h glucose test was evaluated against a diagnosis of GDM defined by the 2-h glucose test, the AUC was 0.903 (0.886-0.919). The cut-off point that maximized sensitivity (83%) and specificity (83%) was 141 mg/dL, identifying 21% of the women as positive. A cut-off point of 160 mg/dL, with lower sensitivity (62%), had higher specificity (94%), labeling 8.6% as positive. Detection of GDM can be done with a 1-h 75-g OGTT: the value of 160 mg/dL has the same diagnostic performance as the conventional 2-h value (140 mg/dL). The simplification of the test may improve coverage and timing of the diagnosis of GDM.

Cognitive performance of young and elderly subjects on the free word recall memory test: effect of presentation order on recall order

The influence of aging on memory has been extensively studied, but the importance of short-term memory and recall sequence has not. The objective of the current study was to examine the recall order of words presented on lists and to determine if age affects recall sequence. Physically and psychologically healthy male subjects were divided into two groups according to age, i.e., 23 young subjects (20 to 30 years) and 50 elderly subjects (60 to 70 years) submitted to the Wechsler Adult Intelligence Scale-Revised and the free word recall test. The order of word presentation significantly affected the 3rd and 4th words recalled (P < 0.01; F = 14.6). In addition, there was interaction between the presentation order and the type of list presented (P < 0.05; F = 9.7). Also, both groups recalled the last words presented from each list (words 13-15) significantly more times 3rd and 4th than words presented in all remaining positions (P < 0.01). The order of word presentation also significantly affected the 5th and 6th words recalled (P = 0.05; F = 7.5) and there was a significant interaction between the order of presentation and the type of list presented (P < 0.01; F = 20.8). The more developed the cognitive functions, resulting mainly from formal education, the greater the cognitive reserve, helping to minimize the effects of aging on the long-term memory (episodic declarative).