Preliminary results of random amplification of polymorphic DNA among Triatominae of the phyllosoma complex (Hemiptera, Reduviidae)

In Mexico, Triatoma longipennis (Usinger), Triatoma picturata (Usinger), and Triatoma pallidipennis (Stal), primary Chagas disease vector species of the phyllosoma complex, were analyzed by randomly amplified polymorphic DNA (RAPD). Sixteen decametric primers resolved individual profiles not identical, but partially discriminative between species. Analysis based on pairwise presence/absence comparisons between the three species was performed using three primers and two outgroup species Triatoma infestans (Klug) and Triatoma barberi (Usinger). Fifty-three bands in total were scored, although only two bands were constant among the three phyllosoma complex species. Two other bands were constant only for T. longipennis and T. picturata together, and not present in T. pallidipennis. Neighbor Joining tree and the multiple correspondence analysis discriminated T. pallidipennis clearly from the other two species, although there was overlap between T. longipennis and T. picturata. The results indicate a close relationship between the studied species and support the hypothesis of their recent evolution. The suitability of RAPD to discern populations within the species is discussed.

Random amplified polymorphic DNA analysis of DNA extracted from Trichuris trichiura (Linnaeus, 1771) eggs and its prospective application to paleoparasitological studies

Random amplified polymorphic DNA analysis was applied to DNAs extracted from Trichuris trichiura eggs recovered from human fecal samples. Four out of 6 primers tested displayed 18 distinct and well defined polymorphic patterns, ranging from 650 to 3200 base pairs. These results, upon retrieval and DNA sequencing of some of these bands from agarose gels, might help in establishing T. trichiura specific genetic markers, not available yet, and an important step to design primers to be used in molecular diagnosis approaches.

Analysis of ancient DNA from coprolites: a perspective with random amplified polymorphic DNA-polymerase chain reaction approach

The aim of this work was to determine approaches that would improve the quality of ancient DNA (aDNA) present in coprolites to enhance the possibility of success in retrieving specific sequence targets. We worked with coprolites from South American archaeological sites in Brazil and Chile dating up to 7,000 years ago. Using established protocols for aDNA extraction we obtained samples showing high degradation as usually happens with this kind of material. The reconstructive polymerization pretreatment was essential to overcome the DNA degradation and the serial dilutions helped with to prevent polymerase chain reaction (PCR) inhibitors. Moreover, the random amplified polymorphic DNA-PCR has been shown to be a reliable technique for further experiments to recover specific aDNA sequences.

Genetic variability analysis among clinical Candida spp. isolates using random amplified polymorphic DNA

The patterns of genetic variation of samples of Candida spp. isolated from patients infected with human immunodeficiency virus in Vitória, state of Espírito Santo, Brazil, were examined. Thirty-seven strains were isolated from different anatomical sites obtained from different infection episodes of 11 patients infected with the human immunodeficiency virus (HIV). These samples were subjected to randomly amplified polymorphic DNA (RAPD) analysis using 9 different primers. Reproducible and complex DNA banding patterns were obtained. The experiments indicated evidence of dynamic process of yeast colonization in HIV-infected patients, and also that certain primers are efficient in the identification of species of the Candida genus. Thus, we conclude that RAPD analysis may be useful in providing genotypic characters for Candida species typing in epidemiological investigations, and also for the rapid identification of pathogenic fungi.

Identification of a genetic marker associated with the resistance to Schistosoma mansoni infection using random amplified polymorphic DNA analysis

In schistosomiasis, the host/parasite interaction remains not completely understood. Many questions related to the susceptibility of snails to infection by respective trematode still remain unanswered. The control of schistosomiasis requires a good understanding of the host/parasite association. In this work, the susceptibility/resistance to Schistosoma mansoni infection within Biomphalaria alexandrina snails were studied starting one month post infection and continuing thereafter weekly up to 10 weeks after miracidia exposure. Genetic variations between susceptible and resistant strains to Schistosoma infection within B. alexandrina snails using random amplified polymorphic DNA analysis technique were also carried out. The results showed that 39.8% of the examined field snails were resistant, while 60.2% of these snails showed high infection rates.In the resistant genotype snails, OPA-02 primer produced a major low molecular weight marker 430 bp. Among the two snail strains there were interpopulational variations, while the individual specimens from the same snail strain, either susceptible or resistant, record semi-identical genetic bands. Also, the resistant character was ascendant in contrast to a decline in the susceptibility of snails from one generation to the next.

A population genetics study of Anopheles darlingi (Diptera: Culicidae) from Colombia based on random amplified polymorphic DNA-polymerase chain reaction and amplified fragment lenght polymorphism markers

The genetic variation and population structure of three populations of Anopheles darlingi from Colombia were studied using random amplified polymorphic markers (RAPDs) and amplified fragment length polymorphism markers (AFLPs). Six RAPD primers produced 46 polymorphic fragments, while two AFLP primer combinations produced 197 polymorphic fragments from 71 DNA samples. Both of the evaluated genetic markers showed the presence of gene flow, suggesting that Colombian An. darlingi populations are in panmixia. Average genetic diversity, estimated from observed heterozygosity, was 0.374 (RAPD) and 0.309 (AFLP). RAPD and AFLP markers showed little evidence of geographic separation between eastern and western populations; however, the F ST values showed high gene flow between the two western populations (RAPD: F ST = 0.029; Nm: 8.5; AFLP: F ST = 0.051; Nm: 4.7). According to molecular variance analysis (AMOVA), the genetic distance between populations was significant (RAPD:phiST = 0.084; AFLP:phiST = 0.229, P < 0.001). The F ST distances and AMOVAs using AFLP loci support the differentiation of the Guyana biogeographic province population from those of the Chocó-Magdalena. In this last region, Chocó and Córdoba populations showed the highest genetic flow.

Genetic diversity of environmental Aspergillus flavus strains in the state of São Paulo, Brazil by random amplified polymorphic DNA

Aspergillus flavus is a very important toxigenic fungus that produces aflatoxins, a group of extremely toxic substances to man and animals. Toxigenic fungi can grow in feed crops, such as maize, peanuts, and soybeans, being thus of high concern for public health. There are toxigenic and non-toxigenic A. flavus variants, but the necessary conditions for expressing the toxigenic potential are not fully understood. Therefore, we have studied total-DNA polymorphism from toxigenic and non toxigenic A. flavus strains isolated from maize crops and soil at two geographic locations, 300 km apart, in the Southeast region of Brazil. Total DNA from each A. flavus isolate was extracted and subjected to polymerase chain reaction amplification with five randomic primers through the RAPD (random amplified polymorphic DNA) technique. Phenetic and cladistic analyses of the data, based on bootstrap analyses, led us to conclude that RAPD was not suitable to discriminate toxigenic from non toxigenic strains. But the present results support the use of RAPD for strain characterization, especially for preliminary evaluation over extensive collections.

Random regression models to estimate genetic parameters for milk production of Guzerat cows using orthogonal Legendre polynomials

The objective of this work was to compare random regression models for the estimation of genetic parameters for Guzerat milk production, using orthogonal Legendre polynomials. Records (20,524) of test-day milk yield (TDMY) from 2,816 first-lactation Guzerat cows were used. TDMY grouped into 10-monthly classes were analyzed for additive genetic effect and for environmental and residual permanent effects (random effects), whereas the contemporary group, calving age (linear and quadratic effects) and mean lactation curve were analized as fixed effects. Trajectories for the additive genetic and permanent environmental effects were modeled by means of a covariance function employing orthogonal Legendre polynomials ranging from the second to the fifth order. Residual variances were considered in one, four, six, or ten variance classes. The best model had six residual variance classes. The heritability estimates for the TDMY records varied from 0.19 to 0.32. The random regression model that used a second-order Legendre polynomial for the additive genetic effect, and a fifth-order polynomial for the permanent environmental effect is adequate for comparison by the main employed criteria. The model with a second-order Legendre polynomial for the additive genetic effect, and that with a fourth-order for the permanent environmental effect could also be employed in these analyses.

Random amplified polymorphic DNA profiles as a tool for the characterization of Brazilian keratitis isolates of the genus Acanthamoeba

The genus Acanthamoeba comprises free-living amebae identified as opportunistic pathogens of humans and other animal species. Morphological, biochemical and molecular approaches have shown wide genetic diversity within the genus. In an attempt to determine the genetic relatedness among isolates of Acanthamoeba we analyzed randomly amplified polymorphic DNA (RAPD) profiles of 11 Brazilian isolates from cases of human keratitis and 8 American type culture collection (ATCC) reference strains. We found that ATCC strains belonging to the same species present polymorphic RAPD profiles whereas strains of different species show very similar profiles. Although most Brazilian isolates could not be assigned with certainty to any of the reference species, they could be clustered according to pattern similarities. The results show that RAPD analysis is a useful tool for the rapid characterization of new isolates and the assessment of genetic relatedness of Acanthamoeba spp. A comparison between RAPD analyses and morphological characteristics of cyst stages is also discussed.

Random amplification of polymorphic DNA reveals clonal relationships among enteropathogenic Escherichia coli isolated from non-human primates and humans

Enteropathogenic Escherichia coli (EPEC) strains are important agents of infantile diarrhea all over the world, gaining even greater importance in developing countries. EPEC have also been isolated from various animal species, but most isolates belong to serotypes that differ from those recovered from humans. However, it has been demonstrated that several isolates from non-human primates belong to the serogroups and/or serotypes related to those implicated in human disease. The objective of this study was to evaluate the genetic differences between thirteen strains isolated from non-human primates and the same number of strains isolated from human infections. Human isolates belonged to the same serogroup/serotype as the monkey strains and the evaluation was done by analysis of random amplified polymorphic DNA. Dendrogram analysis showed that there was no clustering between human and monkey strains. Human and non-human isolates of the EPEC serotypes O127:H40 and O128:H2 shared 90 and 87% of their bands, respectively, indicating strong genomic similarity between the strains, leading to the speculation that they may have arisen from the same pathogenic clone. To our knowledge, this study is the first one comparing genomic similarity between human and non-human primate strains and the results provide further evidence that monkey EPEC strains correlate with human EPEC, as suggested in a previous investigation.

The six-minute walk test and body weight-walk distance product in healthy Brazilian subjects

We assessed the 6-min walk distance (6MWD) and body weight x distance product (6MWw) in healthy Brazilian subjects and compared measured 6MWD with values predicted in five reference equations developed for other populations. Anthropometry, spirometry, reported physical activity, and two walk tests in a 30-m corridor were evaluated in 134 subjects (73 females, 13-84 years). Mean 6MWD and 6MWw were significantly greater in males than in females (622 ± 80 m, 46,322 ± 10,539 kg.m vs 551 ± 71 m, 36,356 ± 8,289 kg.m, P < 0.05). Four equations significantly overestimated measured 6MWD (range, 32 ± 71 to 137 ± 74 m; P < 0.001), and one significantly underestimated it (-36 ± 86 m; P < 0.001). 6MWD significantly correlated with age (r = -0.39), height (r = 0.44), body mass index (r = -0.24), and reported physical activity (r = 0.25). 6MWw significantly correlated with age (r = -0.21), height (r = 0.66) and reported physical activity (r = 0.25). The reference equation devised for walk distance was 6MWDm = 622.461 - (1.846 x Ageyears) + (61.503 x Gendermales = 1; females = 0); r2 = 0.300. In an additional group of 85 subjects prospectively studied, the difference between measured and the 6MWD predicted with the equation proposed here was not significant (-3 ± 68 m; P = 0.938). The measured 6MWD represented 99.6 ± 11.9% of the predicted value. We conclude that 6MWD and 6MWw variances were adequately explained by demographic and anthropometric attributes. This reference equation is probably most appropriate for evaluating the exercise capacity of Brazilian patients with chronic diseases.

Reliability and validity of heart rate variability threshold assessment during an incremental shuttle-walk test in middle-aged and older adults

Studies on the assessment of heart rate variability threshold (HRVT) during walking are scarce. We determined the reliability and validity of HRVT assessment during the incremental shuttle walk test (ISWT) in healthy subjects. Thirty-one participants aged 57 ± 9 years (17 females) performed 3 ISWTs. During the 1st and 2nd ISWTs, instantaneous heart rate variability was calculated every 30 s and HRVT was measured. Walking velocity at HRVT in these tests (WV-HRVT1 and WV-HRVT2) was registered. During the 3rd ISWT, physiological responses were assessed. The ventilatory equivalents were used to determine ventilatory threshold (VT) and the WV at VT (WV-VT) was recorded. The difference between WV-HRVT1 and WV-HRVT2 was not statistically significant (median and interquartile range = 4.8; 4.8 to 5.4 vs4.8; 4.2 to 5.4 km/h); the correlation between WV-HRVT1 and WV-HRVT2 was significant (r = 0.84); the intraclass correlation coefficient was high (0.92; 0.82 to 0.96), and the agreement was acceptable (-0.08 km/h; -0.92 to 0.87). The difference between WV-VT and WV-HRVT2 was not statistically significant (4.8; 4.8 to 5.4 vs 4.8; 4.2 to 5.4 km/h) and the agreement was acceptable (0.04 km/h; -1.28 to 1.36). HRVT assessment during walking is a reliable measure and permits the estimation of VT in adults. We suggest the use of the ISWT for the assessment of exercise capacity in middle-aged and older adults.

Intensity and physiological responses to the 6-minute walk test in middle-aged and older adults: a comparison with cardiopulmonary exercise testing

The 6-minute walk test (6MWT) is a simple field test that is widely used in clinical settings to assess functional exercise capacity. However, studies with healthy subjects are scarce. We hypothesized that the 6MWT might be useful to assess exercise capacity in healthy subjects. The purpose of this study was to evaluate 6MWT intensity in middle-aged and older adults, as well as to develop a simple equation to predict oxygen uptake ( V ˙ O 2 ) from the 6-min walk distance (6MWD). Eighty-six participants, 40 men and 46 women, 40-74 years of age and with a mean body mass index of 28±6 kg/m2, performed the 6MWT according to American Thoracic Society guidelines. Physiological responses were evaluated during the 6MWT using a K4b2 Cosmed telemetry gas analyzer. On a different occasion, the subjects performed ramp protocol cardiopulmonary exercise testing (CPET) on a treadmill. Peak V ˙ O 2 in the 6MWT corresponded to 78±13% of the peak V ˙ O 2 during CPET, and the maximum heart rate corresponded to 80±23% of that obtained in CPET. Peak V ˙ O 2 in CPET was adequately predicted by the 6MWD by a linear regression equation: V ˙ O 2 mL·min-1·kg-1 = -2.863 + (0.0563×6MWDm) (R2=0.76). The 6MWT represents a moderate-to-high intensity activity in middle-aged and older adults and proved to be useful for predicting cardiorespiratory fitness in the present study. Our results suggest that the 6MWT may also be useful in asymptomatic individuals, and its use in walk-based conditioning programs should be encouraged.

Random-start controlled ovarian stimulation for emergency fertility preservation in a patient with myelodysplastic syndrome: a case report

This study reports a case of a gonadotropin-releasing hormone agonist trigger in a young female with myelodysplastic syndrome (MDS) who underwent fertility preservation using random-start controlled ovarian stimulation. This method involves the stimulation of the ovary regardless of a patient's menstrual-cycle phase. A review of the related literature is also provided. A 17-year-old patient was diagnosed with MDS and required initiation of peripheral blood stem cell transplantation within a maximum of 3 weeks and was in the luteal phase of the menstrual cycle when the possibility of attempting preservation of fertility was presented to her. She opted for a random-start controlled ovarian stimulation with gonadotropins. With successful hemorrhagic prophylaxis, 17 oocytes were retrieved including 10 mature and 7 immature oocytes. Of the immature oocytes, 3 were successfully matured in vitro and a vitrification protocol was used to freeze the 13 mature oocytes.

Optimization of random amplified polymorphic DNA protocol for molecular identification of Lophius gastrophysus

Lophius gastrophysus has important commercial value in Brazil particularly for foreign trade. In this study, we described the optimization of Random Amplified Polymorphic DNA (RAPD) protocol for identification of L. gastrophysus. Different conditions (annealing temperatures, MgCl concentrations, DNA quantity) were tested to find reproducible and adequate profiles. Amplifications performed with primers A01, ² A02 and A03 generate the best RAPD profiles when the conditions were annealing temperature of 36ºC, 25 ng of DNA quantity and 2.5 mM MgCl2. Exact identification of the species and origin of marine products is necessary and RAPD could be used as an accurate, rapid tool to expose commercial fraud.