Development and validation of a pentaplex assay for the identification of antibodies against common viral diseases in cattle.

Animal welfare and economic implications of infectious diseases in cattle demand an efficient surveillance as the foundation for control and eradication programmes. Bovine respiratory syncytial virus (BRSV), Parainfluenza virus type 3 (PI3V), Bovine herpes virus-1 (BoHV-1), Bovine viral diarrhoea virus (BVDV), and Enzootic bovine leukosis virus (EBLV) cause common and often underdiagnosed diseases in cattle that are endemic in most countries [1]. A hallmark of individual exposure to a viral pathogen is the presence of antibodies directed towards that virus. The aim of this study was to develop and validate a pentaplex assay to simultaneously detect and quantify antibodies against BRSV, PI3V, BoHV-1, BVDV and EBLV in serum, as an efficient tool to yield epidemiological data. Monoplex assays were initially developed using either complete BRSV or BoHV-1 viral lysates, or recombinant proteins for BVDV, EBLV or PI3V as capture antigens. In addition, 125 serum samples from unvaccinated cattle, which were classified as positive or negative for each of the viruses by commercial ELISA kits, were used for validation. Conditions established for the Luminex monoplex assays were adopted for the pentaplex assay. The accuracy, determined by the area under the ROC curve, was greater than 0.97, and assay diagnostic sensitivities and specificities were over 95 and 90%, respectively, for all antigens. Intra (r) and interassay (R) coefficients of variation were under 10 and 20 %, respectively. Selectivity towards target viruses was shown by binding inhibition assays where unbound viruses reduced fluorescence intensities. Diagnostic agreement for samples analysed simultaneously in the monoplex and multiplex assays was almost perfect. In conclusion, a highly sensitive pentaplex assay was validated for the simultaneous identification of antibodies directed against BVDV, BoHV-1, PI3V, BRSV and EBLV in serum. The developed pentaplex assay complies with performance characteristics established by international guidelines for diagnostic tests and may be used as a tool for the implementation of epidemiological surveillance.

Percutaneous closure of a ruptured right coronary sinus of Valsalva aneurysm that mimics severe aortic regurgitation.

A 34-year-old male was admitted with presumed acute, severe aortic regurgitation. Multimodal imaging was performed and showed a ruptured right coronary sinus of Valsalva aneurysm into the right atrium. He underwent a percutaneous closure of the ruptured sinus of Valsalva aneurysm. The patient had major clinical improvement.

Identification of Zika virus in immature phases of Aedes aegypti and Aedes albopictus: a surveillance strategy for outbreak anticipation.

A progressive increase in the circulation of arboviruses in tropical countries has been observed, accounting for 700,000 yearly deaths in the world. The main objective of this article was to identify the presence of Zika (ZIKV), dengue (DENV), and Chikungunya (CHIKV) viruses in immature stages of Aedes aegypti and Ae. albopictus. Household collections of immature phases of the vectors were carried out in the years 2015 and 2016. A total of 2902 dwellings were visited and the rate of infestation with larvae and pupae of Aedes mosquitoes was 283/1462 (19.4%) in March 2015 and 55/1440 (3.8%) in June 2015. In March 2015, 907 larvae/pupae were collected (583 or 64.3% of Ae. aegypti and 324 or 35.7% of Ae. albopictus) while in June 2015 there was a reduction in the number of immature forms found: 197 larvae/pupae (121 or 61.4% of Ae. aegypti and 76 or 38.6% of Ae. albopictus). This reduction was accompanied by a decrease in suspected human ZIKV cases from March to June 2015. The RT-qPCR performed in 18 pools identified that three (two of Ae. aegypti and one of Ae. albopictus) were positive for ZIKV, and none were positive for DENV or CHIKV. Our findings demonstrated that ZIKV was present in immature stages of insect vectors in the study region at least five months prior to the peak of ZIKV associated cases. Xenomonitoring of immature phases of the vectors may prove useful for predicting outbreaks.

Identification of Zika virus in immature phases of Aedes aegypti and Aedes albopictus: a surveillance strategy for outbreak anticipation

A progressive increase in the circulation of arboviruses in tropical countries has been observed, accounting for 700,000 yearly deaths in the world. The main objective of this article was to identify the presence of Zika (ZIKV), dengue (DENV), and Chikungunya (CHIKV) viruses in immature stages of Aedes aegypti and Ae. albopictus. Household collections of immature phases of the vectors were carried out in the years 2015 and 2016. A total of 2902 dwellings were visited and the rate of infestation with larvae and pupae of Aedes mosquitoes was 283/1462 (19.4%) in March 2015 and 55/1440 (3.8%) in June 2015. In March 2015, 907 larvae/pupae were collected (583 or 64.3% of Ae. aegypti and 324 or 35.7% of Ae. albopictus) while in June 2015 there was a reduction in the number of immature forms found: 197 larvae/pupae (121 or 61.4% of Ae. aegypti and 76 or 38.6% of Ae. albopictus). This reduction was accompanied by a decrease in suspected human ZIKV cases from March to June 2015. The RT-qPCR performed in 18 pools identified that three (two of Ae. aegypti and one of Ae. albopictus) were positive for ZIKV, and none were positive for DENV or CHIKV. Our findings demonstrated that ZIKV was present in immature stages of insect vectors in the study region at least five months prior to the peak of ZIKV associated cases. Xenomonitoring of immature phases of the vectors may prove useful for predicting outbreaks.

Factores de riesgo para sobrepeso y obesidad en lactantes / Risk factors for overweight and obesity in infants

Introduction: At one year of age, factors that favor the development of obesity and other metabolic disorders in later stages of life are present. Objective: To identify perinatal, societal, and dietary risk factors for excess weight and obesity in 12 months infants. Subjects and methods: Case-control studies with infants under the care of Family Medicine Units. These cases studied were overweight or obese children, and control cases were children at a healthy weight. Each group included 152 infants, who were evaluated based on their responses to a questionnaire, their records, and perinatal, societal, and dietary variables. Results: The variables with statistical significance were high pre-gestational weight, high gestational weight gain (OR 1.79-3.88), absence of breastfeeding and exclusive formula feeding (OR 2.02 and 1.75). Conclusion: In the presence of pre-pregnancy obesity and exclusive formula feeding, there is a 64.4% greater risk of becoming overweight or obese at 12 months old.

Introducción: Al año de vida existen factores que favorecen el desarrollo de obesidad y alteraciones metabólicas en etapas posteriores. Objetivo: Identificar factores de riesgo perinatales, sociales y alimentarios de sobrepeso y obesidad en lactantes de 12 meses de edad. Sujetos y método: Estudio de casos y controles en lactantes, que acuden a Unidades de Medicina Familiar, considerando casos; niños con sobrepeso u obesidad y controles: peso adecuado. Se incluyeron 152 lactantes por grupo, a los cuales se les estudió mediante un cuestionario y revisión de expedientes, variables perinatales, sociales y alimentarias. Resultados: Las variables con significancia estadística fueron: peso pre-gestacional de la madre elevado, incremento de peso gestacional elevado; OR 1,79 y 3,88 ausencia de lactancia materna y alimentación con fórmula láctea exclusiva, OR 2,02 y 1,75. Conclusión: en presencia de obesidad pregestacional y alimentación con formula láctea exclusiva, existe un riesgo 64,38% mayor de desarrollar sobrepeso u obesidad a los 12 meses de edad.

Subdominant hierarchical ovarian follicles are needed for steroidogenesis and ovulation in laying hens (Gallus domesticus).

Ovarian follicle development in avian species is characterized by a strict hierarchical arrangement. The hierarchical follicles secrete progesterone, which induces the LH surge, but the capacity to produce other steroids decreases with development. Our aim was to evaluate the complementary action of subdominant follicles (F4-F6) on ovulation and steroidogenesis of the preovulatory follicles (F1-F3) in domestic laying hens. The first study included four groups: control (C); sham-operated (SO); large hierarchical follicles (LHF) from which F4-F6 follicles were extracted; and subdominant hierarchical follicles (SHF) from which F1-F3 follicles were extracted. Blood samples were collected every 2h from 12h before estimated ovoposition until 2h after ovoposition. Egg laying continued at the same rates in C and SO hens, with normal preovulatory surges of oestradiol, testosterone, progesterone and LH. In contrast, in LHF and SHF groups, ovoposition was blocked; oestradiol concentrations were not affected; but no preovulatory surges of testosterone, progesterone or LH were seen. Further, the testosterone surge was required for the occurrence of progesterone and LH surges. In the second study StAR and steroidogenic enzyme mRNA expression was evaluated within F1-F3 follicles from a LHF group and C-14 and C-8 controls groups, in which follicles were collected 14h and 8h before expected ovoposition, respectively. Extraction of F4-F6 follicles caused a significant reduction in StAR and 3ß-HSD expressions within theca, but not in granulosa cells. In conclusion, subdominant hierarchical follicles (F4-F6) are required for the preovulatory release of testosterone, progesterone and LH, which are highly inter-correlated.

Reproduction in hens: is testosterone necessary for the ovulatory process?

Avian reproduction entails complex endocrine interactions at the hypothalamic and ovarian levels. The initiation of the reproductive season is due to the reduction in melatonin and GnIH production as day length increases. The decline in GnIH permits GnRH and gonadotropin secretion starting follicle growth. Follicular steroids stimulate sexual activity and have important roles for the induction of ovulation. Progesterone (P4) is an inductor of the preovulatory surge of LH, while estradiol (E2) acts as a hypothalamic primer to allow P4 receptor development, as well as a stimulator of yolk production. Conversely, the role of testosterone (T) has been more controversial; however, there is now enough evidence, which demonstrates an essential action of T in the ovulatory process. For instance, blockage of endogenous T, by passive or active immunization or by the use of a specific antagonist of T, inhibits ovulation and the preovulatory surges of P4 and LH. This information is supported by the fact that there is a positive correlation between the occurrences of the T preovulatory surge and those of P4 and LH, in which the absence of T caused a lack of P4 and LH increase in almost 90% of the cases. Additionally, it has been observed that T has a paracrine action within the ovary, to promote P4 secretion by granulosa cells from the larger follicles. This has been related with an increased mRNA expression of StAR and P450scc enzymes, which are essential for P4 production, as well as with LH-R mRNA expression in granulosa cells of preovulatory follicles, an effect that should enhance the positive feedback between P4 and LH necessary for ovulation. Lastly, endocrine activity of hierarchical follicles occurs as a result of a complex interaction between the larger follicles (F1-F3) and the smaller follicles (F4-F6), which is necessary to achieve an adequate preovulatory milieu.

Two-year clinical performance of self-etching adhesive systems in composite restorations of anterior teeth.

OBJECTIVE: The aim of this study was to evaluate the two-year clinical performance of Class III, IV, and V composite restorations using a two-step etch-and-rinse adhesive system (2-ERA) and three one-step self-etching adhesive systems (1-SEAs). MATERIAL AND METHODS: Two hundred Class III, IV, and V composite restorations were placed into 50 patients. Each patient received four composite restorations (Amaris, Voco), and these restorations were bonded with one of three 1-SEAs (Futurabond M, Voco; Clearfil S3 Bond, Kuraray; and Optibond All-in-One, Kerr) or one 2-ERA (Adper Single Bond 2/3M ESPE). The four adhesive systems were evaluated at baseline and after 24 months using the following criteria: restoration retention, marginal integrity, marginal discoloration, caries occurrence, postoperative sensitivity and preservation of tooth vitality. After two years, 162 restorations were evaluated in 41 patients. Data were analyzed using the χ(2) test (p<0.05). RESULTS: There were no statistically significant differences between the 2-ERA and the 1-SEAs regarding the evaluated parameters (p>0.05). CONCLUSION: The 1-SEAs showed good clinical performance at the end of 24 months.

Testosterone stimulates progesterone production and STAR, P450 cholesterol side-chain cleavage and LH receptor mRNAs expression in hen (Gallus domesticus) granulosa cells.

The chicken ovary is organized into a hierarchy of yellow yolky follicles that ovulate on successive days. Active or passive immunization of laying hens against testosterone blocks ovulation without affecting follicle development. Testosterone may play a role in pre-ovulatory follicle maturation by stimulating granulosa progesterone production. We assessed whether this stimulus is dose-related and depends on the maturity of the donor follicle, and if it does so by stimulating granulosa cell STAR, P450 cholesterol side-chain cleavage (P450scc), and LH receptor (LHCGR) mRNAs expression. Progesterone production by granulosa cells from F1, F3, and F4 follicles, cultured for 3 h without testosterone was greater in cells collected 11-14 h than 1-4 h after ovulation. These differences in progesterone production were less pronounced after granulosa cells had been cultured for 24 h. Culture of granulosa cells for 3 or 24 h with testosterone (1-100 ng/ml) stimulated progesterone production in cells collected from F4, F3, or F1 follicles 1-4, or 11-14 h after ovulation. Testosterone (0-4000 ng/ml) alone or in combination with LH (0-100 ng/ml) increased progesterone production by F1 granulosa cells, collected 1-4 and 11-14 h after ovulation and cultured for 3 h. Finally, testosterone (10 or 100 ng/ml) increased STAR, P450scc, and LHCGR mRNAs, when added to 3 h cultures of F1 granulosa cells. In conclusion, testosterone stimulates granulosa cell progesterone production in hen pre-ovulatory hierarchical follicles irrespective of maturational state, acting alone or additively with LH. We propose that testosterone promotes granulosa cell maturation to facilitate the pre-ovulatory release of LH.

Agronomic and molecular characterization of introgression lines from the interspecific cross Oryza sativa (BG90-2) x Oryza glumaepatula (RS-16).

The reduced genetic variability of modern rice varieties (Oryza sativa) is of concern because it reduces the possibilities of genetic gain in breeding programs. Introgression lines (ILs) containing genomic fragments from wild rice can be used to obtain new improved cultivars. The objective of the present study was to perform the agronomic and molecular characterizations of 35 BC2F8 ILs from the cross O. glumaepatula x O. sativa, aiming to select high-yielding ILs to be used in rice-breeding programs. All 35 ILs were field evaluated in the season 2002/2003 in three locations and the 15 best performing ones were evaluated in the season 2003/2004 in five locations. In 2003/2004, six ILs (CNAi 9934, CNAi 9931, CNAi 9930, CNAi 9935, CNAi 9936, and CNAi 9937) showed the highest yield means and were statistically superior to the controls Metica 1 and IRGA 417. Molecular characterization of the 35 ILs was performed with 92 microsatellite markers distributed on the 12 rice chromosomes and a simple regression quantitative trait locus analysis was performed using the phenotypic data from 2002/2003. The six high-yielding ILs showed a low proportion of wild fragment introgressions. A total of 14 molecular markers were associated with quantitative trait loci in the three locations. The six high-yielding ILs were incorporated in the Embrapa breeding program, and the line CNAi 9930 is recommended for cultivation due to additional advantages of good grain cooking and milling qualities and high yield stability. The O. glumaepatula-derived ILs proved to be a source of new alleles for the development of high-yielding rice cultivars.

Agronomic and molecular characterization of introgression lines from the interspecific cross Oryza sativa (BG90-2) x Oryza glumaepatula (RS-16)

The reduced genetic variability of modern rice varieties (Oryza sativa) is of concern because it reduces the possibilities of genetic gain in breeding programs. Introgression lines (ILs) containing genomic fragments from wild rice can be used to obtain new improved cultivars. The objective of the present study was to perform the agronomic and molecular characterizations of 35 BC2F8 ILs from the cross O. glumaepatula x O. sativa, aiming to select high-yielding ILs to be used in rice-breeding programs. All 35 ILs were field evaluated in the season 2002/2003 in three locations and the 15 best performing ones were evaluated in the season 2003/2004 in five locations. In 2003/2004, six ILs (CNAi 9934, CNAi 9931, CNAi 9930, CNAi 9935, CNAi 9936, and CNAi 9937) showed the highest yield means and were statistically superior to the controls Metica 1 and IRGA 417. Molecular characterization of the 35 ILs was performed with 92 microsatellite markers distributed on the 12 rice chromosomes and a simple regression Oriza glumaepatula-derived introgression lines quantitative trait locus analysis was performed using the phenotypic data from 2002/2003. The six high-yielding ILs showed a low proportion of wild fragment introgressions. A total of 14 molecular markers were associated with quantitative trait loci in the three locations. The six high-yielding ILs were incorporated in the Embrapa breeding program, and the line CNAi 9930 is recommended for cultivation due to additional advantages of good grain cooking and milling qualities and high yield stability. The O. glumaepatula-derived ILs proved to be a source of new alleles for the development of high-yielding rice cultivars.

Testosterone directly induces progesterone production and interacts with physiological concentrations of LH to increase granulosa cell progesterone production in laying hens (Gallus domesticus).

Blocking testosterone action with immunization or with a specific antagonist blocks the preovulatory surge of progesterone and ovulation in laying hens. Thus, testosterone may stimulate progesterone production in a paracrine fashion within the ovary. To test this hypothesis, we evaluated the effects of testosterone and its interaction with LH on the production of progesterone by granulosa cells in culture. Hen granulosa cells obtained from preovulatory follicles were cultured in 96 well plates. The effects of testosterone (0-100ng/ml) and/or LH (0-100ng/ml) were evaluated. LH-stimulated progesterone production in a dose response manner up to 10ng/ml (p<0.01). Testosterone, up to 10ng/ml, increased progesterone production in a dose response manner in the absence of LH and at all doses of LH up to 1ng/ml (p<0.001). However, at supraphysiological concentrations of LH (10 and 100ng/ml) there was no further increase in progesterone production caused by testosterone (p>0.05). Finally, the addition of 2-hydroxyflutamide (0-1000mug/ml) to hen granulosa cells cultured with 10ng/ml of testosterone reduced progesterone production in a dose response manner (p<0.001). In conclusion, testosterone stimulates progesterone production in preovulatory follicle granulosa cells and interacts with physiological concentrations of LH to increase progesterone production. In addition, testosterone stimulation on granulosa cells is specific since the testosterone antagonist decreased testosterone stimulatory action.

Testosterone antagonist (flutamide) blocks ovulation and preovulatory surges of progesterone, luteinizing hormone and oestradiol in laying hens.

The preovulatory release of luteinizing hormone (LH) in the domestic hen occurs after the initiation of a preovulatory surge of testosterone. The objective of this study was to determine whether this testosterone surge has functional significance in the endocrine control of ovulation. Groups of laying hens (n = 10-22) were treated with the androgen receptor antagonist, flutamide, at 8 h intervals for 24 h at doses of 0, 31.25, 62.5, 125 and 250 mg. All doses reduced egg laying (P < 0.001), with the highest dose being the most effective. In a second study, laying hens (n = 9) were treated with 250 mg flutamide at 8 h intervals for 24 h with a control group being given placebo (n = 10). Blood samples were taken for hormone measurements at 2 h intervals for 18 h starting 4 h before the onset of darkness. The percentage of hens laying per day did not differ between groups before treatment (control, 88% vs flutamide, 86%). Ovulation was blocked in all hens treated with flutamide within 2 days while the control hens continued to lay at the pretreatment rate (80%). Preovulatory surges of plasma testosterone, progesterone, oestradiol and LH were observed in control hens but with the exception of testosterone, flutamide treatment blocked the progesterone, oestradiol and LH surges. LH concentrations declined progressively with time in the flutamide-treated hens. It is concluded that inhibition of testosterone action blocks egg laying and the preovulatory surges of progesterone, luteinizing hormone and oestradiol demonstrating a key role for the preovulatory release of testosterone in the endocrine control of ovulation in the domestic hen.

Testosterone immunization blocks the ovulatory process in laying hens without affecting ovarian follicular development.

The role of testosterone in the ovulatory process in hens has been largely neglected. The aim of the present study was to evaluate if testosterone plays an important role on the ovulatory process in laying hens. The effect of active and passive immunization against testosterone on ovarian follicular development and oviposition was studied. Egg laying percentage was evaluated in hens actively immunized against testosterone-BSA (T-AI; n = 6) or BSA (BSA-AI; n = 6). Oviposition was reduced as antibody titer increased in T-AI hens (r = -0.67; P < 0.01). Ovarian structures were assessed in three animals from each group. Follicles reached preovulatory size in both groups, nonetheless, in T-AI hens follicles at different stages of regression indicated that ovulation was blocked by treatment. In the remaining animals, preovulatory concentrations of progesterone and testosterone were determined. A preovulatory surge release of progesterone, preceded by a testosterone peak, was observed in the BSA-AI group (P < 0.05). In contrast, progesterone in T-AI animals remained at basal concentrations. Whereas, testosterone concentrations were significantly greater in T-AI as compared with BSA-AI animals (P < 0.05). Finally, to study the effect of passive immunization on oviposition, hens were passively immunized (PI) on four occasions, on alternate days with anti-T serum (T-PI; n = 10) or anti-BSA serum (BSA-PI; n = 8). During the 13-day period that preceded treatment, oviposition averaged 94.1%. Forty-eight hours after the first immunization, no egg was laid by 8 out of the 10 T-PI hens. During the 10 days following the first passive immunization, there was a reduction in the laying percentage that was significantly greater in T-PI hens (reduction of 52% in T-PI versus 29% in P-BSA, P < 0.01). In summary, these studies show that testosterone immunization hampers egg-laying without affecting ovarian follicular development, suggesting that testosterone has an important role in the ovulatory process in laying hens.

Characterization of five agricultural by-products as potential biofilter carriers.

Biofiltration is the most commonly used biological gas treatment technology and is extensively used for the treatment of polluted air with gas flow rates of up to 2 x 10(5) m(3)/h. It involves a filter bed of organic matter serving both as carrier for microorganisms and as nutrient supplier. Polluted gas passes through the filter bed and is cleaned by biological activity. Biofiltration is not being developed in Latin America as in the USA, Canada or Europe; the main reason probably being the absence of specific technology and of potential organic carriers locally available. Five different agriculture by-products available in Latin America: peanut shells, rice husk, coconut shells, cane bagasse and maize stubble, were chemically, physically and structurally characterized for their potential use as biofilter carrier. It was found that peanut shells could be used as biofilter carrier and therefore would have potential biological application.

Molecular and kinetic study of catalase-1, a durable large catalase of Neurospora crassa.

Catalase-1 (Cat-1), one of the two monofunctional catalases of Neurospora crassa, increases during asexual spore formation to constitute 0.6% of total protein in conidia. Cat-1 was purified 170-fold with a yield of 48% from conidiating cultures. Like most monofunctional catalases, Cat-1 is a homotetramer, resistant to inactivation by solvents, fully active over a pH range of 4-12, and inactivated by 3-amino-1,2,4-triazole. Unlike most monofunctional catalases, Cat-1 consists of 88 kDa monomers that are glycosylated with alpha-glucose and/or alpha-mannose, is unusually stable, and is not inactivated or inhibited by hydrogen peroxide. Cat-1 was more resistant than other catalases to heat inactivation and to high concentrations of salt and denaturants. Cat-1 exhibited unusual kinetics: at molar concentrations of hydrogen peroxide the apparent V was 10 times higher than at millimolar concentrations. Inactivation of Cat-1 activity with azide and hydroxylamine was according to first order kinetics, while cyanide at micromolar concentrations was a reversible competitive inhibitor.

Development and mapping of Oryza glumaepatula-derived microsatellite markers in the interspecific cross Oryza glumaepatula x O. sativa.

Wild germplasm of domesticated crops is a source of genetic variation little utilized in breeding programs. Interspecific crosses can potentially uncover novel gene combinations that can be important for quantitative trait analysis. The combined use of wide crosses and genetic maps of chromosomal regions associated with quantitative traits can be used to broaden the genetic basis of rice breeding programs. Oryza glumaepatula is a diploid (AA genome) wild rice species native from South and Central America. A genetic map was constructed with 162 PCR-based markers (155 microsatellite and 7 STS markers) using a backcross population derived from the cross O. glumaepatula, accession RS-16 from the Brazilian Amazon Region x O. sativa BG-90-2, an elite rice inbred line. The map included 47 new SSR markers developed from an O. glumaepatula genomic library enriched for AG/TC sequences. All SSR markers were able to amplify the O. sativa genome, indicating a high degree of SSR flanking region conservation between O. glumaepatula and O. sativa species. The map covered 1500.4 cM, with an average of one marker every 10 cM. Despite some chromosomes being more densely mapped, the overall coverage was similar to other maps developed for rice. The advantage to construct a SSR-based map is to permit the combination of the speed of the PCR reaction, and the codominant nature of the SSR marker, facilitating the QTL analysis and marker assisted selection for rice breeding programs.

Analysis of random and specific sequences of nuclear and cytoplasmic DNA in diploid and tetraploid american wild rice species (Oryza spp.).

A sample of American wild rice and other accessions of the genus Oryza were studied at polymorphic regions of nuclear, mitochondrial, and chloroplastic genomes. First, flow cytometry, genome-specific RAPD markers, and chromosome counting were utilized to verify the original ploidy and classification of 230 accessions studied. Based on these methods, 8% of the accessions were considered to be misclassified either taxonomically or as a result of contamination. Second, a fine resolution analysis was conducted at genomic regions sampled at random by RAPD markers and at specific sites of the chloroplast and mitochondrial DNA by cleaved amplified polymorphic sequence (CAPS) analysis. Phylogenetic trees resulting from phenetic and cladistic analyses of RAPD, cpDNA, and mtDNA polymorphisms were obtained. The results indicated that the American diploid species O. glumaepatula should be considered an individual species, distinct from O. rufipogon, and confirmed that the American tetraploid species (O. alta, O. grandiglumis, and O. latifolia) belong to the O. officinalis complex. The data indicate that these species should still be treated as a group rather than as three distinct species and that their closest relative is a CC-genome species. It was estimated that the diploid and tetraploid American species diverged from O. sativa - O. nivara (AA genome) and CC- and BBCC-genome species, respectively, 20 million years ago.

Double-outlet left ventricle. Echocardiographic diagnosis.

This is a case report of a double-outlet left ventricle associated with tricuspid atresia and hypoplasia of the right ventricle, diagnosed during echocardiography with color-flow imaging, in a three-month-old child who presented with fatigue and cyanosis. The child underwent palliative pulmonary arterial banding without an invasive procedure, and showed sustained improvement during follow-up.