Identification of passion fruit (Passiflora edulis) chromosomes using BAC-FISH.

Passiflora edulis, the yellow passion fruit, is the main crop from the Passiflora genus, which comprises 525 species with its diversity center in South America. Genetic maps and a BAC (bacterial artificial chromosome) genomic library are available, but the nine chromosome pairs of similar size and morphology (2n = 18) hamper chromosome identification, leading to different proposed karyotypes. Thus, the aim of this study was to establish chromosome-specific markers for the yellow passion fruit using single-copy and repetitive sequences as probes in fluorescent in situ hybridizations (FISH) to allow chromosome identification and future integration with whole genome data. Thirty-six BAC clones harboring genes and three retrotransposons (Ty1-copy, Ty3-gypsy, and LINE) were selected. Twelve BACs exhibited a dispersed pattern similar to that revealed by retroelements, and one exhibited subtelomeric distribution. Twelve clones showed unique signals in terminal or subterminal regions of the chromosomes, allowing their genes to be anchored to six chromosome pairs that can be identified with single-copy markers. The markers developed herein will provide an important tool for genomic and evolutionary studies in the Passiflora genus.

Activity of Antarctic fungi extracts against phytopathogenic bacteria.

This study aims to obtain secondary metabolites extracts from filamentous fungi isolated from soil and marine sediments from Antarctic ecosystems and to assess its potential antibacterial activity on Xanthomonas euvesicatoria and Xanthomonas axonopodis pv. passiflorae (phytopathogenic bacteria causing diseases in pepper and tomato and passionfruit, respectively). Among the 66 crude intracellular and extracellular extracts obtained from fungi recovered from soil and 79 obtained from marine sediment samples, 25 showed the ability to prevent the growth of X. euvesicatoria in vitro and 28 showed the ability to prevent the growth of X. axonopodis pv. passiflorae in vitro. Intracellular and extracellular extracts from soil fungi inhibited around 97% of X. euvesicatoria and 98% of X. axonopodis pv. passiflorae at 2·1 mg ml-1 . The average inhibition rates against X. euvesicatoria and X. axonopodis pv. passiflorae for intracellular and extracellular extracts from marine sediments fungi were around 96 and 97%, respectively, at 3·0 mg ml-1 . Extracts containing secondary metabolites with antimicrobial activity against X. euvesicatoria and X. axonopodis pv. passiflorae were obtained, containing possible substitutes for the products currently used to control these phytopathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Micro-organisms from extreme ecosystems, such as the Antarctic ecosystem, need to survive in harsh conditions with low temperatures, low nutrients and high UV radiation. Micro-organisms adapt to these conditions evolving diverse biochemical and physiological adaptations essential for survival. All this makes these micro-organisms a rich source of novel natural products based on unique chemical scaffolds. Discovering novel bioactive compounds is essential because of the rise in antibiotic-resistant micro-organisms and the emergence of new infections. Fungi from Antarctic environments have been proven to produce bioactive secondary metabolites against various micro-organisms, but few studies have shown activity against Xanthomonas phytopathogens.

Identification of Stylosanthes guianensis varieties using molecular genetic analysis.

BACKGROUND AND AIMS: The botanical classification of Stylosanthes guianensis is controversial, and few studies have used molecular markers to analyse this species. We used microsatellite markers to study the genetic diversity and population structure of S. guianensis and compare our results with the current infraspecific botanical classification. METHODOLOGY: A representative sample from the S. guianensis Brazilian germplasm collection (150 accessions) was analysed using 20 microsatellite loci. A model-based Bayesian approach implemented in the software STRUCTURE was used to assign accessions into clusters. A dendrogram was constructed based on Roger's genetic distances. PRINCIPAL RESULTS: The number of alleles per locus varied from 2 to 11, with an average of 4.7. The observed (H(O)) and expected (H(E)) heterozygosity values varied from 0 to 0.58 (mean of 0.18) and from 0.04 to 0.83 (mean of 0.55), respectively. Nine groups were assembled in STRUCTURE, and these groups were consistent with clusters inferred from the genetic distances and taxonomic varieties described for S. guianensis. The G(ST) among the nine groups was 0.46. CONCLUSIONS: The low H(O) and the G(ST) values observed are in agreement with the outcrossing rate (26 %) estimated for this species. The data indicate a high genetic diversity among and within the botanical varieties and suggest that microsatellite-based information can be combined with classical taxonomy to elucidate infraspecific levels.

Report of a Large Brazilian Family With a Very Attenuated Form of Hunter Syndrome (MPS II).

Hunter syndrome, or Mucopolysaccharidosis type II (MPS II), is a rare X-linked recessive disorder caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). The phenotypic spectrum varies from severe to attenuated clinical forms. We report a large Brazilian family with 16 affected individuals exhibiting a very attenuated form of MPS II. Fourteen female carriers were also identified. Twelve affected male patients, whose ages ranged from 1 to 35 years, were examined. Molecular analysis showed a novel missense mutation (p.A77D) in the IDS gene, confirming the diagnosis. Nine of the family members presented some degree of heart damage, though only the proband became symptomatic and required heart transplantation. One 19-year-old adult and 1-year-old twin boys each had a normal echocardiogram. Short stature was found in two adults while macrocephaly was found in one; the remaining adults had anthropometric measures within normal range. All affected adults had normal cognitive development and were able to perform normal daily activities, except one who had mild learning disability. Two patients died due to natural causes beyond 70 years of age. The female carriers did not present any signs of disease. In this large family with a mild form of MPS II and variable degree of clinical manifestations, it is noteworthy that several affected individuals have remained asymptomatic even at advanced age and even without enzyme replacement therapy.

Genetic Variability of Beauveria bassiana and a DNA Marker for Environmental Monitoring of a Highly Virulent Isolate Against Cosmopolites sordidus.

The banana weevil Cosmopolites sordidus (Germar) is one of a number of pests that attack banana crops. The use of the entomopathogenic fungus Beauveria bassiana as a biological control agent for this pest may contribute towards reducing the application of chemical insecticides on banana crops. In this study, the genetic variability of a collection of Brazilian isolates of B. bassiana was evaluated. Samples were obtained from various geographic regions of Brazil, and from different hosts of the Curculionidae family. Based on the DNA fingerprints generated by RAPD and AFLP, we found that 92 and 88 % of the loci were polymorphic, respectively. The B. bassiana isolates were attributed to two genotypic clusters based on the RAPD data, and to three genotypic clusters, when analyzed with AFLP. The nucleotide sequences of nuclear ribosomal DNA intergenic spacers confirmed that all isolates are in fact B. bassiana. Analysis of molecular variance showed that variability among the isolates was not correlated with geographic origin or hosts. A RAPD-specific marker for isolate CG 1024, which is highly virulent to C. sordidus, was cloned and sequenced. Based on the sequences obtained, specific PCR primers BbasCG1024F (5'-TGC GGC TGA GGA GGA CT-3') and BbasCG1024R (5'-TGC GGC TGA GTG TAG AAC-3') were designed for detecting and monitoring this isolate in the field.

Genetic diversity and a PCR-based method for Xanthomonas axonopodis detection in passion fruit.

Xanthomonas axonopodis pv. passiflorae causes bacterial spot in passion fruit. It attacks the purple and yellow passion fruit as well as the sweet passion fruit. The diversity of 87 isolates of pv. passiflorae collected from across 22 fruit orchards in Brazil was evaluated using molecular profiles and statistical procedures, including an unweighted pair-group method with arithmetical averages-based dendrogram, analysis of molecular variance (AMOVA), and an assigning test that provides information on genetic structure at the population level. Isolates from another eight pathovars were included in the molecular analyses and all were shown to have a distinct repetitive sequence-based polymerase chain reaction profile. Amplified fragment length polymorphism technique revealed considerable diversity among isolates of pv. passiflorae, and AMOVA showed that most of the variance (49.4%) was due to differences between localities. Cluster analysis revealed that most genotypic clusters were homogeneous and that variance was associated primarily with geographic origin. The disease adversely affects fruit production and may kill infected plants. A method for rapid diagnosis of the pathogen, even before the disease symptoms become evident, has value for producers. Here, a set of primers (Xapas) was designed by exploiting a single-nucleotide polymorphism between the sequences of the intergenic 16S-23S rRNA spacer region of the pathovars. Xapas was shown to effectively detect all pv. passiflorae isolates and is recommended for disease diagnosis in passion fruit orchards.

Impact of continuous positive airway pressure treatment on left atrial volume and function in patients with obstructive sleep apnoea assessed by real-time three-dimensional echocardiography.

BACKGROUND: Obstructive sleep apnoea (OSA) has been reported as a predictor of left ventricle (LV) diastolic dysfunction and left atrium (LA) remodelling. The aim of this study is to evaluate the impact of OSA treatment with a continuous positive airway pressure device (CPAP) on the LA volume and function, as well as on the LV diastolic function. METHODS: In total, 56 OSA patients were studied. All patients underwent real-time three-dimensional (RT3DE) and two-dimensional echocardiogram with tissue Doppler evaluation in order to estimate LA volumes, function and LV diastolic performance. A total of 30 patients with an apnoea-hypopnoea index greater than 20 were randomly selected to receive sham CPAP (n = 15) or effective CPAP (n = 15) for 24 weeks. They underwent echo examination on three different occasions: at baseline, after 12 weeks and 24 weeks of CPAP or sham CPAP. RESULTS: In the effective CPAP group we observed the following changes from the baseline to the 24-week echo evaluation: (a) a reduction in the E/E' ratio (10.3 (1.9) to 7.9 (1.3), p = 0.03); (b) an increase in the LA passive emptying fraction (28.8% (11.9%) to 46.8% (9.3%), p = 0.01); and (c) a reduction in the LA active emptying fraction (42.7% (11.5%) to 25.7 (15.7), p<0.01). In the sham group, there were no changes from the baseline to the 24-week echo. We found a positive correlation between 24 week/baseline LA active emptying volume and 24 week/baseline E/E' ratios (r = 0.40, p<0.05) and a negative correlation between 24 week/baseline LA passive emptying volume and 24 week/baseline E/E' ratios (r = -0.53, p<0.05). No significant changes were found on LA total emptying fraction. CONCLUSION: CPAP improved LV diastolic function and LA passive emptying, but not LA structural variables in OSA patients. TRIAL REGISTRATION NUMBER: NCT00768807.

Resistance to Passion fruit woodiness virus in Transgenic Passionflower Expressing the Virus Coat Protein Gene.

We report the use of the coat protein (CP) gene from Passion fruit woodiness virus (PWV) to produce resistant transgenic plants of yellow passion fruit. A full-length CP gene from a severe PWV isolate from the state of São Paulo, Brazil (PWV-SP) was cloned into pCAMBIA 2300 binary vector, which was further introduced into Agrobacterium tumefaciens strain EHA 105. Leaf disks were used as explants for transformation assays, e.g., 2,700 and 2,730 disks excised from plants from the Brazilian cultivars IAC-275 and IAC-277, respectively. In vitro selection was performed in kanamycin. After transferring to the elongation medium, 119 and 109 plantlets of IAC-275 and IAC-277, respectively, were recovered. Integration of the PWV CP gene was confirmed in seven of eight plants evaluated by Southern blot analysis, showing different numbers of insertional events for the CP gene. Three transgenic plants (T3, T4, and T7) expressed the expected transcript, but the 32 kDa PWV CP was detected by Western blot in only two plants (T3 and T4). The results of three successive mechanical inoculations against the transgenic plants using three PWV isolates showed that the primary transformant T2 of IAC-277 was immune to all isolates.

Repeated echocardiographic examinations of patients with suspected infective endocarditis.

OBJECTIVE: To study the diagnostic contribution of repeated transthoracic (TTE) and transoesophageal echocardiography (TOE) among patients with suspected infective endocarditis. METHODS: 262 patients with 266 episodes of suspected infective endocarditis were referred for TTE and TOE over three years in a 423 bed university cardiology hospital. Patients were a mean (SD) of 47.6 (17.9) years old. 139 (52.3%) episodes occurred in men and 127 (47.7%) in women. The diagnostic information obtained from repeated TTE and TOE examinations was evaluated relative to the diagnosis of endocarditis. RESULTS: TTE examinations were repeated in 192 (72.2%) and TOE examinations were repeated in 49 (18.4%) of 266 episodes. A mean of 2.4 TTE and 1.2 TOE examinations were performed for each episode of suspected endocarditis. The second and third TTEs added diagnostic information in 34 (26.7%) and the second and third TOEs added diagnostic information in 25 (19.7%) of 127 episodes with definite endocarditis. After the third TTE or TOE no additional diagnostic information was obtained. CONCLUSIONS: The diagnostic contribution of repeated TTE or TOE for the diagnosis of endocarditis decreased as the number of repetitions increased. In this setting, the data do not substantiate more than three TTE or TOE examinations as an efficient strategy to increase the diagnostic yield for all but selected patients with suspected endocarditis.

Cryopreservation of Arachis species by vitrification of in vitro-grown shoot apices and genetic stability of recovered plants.

A storage protocol at cryogenic temperature was established for shoot apices from in vitro plants of the cultivated groundnut (Arachis hypogaea) and wild Arachis species (A. retusa and A. burchellii) using a basic vitrification protocol with direct immersion in liquid nitrogen (LN). The effect of pre-treatments of donor-plants with ABA as well as of different supplements in the post-thaw culture medium was studied. After rapid warming at 40 C, the explants were cultured on MS medium devoid of growth regulators (MS0) or MS supplemented with 4.4(M benzylaminopurine (BAP) and 0.5(M naphthalene acetic acid (NAA) plus 5(M silver nitrate (AgNO3), 0.25% polyvinylpyrrolidone (PVP) or 0.2% activated charcoal. Non-frozen explants from the three species formed one shoot through meristematic amplification when cultured on MS0 medium. These explants also developed callus on MS supplemented with growth regulators (4.4(M BAP and 0.5(M NAA) alone or plus PVP or AgNO3. Callus formation was suppressed in the presence of activated charcoal. Post-thaw regeneration ocurred only through indirect organogenesis on media containing AgNO3 or PVP. Preculturing on medium supplemented with abscisic acid (ABA) improved regrowth rate in these media. Recovery failed to occur in the presence of activated charcoal. The genetic stability of shoots of A. burchellii originated from shoot apices was analyzed through Random Amplified Polymorphic DNA (RAPD) markers.

Factors influencing electroporation-mediated gene transfer to Stylosanthes guianensis (Aubl.) Sw. protoplasts

In order to develop a high-efficiency and reproducible transformation protocol for Stylosanthes guianensis we assessed the biological and physical parameters affecting plant electroporation protoplasts. Energy input, as combinations of electric field strengths discharged by different capacitors, electroporation buffer and DNA form were evaluated. Transformation efficiency was assayed in vivo as transient reporter gene expression, using the GFP-coding gene mgfp5 driven by a CaMV 35S constitutive promoter. Energy input and electric field strength had a critical influence on transgene expression with higher transformation levels being achieved with 250 V.cm-1 discharged by 900 and 1000 muF capacitors. Linear plasmid DNA, the absence of chloride and the presence of calcium ions also increased transient gene expression, albeit not significantly

Trombose em artérias coronárias normais causando infarto agudo do miocárdio em pacientes alcoólatra / Thrombosis in normal coronary arteries causing acute myocardial infarction in an alcoholic patient

Paciente jovem, etilista, que sofreu infarto agudo do miocárdio. Após terapêutica trombolítica sem sucesso, a cineangiocoronariografia na fase aguda evidenciava trombos em múltiplas artérias. Um 2§ CATE, realizado no 7§ dia de internação revelou coronárias normais. Não havia nenhum distúrbio do metabolismo lipídico, assim como outro fator de risco de doença arterial coronária, a não ser o fato de ser tabagista de oito a 10 cigarros ao dia, há dois anos, o que nos levou a acreditar que o abuso de álcool etílico possa ter contribuído para esse evento.