Potential sites of triple-helical nucleic acid formation in chromosomes of Rhynchosciara (Diptera: Sciaridae) and Drosophila melanogaster

Antibodies to specific nucleic acid conformations are amongst the methods that have allowed the study of non-canonical (Watson-Crick) DNA structures in higher organisms. In this work, the structural limitations for the immunological detection of DNA.RNA hybrid duplexes were examined using specific RNA homopolymers as probes for homopolymer polydeoxyadenylic acid (poly(dA)).polydeoxythymidylic acid (poly(dT))-rich regions of Rhynchosciara americana (Diptera: Sciaridae) chromosomes. Anti-DNA.RNA duplexes did not react with the complex formed between chromosomal poly(dA) and exogenous polyuridylic acid (poly(rU)). Additionally, poly(rU) prevented the detection of polyadenylic acid.poly(dT) hybrid duplexes preformed in situ. These results raised the possibility that three-stranded structures rather than duplexes were formed in chromosomal sites. To test this hypothesis, the specificity of antibodies to triple-helical nucleic acids was reassessed employing distinct nucleic acid configurations. These antibodies were raised to the poly(dA).poly(rU).poly(rU) complex and have been used here for the first time in immunocytochemistry. Anti-triplex antibodies recognised the complex poly(dA).poly(rU).poly(rU) assembled with poly(rU) in poly(dA).poly(dT)-rich homopolymer regions of R. americana chromosomes. The antibodies could not detect short triplex stretches, suggesting the existence of constraints for triple-helix detection, probably related to triplex tract length. In addition, anti-poly(dA).poly(rU).poly(rU) antibodies reacted with the pericentric heterochromatin of RNase-treated polytene chromosomes of R. americana and Drosophila melanogaster. In apparent agreement with data obtained in cell types from other organisms, the results of this work suggest that significant triple-helix DNA extensions can be formed in pericentric regions of these species.

The DNA puff 4C expresses a salivary secretion protein in Trichosia pubescens (Diptera; Sciaridae)

DNA puffs are genomic regions of polytene chromosomes that undergo developmentally controlled DNA amplification and transcription in salivary glands of sciarid flies. Here, we tested the hypothesis that DNA puff genes code for salivary proteins in Trichosia pubescens. To do that, we generated antibodies against saliva and immunoscreened a cDNA library made from salivary glands. We isolated clones corresponding to DNA puff regions, including clone D-50 that contained the entire coding sequence of the previously isolated C4B1 gene from puff 4C. Indeed, we showed that puff 4C is a DNA puff region detecting its local transcription and its extra rounds of DNA incorporation compared to neighboring regions. We further confirmed D-50 clone identity in Western blots reacted with the anti-saliva anitiserum. We detected a recombinant protein expressed by this clone that had the expected size for a full-length product of the gene. We end with a discussion of the relationship between DNA puff genes and their products.

Nonrecurrent MECP2 duplications mediated by genomic architecture-driven DNA breaks and break-induced replication repair

Recurrent submicroscopic genomic copy number changes are the result of nonallelic homologous recombination (NAHR). Nonrecurrent aberrations, however, can result from different nonexclusive recombination-repair mechanisms. We previously described small microduplications at Xq28 containing MECP2 in four male patients with a severe neurological phenotype. Here, we report on the fine-mapping and breakpoint analysis of 16 unique microduplications. The size of the overlapping copy number changes varies between 0.3 and 2.3 Mb, and FISH analysis on three patients demonstrated a tandem orientation. Although eight of the 32 breakpoint regions coincide with low-copy repeats, none of the duplications are the result of NAHR. Bioinformatics analysis of the breakpoint regions demonstrated a 2.5-fold higher frequency of Alu interspersed repeats as compared with control regions, as well as a very high GC content (53%). Unexpectedly, we obtained the junction in only one patient by long-range PCR, which revealed nonhomologous end joining as the mechanism. Breakpoint analysis in two other patients by inverse PCR and subsequent array comparative genomic hybridization analysis demonstrated the presence of a second duplicated region more telomeric at Xq28, of which one copy was inserted in between the duplicated MECP2 regions. These data suggest a two-step mechanism in which part of Xq28 is first inserted near the MECP2 locus, followed by breakage-induced replication with strand invasion of the normal sister chromatid. Our results indicate that the mechanism by which copy number changes occur in regions with a complex genomic architecture can yield complex rearrangements.

CONSIDERATIONS ON THE REINTRODUCTION AND RECOVERY OF THE ALAGOAS CURASSOW MITU MITU (LINNAEUS, 1766) FROM EXTINCTION USING A POTENTIALLY HYBRID CAPTIVE STOCK

The Alagoas Curassow Mitu mitu is considered extinct in the wild. Since 1979, two females and a male caught in the wild have bred successfully in captivity, and, in 1990, hybridizations between M. mitu and Razor-billed Mitu M. tuberosum were performed. By June 2008, there were around 130 living birds in two different aviaries. We sequenced two regions of the mitochondrial DNA of both captive stocks of Alagoas Curassows. We unequivocally identified hybrids that have haplotype typical of M. tuberosum. However, unless the original studbook can be recovered there is no confident way to discriminate ""pure"" M. mitu birds for breeding and reintroduction purposes. Allied with morphological data gathered in an independent study, we suggest that conservation actions need to focus on specimens with diagnostic phenotypic characters of M. mitu, and avoid birds with mitochondria, genetic contribution of M. tuberosum. Although we have detected low levels of genetic variability among captive birds, the steady increase of the captive population suggests that inbreeding depression and hybridization are not a reproductive hindrance. Reintroduction of some of these potential hybrid birds in the original area of occurrence of the Alagoas Curassow may be the only hope to fill in the ecological niche left vacant. An educational program involving local communities to conserve future reintroduction of curassows and their restored habitat is highly recommended. Accepted 12 November 2009.

Confinement-induced phase transition in a DNA-lipid hydrated complex

We study the effect of the soft confinement by fluid lipid bilayers on the spatial organisation of DNA molecules in a DNA-zwitterionic lipid hydrated lamellar complex. The confinement is increased by dehydrating the complex in a controlled way, which leads to a decrease of the water channel thickness separating the periodically stacked bilayers. Using grazing-incidence small-angle X-ray scattering on an oriented thin film, we probe in situ as dehydration proceeds the structure of the DNA-lipid complex. A structural phase transition is evidenced, where an apparently disordered phase of DNA rods embedded within the one-dimensionally ordered lipid lamellar phase observed at high hydration is replaced by a 2D hexagonal structure of DNA molecules intercalated between the lipid bilayers. Copyright (C) EPLA, 2010

Microgranitic Enclaves as Products of Self-mixing Events: a Study of Open-system Processes in the Maua Granite, Sao Paulo, Brazil, Based on in situ Isotopic and Trace Elements in Plagioclase

Felsic microgranular enclaves with structures indicating that they interacted in a plastic state with their chemically similar host granite are abundant in the Maua Pluton, SE Brazil. Larger plagioclase xenocrysts are in textural disequilibrium with the enclave groundmass and show complex zoning patterns with partially resorbed An-rich cores (locally with patchy textures) surrounded by more sodic rims. In situ laser ablation-(multi-collector) inductively coupled plasma mass spectrometry trace element and Sr isotopic analyses performed on the plagioclase xenocrysts indicate open-system crystallization; however, no evidence of derivation from more primitive basic melts is observed. The An-rich cores have more radiogenic initial Sr isotopic ratios that decrease towards the outermost part of the rims, which are in isotopic equilibrium with the matrix plagioclase. These profiles may have been produced by either (1) diffusional re-equilibration after rim crystallization from the enclave-forming magma, as indicated by relatively short calculated residence times, or (2) episodic contamination with a decrease of the contaminant ratio proportional to the extent to which the country rocks were isolated by the crystallization front. Profiles of trace elements with high diffusion coefficients would require unrealistically long residence times, and can be modeled in terms of fractional crystallization. A combination of trace element and Sr isotope data suggests that the felsic microgranular enclaves from the Maua Pluton are the products of interaction between end-member magmas that had similar compositions, thus recording `self-mixing` events.

Factors associated with increased prevalence of human papillomavirus infection in a cohort of HIV-Infected Brazilian women

Objectives: Human papillomavirus (HPV) infection is a major risk factor for cervical disease. Using baseline data from the HIV-infected cohort of Evandro Chagas Clinical Research Institute at Fiocruz, Rio de Janeiro, Brazil, factors associated with an increased prevalence of HPV were assessed. Methods: Samples from 634 HIV-infected women were tested for the presence of HPV infection using hybrid capture 11 and polymerase chain reaction. Prevalence ratios (PR) were estimated using Poisson regression analysis with robust variance. Results: The overall prevalence of HPV infection was 48%, of which 94% were infected with a high-risk HPV. In multivariate analysis, factors independently associated with infection with high-risk HPV type were: younger age (<30 years of age; PR 1.5, 95% confidence interval (CI) 1.1-2.1), current or prior drug use (PR 1.3, 95% CI 1.0-1.6), self-reported history of HPV infection (PR 1.2, 95% CI 0.96-1.6), condom use in the last sexual intercourse (PR 1.3, 95% CI 1.1-1.7), and nadir CD4+ T-cell count <100 cells/mm(3) (PR 1.6, 95% CI 1.2-2.1). Conclusions: The estimated prevalence of high-risk HPV-infection among HIV-infected women from Rio de Janeiro, Brazil, was high. Close monitoring of HPV-related effects is warranted in all HIV-infected women, in particular those of younger age and advanced immunosuppression. (C) 2008 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Recovery of DNA for the detection and genotyping of human papillomavirus from clinical cervical specimens stored for up to 2 years in a universal collection medium with denaturing reagent

The recovery and stability of DNA for the detection and genotyping of HPV in UCM-containing specimens, after exposure to denaturing reagents and stored for up to 2 years were evaluated. Samples were collected from 60 women who had cervical cytology specimens harboring cervical intraepithelial neoplasia (CIN) 2 or 3. All samples were stored in UCM and had been frozen at -20 degrees C following the addition of the denaturing reagent (sodium hydroxide) and the removal of the aliquot required for Hybrid Capture 2 testing for the identification of HPV DNA. The samples had been stored for 6, 12 and 24 months (20 samples for each storage time). HPV DNA extraction was performed according to a protocol designed specifically and the presence and quality of DNA was confirmed by human P-globin detection using the consensus primers G73 and G74. HPV DNA was amplified using the consensus primers PGMY09 and PGMY11, and reverse line-blot hybridization was used to detect type-specific amplicons for 37 HPV types. The DNA extracted from the denatured specimen was recovered in 57/60 (95%) of the samples. HPV DNA was detected in 56/57 (98%) of the recovered samples. Twenty-six of the 56 samples recovered (48%) were genotyped successfully. (c) 2007 Elsevier B.V. All rights reserved.

Network genealogy of 195-bp satellite DNA supports the superimposed hybridization hypothesis of Trypanosoma cruzi evolutionary pattern

Trypanosoma cruzi is highly diverse genetically and has been partitioned into six discrete typing units (DTUs), recently re-named T. cruzi I-VI. Although T. cruzi reproduces predominantly by binary division, accumulating evidence indicates that particular DTUs are the result of hybridization events. Two major scenarios for the origin of the hybrid lineages have been proposed. It is accepted widely that the most heterozygous TcV and TcVI DTUs are the result of genetic exchange between TcII and TcIII strains. On the other hand, the participation of a TcI parental in the current genome structure of these hybrid strains is a matter of debate. Here, sequences of the T. cruzi-specific 195-bp satellite DNA of TcI, TcII, Tat, TcV, and TcVI strains have been used for inferring network genealogies. The resulting genealogy showed a high degree of reticulation, which is consistent with more than one event of hybridization between the Tc DTUs. The data also strongly suggest that Tat is a hybrid with two distinct sets of satellite sequences, and that genetic exchange between TcI and TcII parentals occurred within the pedigree of the TcV and TcVI DTUs. Although satellite DNAs belong to the fast-evolving portion of eukaryotic genomes, in >100 satellite units of nine T. cruzi strains we found regions that display 100% identity. No DTU-specific consensus motifs were identified, inferring species-wide conservation. (C) 2010 Elsevier B.V. All rights reserved.

Structure effects of self-assembled Prussian blue confined in highly organized mesoporous TiO(2) on the electrocatalytic properties towards H(2)O(2) detection

Here we report the derivatization of mesoporous TiO(2) thin films for the preparation of H(2)O(2) amperometric sensors. The coordination of the bifunctional ligand 1,10 phenantroline, 5,6 dione on the surface Ti(IV) ions provides open coordination sites for Fe(II) cations which are the starting point for the growth of a layer of Prussian blue polymer. The porous structure of the mesoporous TiO(2) allows the growth, ion by ion of the coordination polymer. Up to four layer of Prussian blue can be deposit without losing the porous structure of the film, which results in an enhanced response of these materials as H(2)O(2) sensors. These porous confined PB modified electrodes are robust sensors that exhibit good reproducibility, environmental stability and high sensitivity towards H(2)O(2) detection. (C) 2010 Elsevier B.V. All rights reserved.

Supramolecular Approach to Gold Nanoparticle/Triruthenium Cluster Hybrid Materials and Interfaces

A systematic and comprehensive study of the interaction of citrate-stabilized gold nanoparticles with triruthenium cluster complexes of general formula [Ru(3)(CH(3)COO)(6)(L)](+) [L = 4-cyanopyridine (4-CNpy), 4,4`-bipyridine (4,4`-bpy) or 4,4`-bis(pyridyl)ethylene (bpe)] has been carried out. The cluster-nanoparticle interaction in solution and the construction of thin films of the hybrid materials were investigated in detail by electronic and surface plasmon resonance (SPR) spectroscopy, Raman scattering spectroscopy and scanning electron microscopy (SEM). Citrate-stabilized gold nanoparticles readily interacted with [Ru(3)O(CH(3)COO)(6)(L)(3)](+) complexes to generate functionalized nanoparticles that tend to aggregate according to rates and extents that depend on the bond strength defined by the characteristics of the cluster L ligands following the sequence bpe > 4,4`-bpy >> 4-CNpy. The formation of compact thin films of hybrid AuNP/[Ru(3)O(CH(3)COO)(6)(L)(3)](+) derivatives with L = bpe and 4,4`-bpy indicated that the stability/lability of AuNP-cluster bonds as well as their solubility are important parameters that influence the film contruction process. Fluorine-doped tin oxide electrodes modified with thin films of these nanomaterials exhibited similar electrocatalytic activity but much higher sensitivity than a conventional gold electrode in the oxidation of nitrite ion to nitrate depending on the bridging cluster complex, demonstrating the high potential for the development of amperometric sensors.