Prevalence of human papillomavirus DNA in female cervical lesions from Rio de Janeiro, Brazil

A hundred-sixty paraffin-embedded specimens from female cervical lesions were examined for human papillomavirus (HPV) types 6, 11, 16 and 18 infections by non-isotopic in situ hybridization. The data were compared with histologic diagnosis. Eighty-eight (55) biopsies contained HPV DNA sequences. In low grade cervical intraepithelial neoplasias (CIN I), HPV infection was detected in 78.7 of the cases, the benign HPV 6 was the most prevalent type. HPV DNA was detected in 58 of CIN II and CIN III cases and in 41.8 of squamous cell carcinomas (SCC). Histologically normal women presented 20 of HPV infection. Oncogenic HPV was found in 10 of these cases, what may indicate a higher risk of developing CINs and cancer. Twenty-five percent of the infected tissues contained mixed infections. HPV 16 was the most common type infecting the cervix and its prevalence raised significantly with the severity of the lesions, pointing its role in cancer pathogenesis. White women presented twice the cervical lesions of mulatto and African origin women, although HPV infection rates were nearly the same for the three groups (approximately 50). Our results showed that HPV typing by in situ hybridization is a useful tool for distinguishing between low and high risk cervical lesions. Further studies are required to elucidate risk factors associated with HPV infection and progression to malignancy in Brazilian population.

Avaluació de la capacitat de segrest de carboni d'un sòl restaurat amb fangs de depuradora: paper de les propietats físiques del sòl

S’ha estudiat el grau de protecció física del carboni dins d’agregats de diferents mides en un sòl d’una pedrera restaurada fa 17 anys amb terres adobades amb fangs de depuradora, i s’ha interpretat el seu paper en el context del segrest de carboni en el sòl. La metodologia aplicada es basa en la d’humitejament ràpid dels agregats del sòl per immersió en aigua (Le Bissonnais, 1996), que simula l’estabilitat d'un sòl sec que es veu sotmès a processos tals com la inundació local o saturació ràpida. També s’ha determinat la quantitat de carboni oxidable present en els agregats del sòl amb el mètode de Nelson i Sommers (1982). Els resultats han mostrat que l’adobat amb fangs de depuradora contribueix a augmentar el contingut de carboni orgànic en els agregats del sòl i n’estimula el segrest a mitjà termini (unes 10 tones ha-1 en 17 anys), aportant estabilitat al sòl i protegint físicament el carboni orgànic dins dels agregats de mida major (5-2 mm). A més s’ha constatat que per determinar l’estabilitat del carboni segrestat en el sòl cal conèixer com es distribueix entre les diferents mides d’agregats. Finalment, l’augment del segrest de carboni en el sòl propiciat per l’aplicació dels fangs de depuradora li dóna més capacitat per fixar CO2 atmosfèric.

DNA polymorphism of schistosomes and their snail hosts

Analysis of the genomes of schistosomes and one of their intermediate hosts, Biomphalaria glabrata, using Random Amplified Polymorphic DNA (RAPD) demonstrated that intraspecific genetic polymorphism in the parasite is limited but in the snail is highly pronounced. This suggests an important role for the snail in the determination of the epidemiology of the disease. In addition to their intraspecific stability, schistosome derived RAPDs exhibit a high level of interspecific polymorphism and are thus ideal for the construction of phylogenetic trees. For the detection of intraspecific polymorphisms extensive variation in the mitochondrial DNA is being exploited for the development of a PCR based test for Schistosoma mansoni. Gene level polymorphisms are being analyzed by Low Stringency Single Specific Primer PCR.

Development of specific ITS markers for plant DNA identification within herbivorous insects

DNA based techniques have proved to be very useful methods to study trophic relationships 17 between pests and their natural enemies. However, most predators are best defined as omnivores, 18 and the identification of plant-specific DNA should also allow the identification of the plant 19 species the predators have been feeding on. In this study, a PCR approach based on the 20 development of specific primers was developed as a self-marking technique to detect plant DNA 21 within the gut of one heteropteran omnivorous predator (Macrolophus pygmaeus) and two 22 lepidopteran pest species (Helicoverpa armigera and Tuta absoluta). Specific tomato primers 23 were designed from the ITS 1-2 region, which allowed the amplification of a tomato DNA 24 fragment of 332 bp within the three insect species tested in all cases (100% of detection at t = 0) 25 and did not detect DNA of other plants nor of the starved insects. Plant DNA half-lives at 25ºC 26 ranged from 5.8h, to 27.7h and 28.7h within M. pygmaeus, H. armigera and T. absoluta, 27 respectively. Tomato DNA detection within field collected M. pygmaeus suggests dietary mixing 28 in this omnivorous predator and showed a higher detection of tomato DNA in females and 29 nymphs than males. This study provides a useful tool to detect and to identify plant food sources 30 of arthropods and to evaluate crop colonization from surrounding vegetation in conservation 31 biological control programs.

Identification of species related to Anopheles (Nyssorhynchus) albitarsis by random amplified polymorphic DNA-Polymerase chain reaction (Diptera: Culicidae)

Species-specific Random Amplified Polymorphic DNA-Polymerase chain Reaction (RAPD-PCR) markers were used to identify four species related to Anopheles (Nyssorhynchus) albitarsis Lynch-Arribàlzaga from 12 sites in Brazil and 4 in Venezuela. In a previous study (Wilkerson et al. 1995), which included sites in Paraguay and Argentina, these four species were designated "A", "B", "C" and "D". It was hypothesized that species A is An. (Nys.) albitarsis, species B is undescribed, species C is An. (Nys) marajoara Galvão and Damasceno and species D is An. (Nys.) deaneorum Rosa-Freitas. Species D, previously characterized by RAPD-PCR from a small sample from northern Argentina and southern Brazil, is reported here from the type locality of An. (Nys.) deaneorum, Guajará-Mirim, state of Rondônia, Brazil. Species C and D were found by RAPD-PCR to be sympatric at Costa Marques, state of Rondônia, Brazil. Species A and C have yet to be encountered at the same locality. The RAPD markers for species C were found to be conserved over 4,620 km; from Iguape, state of São Paulo, Brazil to rio Socuavo, state of Zulia, Venezuela. RAPD-PCR was determined to be an effective means for the identification of unknown species within this species complex.

Early DNA vaccination of puppies against canine distemper in the presence of maternally derived immunity.

Canine distemper (CD) is a disease in carnivores caused by CD virus (CDV), a member of the morbillivirus genus. It still is a threat to the carnivore and ferret population. The currently used modified attenuated live vaccines have several drawbacks of which lack of appropriate protection from severe infection is the most outstanding one. In addition, puppies up to the age of 6-8 weeks cannot be immunized efficiently due to the presence of maternal antibodies. In this study, a DNA prime modified live vaccine boost strategy was investigated in puppies in order to determine if vaccinated neonatal dogs induce a neutralizing immune response which is supposed to protect animals from a CDV challenge. Furthermore, a single DNA vaccination of puppies, 14 days after birth and in the presence of high titers of CDV neutralizing maternal antibodies, induced a clear and significant priming effect observed as early as 3 days after the subsequent booster with a conventional CDV vaccine. It was shown that the priming effect develops faster and to higher titers in puppies preimmunized with DNA 14 days after birth than in those vaccinated 28 days after birth. Our results demonstrate that despite the presence of maternal antibodies puppies can be vaccinated using the CDV DNA vaccine, and that this vaccination has a clear priming effect leading to a solid immune response after a booster with a conventional CDV vaccine.

A protective cocktail vaccine against murine cutaneous leishmaniasis with DNA encoding cysteine proteinases of Leishmania major.

The protection elicited by the intramuscular injection of two plasmid DNAs encoding Leishmania major cysteine proteinase type I (CPb) and type II (CPa) was evaluated in a murine model of experimental cutaneous leishmaniasis. BALB/c mice were immunized either separately or with a cocktail of the two plasmids expressing CPa or CPb. It was only when the cpa and cpb genes were co-injected that long lasting protection against parasite challenge was achieved. Similar protection was also observed when animals were first immunized with cpa/cpb DNA followed by recombinant CPa/CPb boost. Analysis of the immune response showed that protected animals developed a specific Th1 immune response, which was associated with an increase of IFN-gamma production. This is the first report demonstrating that co-injection of two genes expressing different antigens induces a long lasting protective response, whereas the separate injection of cysteine proteases genes is not protective.

Questioning the utility of RNA-DNA chimera in gene repair.

In principle, we should be glad that Eric Kmiec and his colleagues published in Science's STKE (1) a detailed experimental protocol of their gene repair method (2, 3). However, a careful reading of their contribution raises more doubts about the method. The research published in Science five years ago by Kmiec and his colleagues was said to demonstrate that chimeric RNA-DNA oligonucleotides could correct the mutation responsible for sickle cell anemia with 50% efficiency (4). Such a remarkable result prompted many laboratories to attempt to replicate the research or utilize the method on their own systems. However, if the method worked at all, which it rarely did, the achieved efficiency was usually lower by several orders of magnitude. Now, in the Science's STKE protocol, we are given crucial information about the method and why it is so important to utilize these expensive chimeric RNA-DNA constructs. In the introduction we are told that the RNA-DNA duplex is more stable than a DNA-DNA duplex and so extends the half-life of the complexes formed between the targeted DNA and the chimeric RNA-DNA oligonucleotides. This logical explanation, however, conflicts with the statement in the section entitled "Transfection with Oligonucleotides and Plasmid DNA" that Kmiec and colleagues have recently demonstrated that classical single-stranded DNA oligonucleotides with a few protective phosphothioate linkages have a "gene repair conversion frequency rivaling that of the RNA/DNA chimera". Indeed, the research cited for that result actually states that single-stranded DNA oligonucleotides are in fact several-fold more efficient (3.7-fold) than the RNA-DNA chimeric constructs (5). If that is the case, it raises the question of why Kmiec and colleagues emphasize the importance of the RNA in their original chimeric constructs. Their own new results show that modified single-stranded DNA oligonucleotides are more effective than the expensive RNA-DNA hybrids. Moreover, the current efficiency of the gene repair by RNA-DNA hybrids, according to Kmiec and colleagues in their recent paper is only 4×10-4 even after several hours of pre-selection permitting multiplification of bacterial cells with the corrected plasmid (5). This efficiency is much lower than the 50% value reported five years ago, but is assuredly much closer to the reality.

DNA binding specificity of different STAT proteins. Comparison of in vitro specificity with natural target sites.

STAT transcription factors are expressed in many cell types and bind to similar sequences. However, different STAT gene knock-outs show very distinct phenotypes. To determine whether differences between the binding specificities of STAT proteins account for these effects, we compared the sequences bound by STAT1, STAT5A, STAT5B, and STAT6. One sequence set was selected from random oligonucleotides by recombinant STAT1, STAT5A, or STAT6. For another set including many weak binding sites, we quantified the relative affinities to STAT1, STAT5A, STAT5B, and STAT6. We compared the results to the binding sites in natural STAT target genes identified by others. The experiments confirmed the similar specificity of different STAT proteins. Detailed analysis indicated that STAT5A specificity is more similar to that of STAT6 than that of STAT1, as expected from the evolutionary relationships. The preference of STAT6 for sites in which the half-palindromes (TTC) are separated by four nucleotides (N(4)) was confirmed, but analysis of weak binding sites showed that STAT6 binds fairly well to N(3) sites. As previously reported, STAT1 and STAT5 prefer N(3) sites; however, STAT5A, but not STAT1, weakly binds N(4) sites. None of the STATs bound to half-palindromes. There were no specificity differences between STAT5A and STAT5B.

FtsK Is a DNA motor protein that activates chromosome dimer resolution by switching the catalytic state of the XerC and XerD recombinases.

FtsK acts at the bacterial division septum to couple chromosome segregation with cell division. We demonstrate that a truncated FtsK derivative, FtsK(50C), uses ATP hydrolysis to translocate along duplex DNA as a multimer in vitro, consistent with FtsK having an in vivo role in pumping DNA through the closing division septum. FtsK(50C) also promotes a complete Xer recombination reaction between dif sites by switching the state of activity of the XerCD recombinases so that XerD makes the first pair of strand exchanges to form Holliday junctions that are then resolved by XerC. The reaction between directly repeated dif sites in circular DNA leads to the formation of uncatenated circles and is equivalent to the formation of chromosome monomers from dimers.

Characteristic bimodal profiles of RNA polymerase II at thousands of active mammalian promoters.

BACKGROUND: In mammals, ChIP-seq studies of RNA polymerase II (PolII) occupancy have been performed to reveal how recruitment, initiation and pausing of PolII may control transcription rates, but the focus is rarely on obtaining finely resolved profiles that can portray the progression of PolII through sequential promoter states. RESULTS: Here, we analyze PolII binding profiles from high-coverage ChIP-seq on promoters of actively transcribed genes in mouse and humans. We show that the enrichment of PolII near transcription start sites exhibits a stereotypical bimodal structure, with one peak near active transcription start sites and a second peak 110 base pairs downstream from the first. Using an empirical model that reliably quantifies the spatial PolII signal, gene by gene, we show that the first PolII peak allows for refined positioning of transcription start sites, which is corroborated by mRNA sequencing. This bimodal signature is found both in mouse and humans. Analysis of the pausing-related factors NELF and DSIF suggests that the downstream peak reflects widespread pausing at the +1 nucleosome barrier. Several features of the bimodal pattern are correlated with sequence features such as CpG content and TATA boxes, as well as the histone mark H3K4me3. CONCLUSIONS: We thus show how high coverage DNA sequencing experiments can reveal as-yet unnoticed bimodal spatial features of PolII accumulation that are frequent at individual mammalian genes and reminiscent of transcription initiation and pausing. The initiation-pausing hypothesis is corroborated by evidence from run-on sequencing and immunoprecipitation in other cell types and species.