Mitochondrial DNA sequence variation within the remnant populations of the endangered numbat (Marsupialia: Myrmecobiidae: Myrmecobius fasciatus).

The numbat has been reduced to two populations in Western Australia. To better understand the effects of range reduction on gene flow and genetic variation, and to address questions crucial for the species' management, we analysed mitochondrial DNA (mtDNA) sequences of free-ranging individuals and museum specimens. The results suggest recent connectivity between the remnant populations, although one of those may have lost significant amounts of genetic diversity during the recent population size reduction. We propose that for management purposes the remnant populations should be treated as a single historical lineage and that, subject to certain caveats, consideration should be given to population augmentation by translocation.

Multilocus sequence analysis reveals the genetic diversity of European fruit tree phytoplasmas and supports the existence of inter-species recombination

The genetic diversity of three temperate fruit tree phytoplasmas ‘Candidatus Phytoplasma prunorum’, ‘Ca. P. mali’ and ‘Ca. P. pyri’ has been established by multilocus sequence analysis. Among the four genetic loci used, the genes imp and aceF distinguished 30 and 24 genotypes, respectively, and showed the highest variability. Percentage of substitution for imp ranged from 50 to 68% according to species. Percentage of substitution varied between 9 and 12% for aceF, whereas it was between 5 and 6% for pnp and secY. In the case of ‘Ca P. prunorum’ the three most prevalent aceF genotypes were detected in both plants and insect vectors, confirming that the prevalent isolates are propagated by insects. The four isolates known to be hypo-virulent had the same aceF sequence, indicating a possible monophyletic origin. Haplotype network reconstructed by eBURST revealed that among the 34 haplotypes of ‘Ca. P. prunorum’, the four hypo-virulent isolates also grouped together in the same clade. Genotyping of some Spanish and Azerbaijanese ‘Ca. P. pyri’ isolates showed that they shared some alleles with ‘Ca. P. prunorum’, supporting for the first time to our knowledge, the existence of inter-species recombination between these two species.

Integration of Trypanosoma cruzi kDNA minicircle sequence in the host genome may be associated with autoimmune serum factors in Chagas disease patients

Integration of kDNA sequences within the genome of the host cell shown by PCR amplification with primers to the conserved Trypanosoma cruzi kDNA minicircle sequence was confirmed by Southern hybridization with specific probes. The cells containing the integrated kDNA sequences were then perpetuated as transfected macrophage subclonal lines. The kDNA transfected macrophages expressed membrane antigens that were recognized by antibodies in a panel of sera from ten patients with chronic Chagas disease. These antigens barely expressed in the membrane of uninfected, control macrophage clonal lines were recognized neither by factors in the control, non-chagasic subjects nor in the chagasic sera. This finding suggests the presence of an autoimmune antibody in the chagasic sera that recognizes auto-antigens in the membrane of T. cruzi kDNA transfected macrophage subclonal lines.

Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis

Molecular studies of insect disease vectors are of paramount importance for understanding parasite-vector relationship. Advances in this area have led to important findings regarding changes in vectors' physiology upon blood feeding and parasite infection. Mechanisms for interfering with the vectorial capacity of insects responsible for the transmission of diseases such as malaria, Chagas disease and dengue fever are being devised with the ultimate goal of developing transgenic insects. A primary necessity for this goal is information on gene expression and control in the target insect. Our group is investigating molecular aspects of the interaction between Leishmania parasites and Lutzomyia sand flies. As an initial step in our studies we have used random sequencing of cDNA clones from two expression libraries made from head/thorax and abdomen of sugar fed L. longipalpis for the identification of expressed sequence tags (EST). We applied differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR to characterize differentially expressed mRNA from sugar and blood fed insects, and, in one case, from a L. (V.) braziliensis-infected L. longipalpis. We identified 37 cDNAs that have shown homology to known sequences from GeneBank. Of these, 32 cDNAs code for constitutive proteins such as zinc finger protein, glutamine synthetase, G binding protein, ubiquitin conjugating enzyme. Three are putative differentially expressed cDNAs from blood fed and Leishmania-infected midgut, a chitinase, a V-ATPase and a MAP kinase. Finally, two sequences are homologous to Drosophila melanogaster gene products recently discovered through the Drosophila genome initiative.

Genetic variability in Brazilian populations of Biomphalaria straminea complex detected by simple sequence repeat anchored polymerase chain reaction amplification

Biomphalaria glabrata, B. tenagophila and B. straminea are intermediate hosts of Schistosoma mansoni, in Brazil. The latter is of epidemiological importance in the northwest of Brazil and, due to morphological similarities, has been grouped with B. intermedia and B. kuhniana in a complex named B. straminea. In the current work, we have standardized the simple sequence repeat anchored polymerase chain reaction (SSR-PCR) technique, using the primers (CA)8RY and K7, to study the genetic variability of these species. The similarity level was calculated using the Dice coefficient and genetic distance using the Nei and Li coefficient. The trees were obtained by the UPGMA and neighbor-joining methods. We have observed that the most related individuals belong to the same species and locality and that individuals from different localities, but of the same species, present clear heterogeneity. The trees generated using both methods showed similar topologies. The SSR-PCR technique was shown to be very efficient in intrapopulational and intraspecific studies of the B. straminea complex snails.

Design of a Control Slide for Cyanoacrylate Polymerization : Application to the CA-Bluestar Sequence

Casework expercience has shown that, in some cases, long exposures of surfaces subjected to cyanoacrylate (CA) fuming had detrimental effects on the subsequent application of Bluestar. This study aimed to develop a control mechanism to monitor the amount of CA deposited prior to the subsequent treatment. A control slide bearing spots of sodium hydroxide (NaOH) of known concentrations and volume was designed and validated against both scanning electron microscopy (SEM) observations and latent print examiners' assessments of the quality of the developed marks. The control slide allows one to define three levels of development that were used to monitor the Bluestar reaction on depleting footwear marks left in diluted blood. The appropriate conditions for a successful application of both CA and Bluestar were determined.

Descriptive epidemiology of skin cancer in the Swiss Canton of Vaud.

Incidence registration and survival data for non-melanocytic skin neoplasms and cutaneous melanoma have been abstracted from the population-based system of the Cancer Registry of the Swiss Canton of Vaud, which has been operating in a particularly favourable environment, since the large majority of cutaneous lesions resected in the area are examined by a pathologist. Among the 5,712 cases registered, 66.7% were basal-cell carcinomas, 20.6% squamous-cell cancers, 9.3% cutaneous melanomas and 3.4% other miscellaneous histological types. The distribution by histological type did not differ appreciably in the 2 sexes, but there were marked inter-sex differences as regards anatomical site. In both sexes, head and neck was by far the commonest localization for non-melanomatous neoplasms (69 to 81% of all incident cases), followed by trunk for basal-cell cancers (18% in males, 15% in females) and upper limb for squamous-cell (10% in males, 17% in females). The distribution of skin melanomas differed considerably between the 2 sexes, by far the commonest site being the trunk for males (45% of cases) and lower limbs for females (40%), followed by head and neck (22% in both sexes). Incidence rates for both basal- and squamous-cell cancers increased with age, and rates were higher in males for each localization except the lower limb. In contrast, incidence for melanoma was higher in females, and incidence rates did not increase with age above 55 years for all sites except head and neck. This can be interpreted in terms of cohort effect, since mortality from melanoma has substantially increased in Switzerland across subsequent birth cohorts. Although this study is essentially descriptive, accurate inspection of these data provides some support for the major aetiological hypotheses of skin carcinogenesis, i.e., the observation that the large majority of basal- and squamous-cell cancers arise on the head and neck confirms the importance of long-term ultraviolet exposure; the relative excess of squamous-cell as compared to basal-cell neoplasms on the upper limb may suggest the role of exposure to other (chemical) carcinogens; and the proportional excess of melanomas on the trunk in males and lower limb in females further indicates that intermittent exposure to sunlight is probably the relevant aetiologic factor for melanocytic skin neoplasms.

MP2RAGE, a self bias-field corrected sequence for improved segmentation and T-1-mapping at high field

The large spatial inhomogeneity in transmit B, field (B-1(+)) observable in human MR images at hi h static magnetic fields (B-0) severely impairs image quality. To overcome this effect in brain T-1-weighted images the, MPRAGE sequence was modified to generate two different images at different inversion times MP2RAGE By combining the two images in a novel fashion, it was possible to create T-1-weigthed images where the result image was free of proton density contrast, T-2* contrast, reception bias field, and, to first order transmit field inhomogeneity. MP2RAGE sequence parameters were optimized using Bloch equations to maximize contrast-to-noise ratio per unit of time between brain tissues and minimize the effect of B-1(+) variations through space. Images of high anatomical quality and excellent brain tissue differentiation suitable for applications such as segmentation and voxel-based morphometry were obtained at 3 and 7 T. From such T-1-weighted images, acquired within 12 min, high-resolution 3D T-1 maps were routinely calculated at 7 T with sub-millimeter voxel resolution (0.65-0.85 mm isotropic). T-1 maps were validated in phantom experiments. In humans, the T, values obtained at 7 T were 1.15 +/- 0.06 s for white matter (WM) and 1.92 +/- 0.16 s for grey matter (GM), in good agreement with literature values obtained at lower spatial resolution. At 3 T, where whole-brain acquisitions with 1 mm isotropic voxels were acquired in 8 min the T-1 values obtained (0.81 +/- 0.03 S for WM and 1.35 +/- 0.05 for GM) were once again found to be in very good agreement with values in the literature. (C) 2009 Elsevier Inc. All rights reserved.

Complete nucleotide sequence analysis of a Brazilian dengue virus type 2 strain

In the last decade, dengue fever (DF) in Brazil has been recognized as an important public health problem, and an increasing number of dengue haemorrhagic fever (DHF) cases have been reported since the introduction of dengue virus type 2 (DEN-2) into the country in 1990. In order to analyze the complete genome sequence of a DEN-2 Brazilian strain (BR64022/98), we designed primers to amplify contiguous segments of approximately 500 base pairs across the entire sequence of the viral genome. Twenty fragments amplified by reverse transcriptase-PCR were cloned, and the complete nucleotide and the deduced amino acid sequences were determined. This constitutes the first complete genetic characterization of a DEN-2 strain from Brazil. All amino acid changes differentiating strains related to the Asian/American-Asian genotype were observed in BR64022/98, indicating the Asiatic origin of the strain.

Lesion Recognition in Multiple Sclerosis: A Sequence Comparison and Quantification Study at 3T

Introduction Lesion detection in multiple sclerosis (MS) is an essential part of its clinical diagnosis. In addition, radiological characterisation of MS lesions is an important research field that aims at distinguishing different MS types, monitoring drug response and prognosis. To date, various MR protocols have been proposed to obtain optimal lesion contrast for early and comprehensive diagnosis of the MS disease. In this study, we compare the sensitivity of five different MR contrasts for lesion detection: (i) the DIR sequence (Double Inversion Recovery, [4]), (ii) the Dark-fluid SPACE acquisition schemes, a 3D variant of a 2D FLAIR sequence [1], (iii) the MP2RAGE [2], an MP-RAGE variant that provides homogeneous T1 contrast and quantitative T1-values, and the sequences currently used for clinical MS diagnosis (2D FLAIR, MP-RAGE). Furthermore, we investigate the T1 relaxation times of cortical and sub-cortical regions in the brain hemispheres and the cerebellum at 3T. Methods 10 early-stage female MS patients (age: 31.64.7y; disease duration: 3.81.9y; disability score, EDSS: 1.80.4) and 10 healthy controls (age and gender-matched: 31.25.8y) were included in the study after obtaining informed written consent according to the local ethic protocol. All experiments were performed at 3T (Magnetom Trio a Tim System, Siemens, Germany) using a 32-channel head coil [5]. The imaging protocol included the following sequences, (all except for axial FLAIR 2D with 1x1x1.2 mm3 voxel and 256x256x160 matrix): DIR (TI1/TI2/TR XX/3652/10000 ms, iPAT=2, TA 12:02 min), MP-RAGE (TI/TR 900/2300 ms, iPAT=3, TA 3:47 min); MP2RAGE (TI1/TI2/TR 700/2500/5000 ms, iPAT=3, TA 8:22 min, cf. [2]); 3D FLAIR SPACE (only for patient 4-6, TI/TR 1800/5000 ms, iPAT=2, TA=5;52 min, cf. [1]); Axial FLAIR (0.9x0.9x2.5 mm3, 256x256x44 matrix, TI/TR 2500/9000 ms, iPAT=2, TA 4:05 min). Lesions were identified by two experienced neurologist and radiologist, manually contoured and assigned to regional locations (s. table 1). Regional lesion masks (RLM) from each contrast were compared for number and volumes of lesions. In addition, RLM were merged in a single "master" mask, which represented the sum of the lesions of all contrasts. T1 values were derived for each location from this mask for patients 5-10 (3D FLAIR contrast was missing for patient 1-4). Results & Discussion The DIR sequence appears the most sensitive for total lesions count, followed by the MP2RAGE (table 1). The 3D FLAIR SPACE sequence turns out to be more sensitive than the 2D FLAIR, presumably due to reduced partial volume effects. Looking for sub-cortical hemispheric lesions, the DIR contrast appears to be equally sensitive to the MP2RAGE and SPACE, but most sensitive for cerebellar MS plaques. The DIR sequence is also the one that reveals cortical hemispheric lesions best. T1 relaxation times at 3T in the WM and GM of the hemispheres and the cerebellum, as obtained with the MP2RAGE sequence, are shown in table 2. Extending previous studies, we confirm overall longer T1-values in lesion tissue and higher standard deviations compared to the non-lesion tissue and control tissue in healthy controls. We hypothesize a biological (different degree of axonal loss and demyelination) rather than technical origin. Conclusion In this study, we applied 5 MR contrasts including two novel sequences to investigate the contrast of highest sensitivity for early MS diagnosis. In addition, we characterized for the first time the T1 relaxation time in cortical and sub-cortical regions of the hemispheres and the cerebellum. Results are in agreement with previous publications and meaningful biological interpretation of the data.

Sequencing of simple sequence repeat anchored polymerase chain reaction amplification products of Biomphalaria glabrata

Simple sequence repeat anchored polymerase chain reaction amplification (SSR-PCR) is a genetic typing technique based on primers anchored at the 5' or 3' ends of microsatellites, at high primer annealing temperatures. This technique has already been used in studies of genetic variability of several organisms, using different primer designs. In order to conduct a detailed study of the SSR-PCR genomic targets, we cloned and sequenced 20 unique amplification products of two commonly used primers, CAA(CT)6 and (CA)8RY, using Biomphalaria glabrata genomic DNA as template. The sequences obtained were novel B. glabrata genomic sequences. It was observed that 15 clones contained microsatellites between priming sites. Out of 40 clones, seven contained complex sequence repetitions. One of the repeats that appeared in six of the amplified fragments generated a single band in Southern analysis, indicating that the sequence was not widespread in the genome. Most of the annealing sites for the CAA(CT)6 primer contained only the six repeats found within the primer sequence. In conclusion, SSR-PCR is a useful genotyping technique. However, the premise of the SSR-PCR technique, verified with the CAA(CT)6 primer, could not be supported since the amplification products did not result necessarily from microsatellite loci amplification.

Missed appointments in an adolescent outpatient clinic : descriptive analyses of consultations over eight years ; et, Missed appointments in an outpatient clinic for adolescents, an approache to predict the risk of missing

Rapport de synthèse: Les rendez-vous manqués représentent un problème important, tant du point de vue de la santé des patients que du point de vue économique. Pourtant peu d'études se sont penchées sur le sujet, particulièrement dans une population d'adolescents. Les buts de cette étude étaient de caractériser les adolescents qui sont à risque de manquer ou d'annuler leurs rendez-vous dans une clinique ambulatoire de santé pour adolescents, de comparer les taux des rendez-vous manqués et annulés entre les différents intervenants et d'estimer l'efficacité d'une politique de taxation des rendez-vous manqués non excusés. Finalement, un modèle multi-niveau markovien a été utilisé afin de prédire le risque de manquer un rendez-vous. Ce modèle tient compte du passé de l'adolescent en matière de rendez-vous manqués et d'autres covariables et permet de grouper les individus ayant un comportement semblable. On peut ensuite prédire pour chaque groupe le risque de manquer ou annuler et les covariables influençant significativement ce risque. Entre 1999 et 2006, 32816 rendez-vous fixés pour 3577 patients âgés de 12 à 20 ans ont été analysés. Le taux de rendez-vous manqués était de 11.8%, alors que 10.9% avaient été annulés. Soixante pour cent des patients n'ont pas manqué un seul de leur rendezvous et 14% en ont manqué plus de 25%. Nous avons pu mettre en évidence plusieurs variables associées de manière statistiquement significative avec les taux de rendez-vous manqués et d'annulations (genre, âge, heure, jour de la semaine, intervenant thérapeutique). Le comportement des filles peut être catégorisé en 2 groupes. Le premier groupe inclut les diagnostiques psychiatriques et de trouble du comportement alimentaire, le risque de manquer dans ce groupe étant faible et associé au fait d'avoir précédemment manqué un rendez-vous et au délai du rendez-vous. Les autres diagnostiques chez les filles sont associés à un second groupe qui montre un risque plus élevé de manquer un rendez-vous et qui est associé à l'intervenant, au fait d'avoir précédemment manqué ou annulé le dernier rendez-vous et au délai du rendez-vous. Les garçons ont tous globalement un comportement similaire concernant les rendez-vous manqués. Le diagnostic au sein de ce groupe influence le risque de manquer, tout comme le fait d'avoir précédemment manqué ou annulé un rendez-vous, le délai du rendez-vous et l'âge du patient. L'introduction de la politique de taxation des rendez-vous non excusés n'a pas montré de différence significative des tàux de rendez-vous manqués, cependant cette mesure a permis une augmentation du taux d'annulations. En conclusion, les taux de présence des adolescents à leurs rendez-vous sont dépendants de facteurs divers. Et, même si les adolescents sont une population à risque concernant les rendez-vous manqués, la majorité d'entre eux ne manquent aucun de leurs rendez-vous, ceci étant vrai pour les deux sexes. Etudier les rendez-vous manqués et les adolescents qui sont à risque de rater leur rendez-vous est un pas nécessaire vers le contrôle de ce phénomène. Par ailleurs, les moyens de contrôle concernant les rendez-vous manqués devraient cibler les patients ayant déjà manqué un rendez-vous. La taxation des rendez-vous manqués permet d'augmenter les rendez-vous annulés, ce qui a l'avantage de permettre de fixer un nouveau rendez-vous et, de ce fait, d'améliorer la continuité des soins.