Accuracy of qPCR for quantifying Leishmania kDNA in different skin layers of patients with American tegumentary leishmaniasis.

OBJECTIVES: Superficial swab sampling of American tegumentary leishmaniasis (ATL) lesions shows higher amounts of Leishmania than those from biopsy. Subcutaneous involvement is also important in ATL, but parasite quantification according to lesion depth has not been evaluated. We aim to present the best depth at which sampling should be performed for molecular exams of ATL. METHODS: Patients with a clinical presentation compatible with ATL were allocated to ATL and control groups. Qualitative and quantitative qPCR assays were performed using SYBR Green and primers amplifying the kDNA minicircle of Leishmania spp. in different skin layers, including the epidermis, the superior dermis, the inferior dermis, and the hypodermis. RESULTS: Fifty-nine patients were included in this study, including 40 who had been diagnosed with ATL and 19 controls. The number of parasites was greater in samples of the epidermis and superior dermis (159.1 × 106, range 4.0-781.7, and 75.4 × 106, range 8.0-244.5, mean Leishmania parasite equivalents per µg of tissue DNA, respectively) than those in samples of the inferior dermis and hypodermis (54.6, range 8.0-256.6, and 16.8 × 106, range 8.0-24.1, mean Leishmania parasite equivalents per µg of tissue DNA, respectively). The best diagnostic accuracy was achieved in the superior dermis (77.9%) and was significantly greater than that in the hypodermis (63.3%; p 0.039). CONCLUSIONS: We conclude that superficial sampling can retrieve a greater quantity of parasites. Future studies of the role of transepidermal elimination as a mechanism of host defence in ATL must be performed as there is a considerable quantity of Leishmania kDNA in the epidermis.

Non-linear conduction due to depinning of charge order domains in Fe3O2BO3.

The oxyborate Fe3O2BO3 presents a charge density wave (CDW) transition close to room temperature. As we show here, this is associated with a well defined anomaly in the specific heat. Below this transition, when applying in a single crystal of Fe3O2BO3 a DC voltage above a temperature dependent threshold, a high current is liberated in this material. We study the conduction in single crystals of Fe3O2BO3 with voltage applied parallel and perpendicular to the crystallographic c axis direction. The observed currents are attributed to the depinning of charge ordered domains above a threshold voltage V T2 that gives rise to a collective conduction due to coherent domains. Compliance limited DC data shows that above a lower threshold voltage depinning is smooth and follows a power law scaling. Similar depinning with power law scaling is also revealed in the AC conductivity.

High sodium intake during postnatal phases induces an increase in arterial blood pressure in adult rats.

Epigenetic studies suggest that diseases that develop in adulthood are related to certain conditions to which the individual is exposed during the initial stages of life. Experimental evidence has demonstrated that offspring born to mothers maintained on high-Na diets during pregnancy have higher mean arterial pressure (MAP) in adulthood. Although these studies have demonstrated the importance of prenatal phases to hypertension development, no evidence regarding the role of high Na intake during postnatal phases in the development of this pathology has been reported. Therefore, in the present study, the effects of Na overload during childhood on induced water and Na intakes and on cardiovascular parameters in adulthood were evaluated. Experiments were carried out in two groups of 21-d-old rats: experimental group, maintained on hypertonic saline (0.3 m-NaCl) solution and food for 60 d, and control group, maintained on tap water and food. Later, both groups were given water and food for 15 d (recovery period). After the recovery period, chronic cannulation of the right femoral artery was performed in unanaesthetised rats to record baseline MAP and heart rate (HR). The experimental group was found to have increased basal MAP (98.6 (sem 2.6) v. 118.3 (sem 2.7) mmHg, P< 0.05) and HR (365.4 (sem 12.2) v. 398.2 (sem 7.5) beats per min, P< 0.05). There was a decrease in the baroreflex index in the experimental group when compared with that in the control group. A water and Na intake test was performed using furosemide. Na depletion was found to induce an increase in Na intake in both the control and experimental groups (12.1 (sem 0.6) ml and 7.8 (sem 1.1), respectively, P< 0.05); however, this increase was of lower magnitude in the experimental group. These results demonstrate that postnatal Na overload alters behavioural and cardiovascular regulation in adulthood.

The presence of resistance mutations to protease and polymerase inhibitors in Hepatitis C virus sequences from the Los Alamos databank.

Several new direct-acting antiviral (DAA) drugs are in development for chronic hepatitis C viral (HCV) infection, and NS3-NS4A serine protease and the NS5B RNA-dependent RNA polymerase have been the major targets. HCV variants displaying drug-resistant phenotypes have been observed both in vitro and during clinical trials. Our aim was to characterize amino acid changes at positions previously associated with resistance in protease (NS3) and polymerase (NS5B) regions from treatment-naïve HCV patients infected with genotypes 1a, 1b and 3a. All 1383 NS3 protease sequences (genotype 1a = 680, 1b = 498 and 3a = 205) and 806 NS5B polymerase sequences (genotypes 1a = 471, 1b = 329, 3a = 6) were collected from Los Alamos databank. Genotype 3a protease sequences showed the typical low-level resistance mutation V36L. NS3 sequences from other genotypes presented mutations on positions 36, 39, 41, 43, 54, 80, 109, 155 and 168 in a frequency lower than 2%, except for the mutation Q80R found in 35% of genotype 1a isolates. Polymerase sequences from genotype 3a patients showed five typical mutations: L419I, I424V, I482L, V499A and S556G. Two positions presented high polymorphism in the NS5B region from genotype 1a (V499A) and genotype 1b (C316N) subjects. Our results demonstrated a natural profile of genotype 3a that can be associated with the pre-existence of HCV variants resistant to first-generation protease inhibitors and to non-nucleoside polymerase inhibitors. Likewise, genotype 1b isolates and genotype 1a sequences exhibited pre-existing mutations associated with resistance to Palm II and Thumb I polymerase inhibitors, respectively.

In vitro and in vivo activities of 1,3,4-thiadiazole-2-arylhydrazone derivatives of megazol against Trypanosoma cruzi.

From a series of 1,3,4-thiadiazole-2-arylhydrazone derivatives of megazol screened in vitro against Trypanosoma cruzi, eight (S1 to S8) were selected for in vivo screening by single-dose oral administration (200 mg/kg of body weight) to infected mice at 5 days postinfection (dpi). Based on significant decreases in both parasitemia levels and mortality rates, S2 and S3 were selected for further assays. Despite having no in vivo effect, S1 was included since it was 2-fold more potent against trypomastigotes than megazol in vitro. Trypomastigotes treated with S1, S2, or S3 showed alterations of the flagellar structure and of the nuclear envelope. When assayed on intracellular amastigotes, the selectivity index (SI) for macrophages was in the range of >27 to >63 and for cardiac cells was >32 for S1 and >48 for megazol. In noninfected mice, S1 did not alter the levels of glutamic oxalacetic transaminase (GOT), glutamate pyruvate transaminase (GPT), or urea. S2 led to an increase in GOT, S3 to increases in GOT and GPT, and megazol to an increase in GOT. Infected mice were treated with each derivative at 50 and 100 mg/kg from dpi 6 to 15: S1 did not interfere with the course of infection or reduce the number of inflammatory foci in the cardiac tissue, S2 led to a significant decrease of parasitemia, and S3 decreased mortality. There was no direct correlation between the in vitro effect on trypomastigotes and amastigotes and the results of the treatment in experimental models, as S1 showed a high potency in vitro while, in two different schemes of in vivo treatment, no decrease of parasitemia or mortality was observed.

Leishmanicidal activity of new megazol derivatives.

A series of thirteen new megazol derivatives, designed exploring the molecular hybridization approach between megazol (3) and heterocombretastatins (2), was synthesized. These new compounds were tested for in vitro antiparasitic activity upon axenic amastigotes of Leishmania donovani. Biological results led us to identify a new potent megazol derivative (4g), which presents an IC(50) = 0.081microg/mL, more active tham the reference drug miltefosine (IC(50) = 0.131microg/mL).

Precise dose evaluation using a commercial phototransistor as a radiation detector.

An experimental arrangement and a circuitry based on an NPN phototransistor-type silicon radiation detector have been used for evaluating the X-ray beam dose in the diagnostic range. The circuitry was built to allow alteration of the electric field in the phototransistor internal structure, with some devices that have an available base connection. By changing the transistor base bias it is possible to alter its operation point to obtain a response gain from the selected photon energy range. In this way we have made an electronic energy-domain discretisation and we are investigating a model to calculate the dose contribution from each energy discretised into 10 keV steps. The method has been tested in filtered radiation beams generated from an HF-160 Pantak X-ray unit and compared with the usual dosimetry method. Our results have demonstrated that it is possible to make such a dose deconvolution from 40 to 140 keV energies by controlling the phototransistor base bias properly.

Highly sensitive thermoluminescent carbon doped nanoporous aluminium oxide detectors.

In this work we present the synthesis, characterisation and the thermoluminescence (TL) response of nanoporous carbon doped aluminium oxide Al2O3:C produced by anodic oxidation of aluminium in organic and inorganic solvents. The X-ray and scanning electron microscopy (SEM) measurements reveal that the synthesised samples are amorphous and present highly ordered structures with uniform pore distribution with diameter of the order 50 nm. The photoluminescence and spectroscopic analysis in the visible and infrared regions show that the luminescence properties presented by the samples prepared in organic acid are due to carboxylate species, incorporated in anodic alumina films during the synthesis process. After an annealing treatment, part of the incorporated species decomposes and is incorporated into the structure of the aluminium oxide yielding a highly thermoluminescent detector (TL) . The results for X-ray irradiation in the range from 21 to 80 keV indicate a linear TL response with the dose in the range from 5 mGy to 1 Gy, suggesting that nanoporous aluminium oxide produced in the present route of synthesis is a suitable detector for radiation measurements.

The performance of commercial photodiodes for dosimetry in mammography.

This paper reports the results of an investigation carried out to determine the suitability of using a commercially available photodiode for dosimetry in mammography thereby providing a low cost dosemeter with a direct reading of the radiation dose. A mammographic X-ray generator (Siemens Mammomat 1000) with tube potential in the 23-30 kV range and a constant potential X-ray generator (Pantak) with a W/Mo anode/filter combination were used in this study. The results showed that the photodiode response is highly linear within mammographic dose ranges and that the energy dependence was <3% at tube potentials of 25-30 kV. Good agreement was observed between the incident air kerma measured with both the photodiode and the ionization chamber. These results show the viability of using the photodiode as a dosemeter system in mammography.

Filtered X ray beam dosimetry from 10(-3) to 10(2) Gy dose range by using phototransistors.

Low-cost, commercially available phototransistor-type semiconductor devices have been tested for monitoring filtered X ray beam dose. A Pantak X-ray unit was used to generate aluminium filtered X ray beams from 60 to 120 kV potentials. The analysis of the radiation detection behaviour as a function of the X ray tube parameters (peak kilovoltage and electrical current) are presented. The changes of the phototransistor electronic parameters have been evaluated and the results indicate that phototransistors can be used as X ray detectors for dose estimation in two different ways: electrical current read-out from 1 to 100 mGy dose range, and the changing of the radiation detection sensitivity in the dose range from 0.1 to 100 Gy. In addition, the devices show high reliability with no sign of substantial performance degradation with use and, in certain dose ranges, the cumulative dose evaluations could be performed up to 10,000 times with no need for re-calibration.

Gamma ray detection using sol-gel glass doped with lanthanide ions.

In this work we discuss the synthesis, characterization and the use of rare earth doped silica glass prepared by the sol-gel technique as a gamma radiation detector. We obtain a glass material doped with rare earth ions in a high local symmetry, in contrast to the low symmetry found in glasses produced by conventional melting methods and technologies. The behavior of the luminescence spectra of the excited states of rare earth indicates a strong dependence with gamma radiation doses, where the magnetic dipole transition 5D0-->7F1 of the Eu3+ ions presents an huge enhancement of 900% for irradiation doses up to 400 Gy.

Dinucleotide repeat expansion catalyzed by bacteriophage T4 DNA polymerase in vitro.

DNA replication normally occurs with high fidelity, but certain "slippery" regions of DNA with tracts of mono-, di-, and trinucleotide repeats are frequently mutation hot spots. We have developed an in vitro assay to study the mechanism of dinucleotide repeat expansion. The primer-template resembles a base excision repair substrate with a single nucleotide gap centered opposite a tract of nine CA repeats; nonrepeat sequences flank the dinucleotide repeats. DNA polymerases are expected to repair the gap, but further extension is possible if the DNA polymerase can displace the downstream oligonucleotide. We report here that the wild type bacteriophage T4 DNA polymerase carries out gap and strand displacement replication and also catalyzes a dinucleotide expansion reaction. Repeat expansion was not detected for an exonuclease-deficient T4 DNA polymerase or for Escherichia coli DNA polymerase I. The dinucleotide repeat expansion reaction catalyzed by wild type T4 DNA polymerase required a downstream oligonucleotide to "stall" replication and 3' --> 5' exonuclease activity to remove the 3'-nonrepeat sequence adjacent to the repeat tract in the template strand. These results suggest that dinucleotide repeat expansion may be stimulated in vivo during DNA repair or during processing of Okazaki fragments.

[Evaluation of the structures of agreement and disagreement in reliability studies]. / Avaliação das estruturas de concordância e discordância nos estudos de confiabilidade.

The kappa coefficient has been the measurement preferred by epidemiologists for reliability studies. Various articles have demonstrated that the use of the kappa coefficient may have some undesirable features in certain contexts. Recently, methodologies using an ordinal scale for the modelling of interobserver agreement have been developed as an alternative to kappa. To show that there is a class of log-linear statistical models that when analyzed sequentially can be used to rate the patterns of agreement and disagreement. Using data on the comparability of primary and proxy respondent reports with respect to the frequency of alcoholic consumption and its correlation to coronary diseases a nested set of log-linear models was adjusted to find the "best" model. Computed odds ratios to determine the measure of agreement were also computed. The weight kappa was equal 0.685 with 95% CI (0.638-0.732) showing a good agreement. But it does not give any information about the structure of the agreement and disagreement. Among the sequence of models analyzed, the one with the best adjustment showed an agreement estimated at 0.4454 with 95% CI (0.1300-0.7608) and an association estimated at 1.3309 with 95% CI (0.9649-1.6978). The measure tau for adjacent categories was 9.2 with 95% CI (6.0-14.2). Thus, evidence shows that the observers tended to rate many phenomena similarly. Furthermore, high (or low) ratings made by primary respondents tended to be associated with high (or low) ratings made by the proxy respondents. Log-linear models can give us a more informative and more complete analysis with respect to the rating of matched pairs of observers than that given by kappa. In conclusion, the indiscriminate use of kappa as the only agreement index must be questioned. The appendix demonstrates how to use PROC GENMOD in SAS to fit these models.

Cell-surface carbohydrates of Entamoeba invadens.

Cell-surface carbohydrates of Entamoeba invadens trophozoites were analyzed using (a) a panel of highly purified lectins specific for molecules containing N-acetylglucosamine or sialic acid, N-acetylgalactosamine, galactose, mannose-like residues, and fucose; (b) Escherichia coli K-12 with mannose-sensitive fimbria; (c) enzymatic digestion; and (d) scanning electron microscopy. The presence of galactose (D-Gal) and N-acetylgalactosamine (D-GalNAc) was detected in the amoeba. Previous trypsinization induced the appearance of Glycine max (SBA, specific for D-GalNAc residues)-binding sites, whereas such treatment completely abolished the ability of Ricinus communis (RCAI) and Axinalla polypoides (APP, specific for D-Gal) lectins and partially abolished that of Euonymus europaeus (EEL, specific for D-Gal) lectins to agglutinate the trophozoites. The agglutinating activity of E. coli K-12 adheans with the amoeba was markedly increased after trypsin digestion, indicating that mannose units become exposed after enzyme treatment. These findings were essentially confirmed by scanning electron microscopy. After neuraminidase treatment the parasites became strongly agglutinated with SBA and Arachis hypogaea (PNA, specific for D-Gal) and the cell interaction with Wisteria floribunda (WFH, specific for D-GalNAc) was markedly increased. These results suggest that in E. invadens trophozoites, sialic acid residues are linked to D-Gal and D-GalNAc.

Improvement of enantioselective syntheses and chiral high resolution gas chromatographic analyses of (+)-2-allyl-2-carboethoxy-cyclopentanol.

The improvement of the biocatalytic reduction of 2-allyl-carboethoxy-cyclopentanone (2) to the corresponding cyclopentanol derivative (+)-(1R,2R)-(1) was accomplished employing baker's yeast in organic media. This chiral cyclopentanol derivative (1), analyzed by high resolution gas chromatography performed over beta-cyclodextrin stationary phase, was obtained in 38% yield (> 99% e.e.).

Type 2 poliovirus recombinants isolated from vaccine-associated cases and from healthy contacts in Brazil.

In a previous study (Friedrich et al., 1995b) P2/Sabin-derived strains isloated in Brazil from vaccine-associated paralytic poliomyelitis (VAPP) cases and from healthy contacts were analyzed for the presence of mutations at nucleotide (nt) 481 in the 5'-noncoding region (5'NCR) and at the codon of amino acid (aa) 143 of the capsid protein VP1, that are known to increase neurovirulence. In the present study a part of the 3Dpol-coding region of these strains was sequenced (3Dpol seq.) with the aim to find recombinant strains. In the 3Dpol seq., four out of ten strains isolated from VAPP cases turned out to be recombinants: one had 3Dpol seq. from the P1/Sabin strain, while the second had a part of 3Dpol seq. both from the P2/Sabin and P1/Sabin strains; the third and fourth recombinants had 3Dpol seq. from non-vaccine strains. The strains isolated from healthy contacts of the two VAPP cases, from which type 2 vaccine/non-vaccine recombinant strains were isolated, also consisted from recombinant genomes with the same nt sequences as those of the isolates from VAPP cases, confirming the transmission of P2/Sabin-derived recombinants. Comparison of the aa sequence of the viral RNA polymerase of the P2/Sabin strain with the predicted aa sequences of these recombinants in 3Dopl seq. demonstrated that an aa 69 (Asp-->Glu)) substitution was observed in most of the recombinant genomes, while an aa 113 (Thr-->Ser) substitution was observed in all the recombinant genomes. The possibility that the genomic recombination increased the neurovirulence of these strains cannot be excluded.

[Demographic profile and work situation of patients with Chagas disease]. / Perfil demográfico e situação de trabalho de portadores da doença de Chagas.

PURPOSE: To evaluate the social profile and work habits in patients with Chagas' disease. METHODS: We studied 284 ambulatory patients age ranged 21 to 74 years, female (62.3%) and married people (71.8%) divided according to their activities in three groups: active workers--121 (42.7%); under social security--93 (32.8%); housewives--61 (21.4%); unemployed people--9 (3.1%). RESULTS: The majority of them were in stage II of Chagas' disease, 123 (43.3%) for both sexes. Migratory characteristics in this population was observed, with the search for work as the main reason for this. The level of education was low, considerably affecting the kind of profession. CONCLUSION: Patients with Chagas' disease are originally from the rural area and migrate to urban area in order to improve economic conditions. They show unfavourable social conditions, with inadequate level of education and lack of professional gratifications.

The effects of N helix deletion and mutant F29W on the Ca2+ binding and functional properties of chicken skeletal muscle troponin.

To assess the structural and functional significance of the N helix (residues 3-13) of avian recombinant troponin C (rTnC), we have constructed NHdel, in which residues 1-11 have been deleted, both in rTnC and in the spectral probe mutant F29W (Pearlstone, J. R., Borgford, T., Chandra, M., Oikawa, K., Kay, C. M., Herzberg, O., Moult, J., Herklotz, A., Reinach, F. C., and Smillie, L. B. (1992) Biochemistry 31, 6545-6553). Comparison of the far- and near-UV CD spectra (+/- Ca2+) of F29W and F29W/NHdel and titration of the Ca(2+)-induced ellipticity and fluorescence changes indicates that the deletion has little effect on the global fold of the molecule but reduces the Ca2+ affinity of the N domain, but not the C domain, by 1.6-1.8-fold. Comparisons of the mutants NHdel, F29W, and F29W/NHdel with rTnC have been made using several functional assays. In reconstituted troponin-tropomyosin actomyosin subfragment 1 and myofibrillar ATPase systems, both F29W and NHdel have significantly reduced Ca(2+)-activated enzymatic activities. These effects are cumulative in the double mutant F29W/NHdel. On the other hand, maximal isometric tension development in Ca(2+)-activated reconstituted skinned fibers is not affected with F29W and NHdel, although the Ca2+ sensitivity of NHdel in this system is markedly reduced. We conclude that both mutations, NHdel and F29W, are functionally deleterious, possibly affecting interactions of the N domain with troponin I and/or T.