Single step polymerase chain reaction (PCR) for the diagnosis of the Leishmania (Viannia) subgenus

In Brazil, the main etiologic agent of Leishmaniasis that frequently presents with mucosal involvement belongs to the Viannia subgenus. The therapeutic conduct in this disease depends on the parasitological diagnosis, and classical methods are restricted in identifying the agent. In this paper we describe a polymerase chain reaction (PCR), which uses primers designed from mini-exons repetitive sequences. The PCR amplifies a 177bp fragment that can distinguish (Viannia) from (Leishmania) subgenus. This test could be a useful diagnostic tool.

Single-step purification of crotapotin and crotactine from Crotalus durissus terrificus venom using preparative isoelectric focusing

We describe the isolation of crotoxin, a presynaptic B-neurotoxin, as well as its subunits B (crotactine) and A (crotapotin) from lyophilized Crotalus durissus terrificus venom by a single-step preparative isoelectric focusing procedure. From 98 mg of dried venom protein 20.1 mg of crotactine and 13.1 mg of crotapotin were recovered in the first step of focalization and 4.2 mg in a second run. These values correspond to 35.7% of the total venom protein applied. Crotactine separated in the 9.3-7.0 pH range (tubes 1-6) and crotapotin in the 1.8-2.8 pH range (tubes 15-19) and both were homogeneous by SDS-PAGE and N-terminal amino acid analysis. Crotactine, a 12-kDa protein, presented hemolytic and phospholipase A2 activity. Thus, using isoelectric focusing we simultaneously purified both toxins in high yields. This method can be used as an alternative for the purification and characterization of proteins from other snake venoms under conditions in which biological activity is retained

A single-step purification of bothropstoxin-1

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A2-like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications.

Optimisation of an asymmetric polymerase chain reaction assay for the amplification of single-stranded DNA from Wuchereria bancrofti for electrochemical detection

Single-stranded DNA (ssDNA) is a prerequisite for electrochemical sensor-based detection of parasite DNA and other diagnostic applications. To achieve this detection, an asymmetric polymerase chain reaction method was optimised. This method facilitates amplification of ssDNA from the human lymphatic filarial parasite Wuchereria bancrofti. This procedure produced ssDNA fragments of 188 bp in a single step when primer pairs (forward and reverse) were used at a 100:1 molar ratio in the presence of double-stranded template DNA. The ssDNA thus produced was suitable for immobilisation as probe onto the surface of an Indium tin oxide electrode and hybridisation in a system for sequence-specific electrochemical detection of W. bancrofti. The hybridisation of the ssDNA probe and target ssDNA led to considerable decreases in both the anodic and the cathodic currents of the system's redox couple compared with the unhybridised DNA and could be detected via cyclic voltammetry. This method is reproducible and avoids many of the difficulties encountered by conventional methods of filarial parasite DNA detection; thus, it has potential in xenomonitoring.

Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients) and 18.7% (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7%), followed by atypical EPEC (9.4%), ETEC (3.7%), and STEC (0.6%). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.

Helicobacter pylori transiently in the mouth may participate in the transmission of infection

Helicobacter pylori infection is associated with peptic ulcer and gastric carcinoma. The oral cavity may be a reservoir for H. pylori; however, the results of studies on this subject are controversial. We employed single-step and nested polymerase chain reactions (PCR) to detect the presence of the vacA, ureA and 16S rDNA genes of H. pylori in the stomach, saliva and dental plaque of 30 subjects. The results were confirmed by sequencing. Nested 16S rDNA and ureA amplification was achieved in 80% of gastric, 30% of saliva and 20% of dental plaque specimens. Sequencing of 10, seven and four 16S rDNA products from stomach, saliva and dental plaque, respectively, showed > 99% identity with H. pylori. Sequencing of the other four oral cavity PCR products showed similarity with Campylobacter and Wolinella species. Additionally, the vacA genotype identified in the samples of different sites was the same within a given subject.H. pylori may be found in the oral cavity of patients with gastric infection, thus it could be a source of transmission. However, results obtained with detection methods based only on PCR should be interpreted with caution because other microorganisms that are phylogenetically very close to H. pylori are also present in the mouth.

Molecular diagnosis of eosinophilic meningitis due to Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) by polymerase chain reaction-DNA sequencing of cerebrospinal fluids of patients

Cerebrospinal fluid (CSF) samples from clinically diagnosed patients with detectable Angiostrongylus canto-nensis-specific antibodies (n = 10), patients with clinically suspected cases that tested negative for A. cantonensis-an-tibodies (n = 5) and patients with cerebral gnathostomiasis (n = 2) and neurocysticercosis (n = 2) were examined by a single-step polymerase chain reaction (PCR) method using the AC primers for the 66-kDa native protein gene. The PCR method detected A. cantonensis DNA in CSF samples from four of 10 serologically confirmed angiostrongyliasis cases. The PCR results were negative for the remaining CSF samples. The nucleotide sequences of three positive CSF-PCR samples shared 98.8-99.2% similarity with the reference sequence of A. cantonensis. These results indicate the potential application of this PCR assay with clinical CSF samples for additional support in the confirmation of eosinophilic meningitis due to A. cantonensis.

Recuperação de metais de catalisadores mássicos e monometálicos

This work presents a study on the dissolution of some commercial monometallic and non-supported deactivated catalysts in HF + H2O2 mixtures (and, eventually, other media) under mild experimental conditions, after a previous oxidation step. The samples were neither crushed nor grinded. The best experimental conditions were dependent on the nature of the support and of the active phase. For example, the Pt/Al2O3 catalyst was dissolved in about 10 minutes, without agitation and heating; however, dissolution of the Pd/Al2O3, Ni/Al2O3, Ni/SiO2, Cu/Al2O3 and V2O5 samples required a temperature of 60 ºC and an agitation of 400 rpm. A careful addition of a NaOH solution allowed a quantitative precipitation of aluminium as criolite (Na3AlF6) or precipitation of Si as Na2SiF6; NaF was obtained as a by-product. As expected, processing of Pd/C, V2O5 and CuO.Cr2O3 samples was relatively simple. Metals recovery from catalysts reached a quantitative level in all samples studied; it is particularly interesting that platinum and palladium could be easily recovered in a single step process, thus separing them from aluminium.

Comportamento eletroquímico da N-nitrosotiazolidina ácido carboxílico sobre eletrodos de ouro e mercúrio

The electrochemical behavior of N-nitrosothiazolidine carboxylic acid (NTAC) on gold and hanging mercury electrodes, using the cyclic and square wave voltammetries, was studied. Whereas NTAC suffer reduction in a single step on the mercury electrode, two peaks appears on the cyclic voltammograms on the gold electrode, one anodic peak overlaying the gold oxide process at 1.2 V and one cathodic peak at -0.41 V vs Ag/AgCl, KCl 3.0 mol L-1. The cathodic peak depends on the previous oxidation of NTAC at the electrode surface, presents irreversible and adsorption controlled characteristics and it is suitable for quantitative purposes.

Síntese de 5-nitro-isatina e 5-cloro-isatina a partir da isonitrosoacetanilida

This article describes the preparation of 5-nitroisatin and of 5-chloroisatin from isonitrosoacetanilide in a single step, using readily available and inexpensive reagents. These reactions require around 90 minutes and may be carried out as an undergraduate experiment, providing an opportunity to discuss the electrophilic aromatic substitution mechanism, as well as spectroscopic techniques for product identification.

DERIVATIZACIÓN Y CARACTERIZACIÓN ESPECTROSCÓPICA DE UN BIOPOLÍMERO A BASE DE L -LISINA CON ANÁLOGOS DE LA D-BIOTINA: CO -POLI(L-LISINA)-GRAFT -(ε-N-[X-D -BIOTINIL]-L-LISINA)

We report the single-step derivatization reaction of a biopolymer based onL -lysine with D -biotin analogs:Co -poly(L -lysine)-graft-(ε-N -[X-D-biotinyl]-L -lysine) (PLL-X-Biotin). The valeric acid carboxylate of D -biotin is activated to an NHS ester for direct modification of amine groups in proteins and other macromolecules. NHS esters react by nucleophilic attack of an amine in the carbonyl group, releasing the NHS group, and forming a stable amide linkage. NHS-X-Biotin is the simplest biotinylation reagent commercially available. In contrast withD -biotin, it has a longer spacer arm off the valeric acid side chain allowing better binding potential for avidin or streptavidin probes. Derivatization of poly(L -lysine) (PLL) with NHS-X-Biotin led to a copolymer PLL-X-Biotin. UV-Visible, IR-FT and 1H NMR characteristics derived from synthesis are briefly discussed.

Soybean growth and yield after single tillage and species mixture of cover plants

The use of cover crops is important for the agricultural crop and soil management in order to improve the system and, consequently, to increase yield. Therefore, the present study analyzed the effect of crop residues of black oat (Avena strigosa Schreb.) (BO) and a cocktail (CO) of BO, forage turnip (Raphanus sativus L.) (FT) and common vetch (Vicia sativa L.) (V) on the emergence speed index (ESI), seedling emergence speed (SES) plant height and soybean yield in different intervals between cover crop desiccation with glyphosate 480 (3 L ha-1) and BRS 232 cultivar sowing. Plots of 5 x 2.5 m with 1 m of border received four treatments with BO cover crops and four with CO as well as a control for each cover crop, at random, with five replications. The plots were desiccated in intervals of 1, 10, 20 and 30 days before soybean seeding. The harvest was manual while yield was adjusted to 13% of moisture content. The experimental design was completely randomized with splitplots and means compared by the Scott and Knott test at 5% of significance. The results showed that CO of cover crops can be recommended for soybean to obtain a more vigorous seedling emergence, from 10 days after cover crop desiccation.

Urease inhibitor (NBPT) and efficiency of single or split application of urea in wheat crop

NBPT (N-(n-butyl) thiophosphoric triamide), a urease inhibitor, has been reported as one of the most promising compounds to maximize urea nitrogen use in agricultural systems. The objective of this study was to evaluate the performance of irrigated wheat fertilized with urea or urea + NBPT as single or split application. The experiment was conducted from June to October 2006 in Viçosa, MG, Brazil. The experimental design followed a 2×2 factorial scheme, in which urea or urea + NBPT were combined with two modes of application: full dose at sowing (60kg ha-1) or split (20kg ha-1 at sowing + 40kg ha-1 as topdressing at tillering), in randomized blocks with ten replications. The split application of nitrogen fertilization does not improve the yield wheat under used conditions. The use of urease inhibitor improves the grain yield of wheat crop when urea is applied in topdressing at tillering, but its use does not promote difference when urea is applied in the furrow at planting.

WHAT IS THE USE OF A SINGLE-CASE STUDY IN MANAGEMENT RESEARCH?

The purpose of this article is to present a defense of the use of single case studies in management research. The defense is necessary because this type of research has been relegated to a secondary role, or even rejected, by many researchers, who consider it unscientific. Evidence of this low status is the fact that most reputable academic journals in management publish few articles based on single-case studies. In this paper, we examine in detail the objections to the use of such cases in management research. We show the efforts made by some researchers to answer these objections and we show quality criteria for research that are alternatives to the criteria used in the so-called "scientific method." Our analysis suggests that a better understanding - by researchers with different methodological preferences - of the arguments for each particular use of the single-case study as a research method would allow a better dialogue between researchers and benefit management research as a whole.

Sustainable Tourism and Eradication of Poverty (Step): impact assessment of a tourism development program in Brazil

This paper aims to evaluate the social impacts of the Tourism Development Program (Prodetur) in the northeastern town of Porto Seguro, Bahia, Brazil. The method used is based on the difference in difference technique applied to the 1991 and 2000 Census microdata. The results suggest social advances following from poverty relief based on income - where the benefits are distributed, generally, in a relatively equal manner between the native and migrant population. There is a relative deterioration in the sanitary situation, which consists of a very serious problem in the mid- and long-term, whose costs are mostly borne by the native population. Therefore, maintaining the natural capital is the main aspect that distances Porto Seguro’s tourism supply from the concept of sustainability. The article also relies on difference in difference estimators to assess the impacts of local public policies related to the sector.