PERFORMANCE OF CONVENTIONAL PCRs BASED ON PRIMERS DIRECTED TO NUCLEAR AND MITOCHONDRIAL GENES FOR THE DETECTION AND IDENTIFICATION OF Leishmania spp.

In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs.

Human antibody responses to Schistosoma mansoni: does antigen directed, isotype restriction result in the production of blocking antibodies?

After treatment young Kenyan schoolchildren are highly susceptible to reinfection with Schistosoma mansoni. Older children and adults are resistant to reinfection. There is no evidence that this age related resistance is due to a slow development of protective immunological mechanisms, rather, it appears that young children are susceptible because of the presence of blocking antibodies which decline with age, thus allowing the expression of protective responses. Correlations between antibody responses to different stages of the parasite life-cycle suggest that, in young children, antigen directed, isotype restriction of the response against cross-reactive polysaccharide egg antigens results in an ineffectual, or even blocking antibody response to the schistosomulum.

A directed approach for the identification of transcripts harbouring the spliced leader sequence and the effect of trans-splicing knockdown in Schistosoma mansoni

Schistosomiasis is a major neglected tropical disease caused by trematodes from the genus Schistosoma. Because schistosomes exhibit a complex life cycle and numerous mechanisms for regulating gene expression, it is believed that spliced leader (SL) trans-splicing could play an important role in the biology of these parasites. The purpose of this study was to investigate the function of trans-splicing in Schistosoma mansoni through analysis of genes that may be regulated by this mechanism and via silencing SL-containing transcripts through RNA interference. Here, we report our analysis of SL transcript-enriched cDNA libraries from different S. mansoni life stages. Our results show that the trans-splicing mechanism is apparently not associated with specific genes, subcellular localisations or life stages. In cross-species comparisons, even though the sets of genes that are subject to SL trans-splicing regulation appear to differ between organisms, several commonly shared orthologues were observed. Knockdown of trans-spliced transcripts in sporocysts resulted in a systemic reduction of the expression levels of all tested trans-spliced transcripts; however, the only phenotypic effect observed was diminished larval size. Further studies involving the findings from this work will provide new insights into the role of trans-splicing in the biology of S. mansoni and other organisms. All Expressed Sequence Tags generated in this study were submitted to dbEST as five different libraries. The accessions for each library and for the individual sequences are as follows: (i) adult worms of mixed sexes (LIBEST_027999: JZ139310 - JZ139779), (ii) female adult worms (LIBEST_028000: JZ139780 - JZ140379), (iii) male adult worms (LIBEST_028001: JZ140380 - JZ141002), (iv) eggs (LIBEST_028002: JZ141003 - JZ141497) and (v) schistosomula (LIBEST_028003: JZ141498 - JZ141974).

Polymers as carriers for rhizobial inoculant formulations

The aim of this work was to evaluate the efficiency of carboxymethyl cellulose (CMC) and starch blends as carrier materials of rhizobial inoculants regarding their capacity to maintain viable cells and promote cowpea (Vigna unguiculata) nodulation. The experimental design adopted was completely randomized, with three replicates. Forty different compositions of carboxymethyl cellulose (CMC) with starch, compatibilized or not with different proportions of MgO or ZnO, were evaluated regarding their ability of maintaining rhizobial viable cells during the storage period of one month at room temperature, in an initial screening. Thereafter, selected inoculant carrier blends were evaluated regarding their ability to maintain viable rhizobial cells for a period of 165 days, and their performance as inoculant carriers was compared to a peat-based inoculant carrier under greenhouse conditions. Rhizobial cells were better maintained in blends containing 50-60% CMC. Compatibilizing agents did not increase survival of rhizobial cells for 30 days of storage. The cowpea nodulation of polymer blends was statistically the same of peat-based inoculants. CMC/starch polymer blends are efficient carriers to rhizobial inoculants for up to 165 days of storage, when compatibilized with MgO (1%).

Polímeros biomiméticos em química analítica. Parte 1: preparo e aplicações de MIP ("Molecularly Imprinted Polymers") em técnicas de extração e separação

MIPs are synthetic polymers that are used as biomimetic materials simulating the mechanism verified in natural entities such as antibodies and enzymes. Although MIPs have been successfully used as an outstanding tool for enhancing the selectivity or different analytical approaches, such as separation science and electrochemical and optical sensors, several parameters must be optimized during their synthesis. Therefore, the state-of-the-art of MIP production as well as the different polymerization methods are discussed. The potential selectivity of MIPs in the extraction and separation techniques focusing mainly on environmental, clinical and pharmaceutical samples as applications for analytical purposes is presented.

Polímeros biomiméticos em química analítica. Parte 2: aplicações de MIP ("Molecularly Imprinted Polymers") no desenvolvimento de sensores químicos

The aim of this paper is the description of the strategies and advances in the use of MIP in the development of chemical sensors. MIP has been considered an emerging technology, which allows the synthesis of materials that can mimic some highly specific natural receptors such as antibodies and enzymes. In recent years a great number of publications have demonstrated a growth in their use as sensing phases in the construction of sensors . Thus, the MIP technology became very attractive as a promising analytical tool for the development of sensors.

Use of new silica fillers as additives for polymers used in packaging of fruit

The objective of this work was to synthesize nanosilicas with different degree of hydrophobicity by the sol-gel method, using tetraethyl orthosilicate as a precursor. For this purpose, 3-aminopropyl triethoxysilane (APS) and 1,1,1,3,3,3 - hexamethyldisilazane (HMDS), were added during synthesis as modifiers. A commercial biopolymer (Hexamoll Dinch, BASF) intended for packaging of apples, was added to the new nanosilicas. The materials obtained were characterized by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, potentiometric titration, porosity, specific surface area and hydrophobicity/hydrophilicity by wetting test. Colorimetry was used to evaluate change in apple pulp color after contact with the different silicas.

Chiral separations of mandelic acid by HPLC using molecularly imprinted polymers

Styrene is used in a variety of chemical industries. Environmental and occupational exposures to styrene occur predominantly through inhalation. The major metabolite of styrene is present in two enantiomeric forms, chiral R- and S- hydroxy-1-phenyl-acetic acid (R-and S-mandelic acid, MA). Thus, the concentration of MA, particularly of its enantiomers, has been used in urine tests to determine whether workers have been exposed to styrene. This study describes a method of analyzing mandelic acid using molecular imprinting techniques and HPLC detection to perform the separation of diastereoisomers of mandelic acid. The molecularly imprinted polymer (MIP) was prepared by non-covalent molecular imprinting using (+) MA, (-) MA or (+) phenylalanine, (-) phenylalanine as templates. Methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) were copolymerized in the presence of the template molecules. The bulk polymerization was carried out at 4ºC under UV radiation. The resulting MIP was grounded into 25~44¼m particles, which were slurry packed into analytical columns. After the template molecules were removed, the MIP-packed columns were found to be effective for the chromatographic resolution of (±)-mandelic acid. This method is simpler and more convenient than other chromatographic methods.

Self-assembly of supermolecular species directed by hydrogen bonding and aromatic π-π stacking interactions

A new Cu(II) trimers, [Cu3(dcp)2(H2O)8]. 4DMF, with the ligand 3,5-pyrazoledicarboxylic acid monohydrate (H3dcp) has been prepared by solvent method. Its solid-state structure has been characterized by elemental analysis, thermal analysis (TGA and DSC), and single crystal X-ray diffraction. X-ray crystallographic studies reveal that this complex has extended 1-D,2-D and 3-D supramolecular architectures directed by weak interactions (hydrogen bond and aromatic π-π stacking interaction) leading to a sandwich solid-state structure.

Directed-spray application of paraquat and diuron in physic nut plants

There is little information about the selectivity of herbicides in physic nut (Jatropha curcas) in Brazil. Therefore, this study aimed to evaluate the selectivity of different doses and mixtures of paraquat and diuron in direted-spray applications in physic nut plants in greenhouse conditions. The study used a randomized block design, with five replicates. The treatments were: paraquat (200 and 600 g ha-1), diuron (1,000 and 2,000 g ha-1), paraquat + diuron (200 + 1,000 g ha-1), paraquat + diuron (200 + 2,000 g ha-1), paraquat + diuron (600 + 1,000 g ha-1), paraquat + diuron (600 + 2,000 g ha-1) and a control (no application). Directed-spray application was performed at 70 days after sowing by the lower third of the plants. The treatments of diuron and paraquat + diuron mixtures affected the growth and photosynthetic activity of physic nut plants, injuries being more pronounced at doses of diuron of 2,000 g ha‑1, while the isolated application of paraquat at doses of 200 and 600 g ha-1 showed good selectivity potential for physic nut plants.

Fingerprinting of cell lines by directed amplification of minisatellite-region DNA (DAMD)

The development of in vitro propagation of cells has been an extraordinary technical advance for several biological studies. The correct identification of the cell line used, however, is crucial, as a mistaken identity or the presence of another contaminating cell may lead to invalid and/or erroneous conclusions. We report here the application of a DNA fingerprinting procedure (directed amplification of minisatellite-region DNA), developed by Heath et al. [Nucleic Acids Research (1993) 21: 5782-5785], to the characterization of cell lines. Genomic DNA of cells in culture was extracted and amplified by PCR in the presence of VNTR core sequences, and the amplicons were separated by agarose gel electrophoresis. After image capture with a digital camera, the banding profiles obtained were analyzed using a software (AnaGel) specially developed for the storage and analysis of electrophoretic fingerprints. The fingerprints are useful for construction of a data base for identification of cell lines by comparison to reference profiles as well as comparison of similar lines from different sources and periodic follow-up of cells in culture.

Target-directed catalytic metallodrugs

Most drugs function by binding reversibly to specific biological targets, and therapeutic effects generally require saturation of these targets. One means of decreasing required drug concentrations is incorporation of reactive metal centers that elicit irreversible modification of targets. A common approach has been the design of artificial proteases/nucleases containing metal centers capable of hydrolyzing targeted proteins or nucleic acids. However, these hydrolytic catalysts typically provide relatively low rate constants for target inactivation. Recently, various catalysts were synthesized that use oxidative mechanisms to selectively cleave/inactivate therapeutic targets, including HIV RRE RNA or angiotensin converting enzyme (ACE). These oxidative mechanisms, which typically involve reactive oxygen species (ROS), provide access to comparatively high rate constants for target inactivation. Target-binding affinity, co-reactant selectivity, reduction potential, coordination unsaturation, ROS products (metal-associated vsmetal-dissociated; hydroxyl vs superoxide), and multiple-turnover redox chemistry were studied for each catalyst, and these parameters were related to the efficiency, selectivity, and mechanism(s) of inactivation/cleavage of the corresponding target for each catalyst. Important factors for future oxidative catalyst development are 1) positioning of catalyst reduction potential and redox reactivity to match the physiological environment of use, 2) maintenance of catalyst stability by use of chelates with either high denticity or other means of stabilization, such as the square planar geometric stabilization of Ni- and Cu-ATCUN complexes, 3) optimal rate of inactivation of targets relative to the rate of generation of diffusible ROS, 4) targeting and linker domains that afford better control of catalyst orientation, and 5) general bio-availability and drug delivery requirements.

The adherence of Pseudomonas fluorescens to marble, granite, synthetic polymers, and stainless steel

The adherence of Pseudomonas fluorescens cells to nine food-processing contact surfaces was evaluated using the plate-count method. The surfaces include marble, granite, stainless steel, polyvinyl chloride, polyurethane, and silicone-coated cloth, which have been used only in a few studies concerning bacterial adherence. The number of cells adhered to the surfaces increased with contact time reaching 5.0-6.1 log CDM.cm-2 after 10 hours, which can be considered a well established adherence process. The number of adhered cells doubled in 29.5 minutes and 23.5 minutes on stainless steel and thin polyvinyl chloride-coated cloth, respectively. For the other surfaces, this value was 9.8 minutes on average. Marble, granite, thick polyvinyl-coated cloth, double-faced rugous polyurethane, and silicone-coated cloth were not different (p < 0.05) in their ability to adhere cells (CFU/cm²) after 2 and 10 hours. The surfaces that had higher percentage of similarity in the adhesion level and higher log CFU/cm² of adhered cells were double-faced rugous polyurethane, silicone-coated cloth, and granite. The surfaces showed very different microtopography characteristics when viewed using scanning electron microscopy. This experiment showed the importance of using appropriate materials for food contact during processing, which will affect the cleaning and sanitation procedures.