Escherichia coli phylogenetic group determination and its application in the identification of the major animal source of fecal contamination

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expressed sequence tags from Atta laevigata and identification of candidate genes for the control of pest leaf-cutting ants.

Leafcutters are the highest evolved within Neotropical ants in the tribe Attini and model systems for studying caste formation, labor division and symbiosis with microorganisms. Some species of leafcutters are agricultural pests controlled by chemicals which affect other animals and accumulate in the environment. Aiming to provide genetic basis for the study of leafcutters and for the development of more specific and environmentally friendly methods for the control of pest leafcutters, we generated expressed sequence tag data from Atta laevigata, one of the pest ants with broad geographic distribution in South America. Results: The analysis of the expressed sequence tags allowed us to characterize 2,006 unique sequences in Atta laevigata. Sixteen of these genes had a high number of transcripts and are likely positively selected for high level of gene expression, being responsible for three basic biological functions: energy conservation through redox reactions in mitochondria; cytoskeleton and muscle structuring; regulation of gene expression and metabolism. Based on leafcutters lifestyle and reports of genes involved in key processes of other social insects, we identified 146 sequences potential targets for controlling pest leafcutters. The targets are responsible for antixenobiosis, development and longevity, immunity, resistance to pathogens, pheromone function, cell signaling, behavior, polysaccharide metabolism and arginine kynase activity. Conclusion: The generation and analysis of expressed sequence tags from Atta laevigata have provided important genetic basis for future studies on the biology of leaf-cutting ants and may contribute to the development of a more specific and environmentally friendly method for the control of agricultural pest leafcutters.

Primary cutaneous amyloidosis: Clinical, laboratorial and histopathological study of 25 cases. Identification of gammaglobulins and C3 in the lesions by immunofluorescence

25 cases of primary cutaneous amyloidosis are studied. 16 patients had macular amyloidosis (MPA) and 9 lichen amyloidosus (LPA). γ-Globulins were increased in 50% of the patients. IgG and IgA were increased in the serum of 5 and 3 patients with MPA and 4 and 2 patients with LPA, respectively. Volume of amyloid deposits was similar in both forms. By direct immunofluorescence we demonstrated IgG in the amyloid deposits of 21 of the 25 cases and C3 in 13; IgM was present in 9 cases of MPA and in 3 cases of LPA. MPA was more frequent than LPA; histologically, it was impossible to distinguish MPA from LPA; correlation between serum levels of γ-globulins and their presence in amyloid deposits was weak; MPA and LPA seem to be distinct clinical manifestations of the same disease and itching does not cause transformation of MPA in LPA.

Western blot analysis of tick antigens from a Rhipicephalus sanguineus unfed larval extract and identification of antigenic sites in tick sections using immunohistochemistry. A comparative study between resistant and susceptible host species

Most parasite-host relationships are characterized by the development of resistance by the host, thus limiting the number of parasites. However, some cases are very unusual. In the relationship of the domestic dog with the brown dog-tick Rhipicephalus sanguineus this does not occur, whereas guinea pigs develop efficient resistance. Sera from domestic dogs, crab-eating foxes and guinea pigs collected before and after infestation with R. sanguineus ticks, and after immunization with a whole tick adult or larval homogenate, were used in Western blot analysis to compare and identify potential important antigens from a tick larval homogenate. The same sera were tested in an indirect immunohistochemistry assay in an attempt to compare relevant antigenic sites on histological tick sections. The immunoblotting displayed antigens recognized only by the guinea pigs, as well as several shared antigens between host species, depending on the kind of immunization. Immunohistochemistry revealed probable antigenic sites on the cells and tissues of ticks, which varied depending on the kind of immunization (infestation or vaccination) and the animal species involved.

The diagnostic value of Gram stain for initial identification of the etiologic agent of peritonitis in CAPD patients

Objective: To evaluate the effectiveness of the Gram stain in the initial diagnosis of the etiologic agent of peritonitis in continuous ambulatory peritoneal dialysis (CAPD). Design: Retrospective study analyzing the sensitivity (S), specificity (SS), positive predictive value (+PV), and negative predictive value (-PV) of the Gram stain relating to the results of cultures in 149 episodes of peritonitis in CAPD. The data were analyzed in two studies. In the first, only the cases with detection of a single agent by Gram stain were taken (Study 1). In the second, only the cases with two agents in Gram stain were evaluated (Study 2). Setting: Dialysis Unit and Laboratory of Microbiology of a tertiary medical center. Patients: Sixty-three patients on regular CAPD who presented one or more episodes of peritonitis from May 1992 to May 1995. Results: The positivity of Gram stain was 93.2% and the sensitivity was 95.7%. The values of S, SS, +PV, and -PV were respectively: 94.9%, 53.5%, 68.3%, and 90.9% for gram-positive cocci and 83.3%, 98.8%, 95.2%, and 95.6% for gram-negative bacilli. The association of gram-positive cocci plus gram-negative bacilli were predictive of growth of both in 6.8%, growth of gram-positive cocci in 13.7%, and growth of gram-negative bacilli in 72.5%. Conclusions: The Gram stain is a method of great value in the initial diagnosis of the etiologic agent of peritonitis in CAPD, especially for gram-negative bacilli.

A SequenceSpace analysis of Lys49 phospholipases A2: Clues towards identification of residues involved in a novel mechanism of membrane damage and in myotoxicity

'SequenceSpace' analysis is a novel approach which has been used to identify unique amino acids within a subfamily of phospholipases A2 (PLA2) in which the highly conserved active site residue Asp49 is substituted by Lys (Lys49-PLA2s). Although Lys49-PLA2s do not bind the catalytic co-factor Ca2+ and possess extremely low catalytic activity, they demonstrate a Ca2+-independent membrane damaging activity through a poorly understood mechanism, which does not involve lipid hydrolysis. Additionally, Lys49-PLA2s possess combined myotoxic, oedema forming and cardiotoxic pharmacological activities, however the structural basis of these varied functions is largely unknown. Using the 'SequenceSpace' analysis we have identified nine residues highly unique to the Lys49-PLA2 sub-family, which are grouped in three amino acid clusters in the active site, hydrophobic substrate binding channel and homodimer interface regions. These three highly specific residue clusters may have relevance for the Ca2+-independent membrane damaging activity. Of a further 15 less stringently conserved residues, nine are located in two additional clusters which are well isolated from the active site region. The less strictly conserved clusters have been used in predictive sequence searches to correlate amino acid patterns in other venom PLA2s with their pharmacological activities, and motifs for presynaptic and combined toxicities are proposed.

Evaluation and identification of lightning models by artificial neural networks

This paper describes a novel approach for mapping lightning models using artificial neural networks. The networks acts as identifier of structural features of the lightning models so that output parameters can be estimated and generalized from an input parameter set. Simulation examples are presented to validate the proposed approach. More specifically, the neural networks are used to compute electrical field intensity and critical disruptive voltage taking into account several atmospheric and structural factors, such as pressure, temperature, humidity, distance between phases, height of bus bars, and wave forms. A comparative analysis with other approaches is also provided to illustrate this new methodology.

Application of HPLC-NMR coupling using C30 phase in the separation and identification of flavonoids of taxonomic relevance

The on-line separation and identification of two important taxonomic markers for plant species of the Paepalanthus genus, the flavonoids 6-methoxykaempferol-3-O-β-D-glucopyranoside and 6-methoxykaempferol-3-O-β-D-6″(p-coumaroyl)glucopyranoside, has been performed with an HPLC-NMR coupling using C30 phase. 1D spectra have been recorded in the stopped-flow mode for the two predominant chromatographic peaks. This is the first application of HPLC-NMR coupling using C30 phase to a taxonomic problem. The technique drastically reduces the required amount of sampling for structure determination. © Springer-Verlag 2000.

Modeling and identification of fertility maps using artificial neural networks

The application of agricultural fertilizers using variable rates along the field can be made through fertility maps previously elaborated or through real-time sensors. In most of the cases applies maps previously elaborated. These maps are identified from analyzes done in soil samples collected regularly (a sample for each field cell) or irregularly along the field. At the moment, mathematical interpolation methods such as nearest neighbor, local average, weighted inverse distance, contouring and kriging are used for predicting the variables involved with elaboration of fertility maps. However, some of these methods present deficiencies that can generate different fertility maps for a same data set. Moreover, such methods can generate inprecise maps to be used in precision farming. In this paper, artificial neural networks have been applied for elaboration and identification of precise fertility maps which can reduce the production costs and environmental impacts.

Identification of synchronous machine parameters using load rejection test data

This work shows a computational methodology for the determination of synchronous machines parameters using load rejection test data. By machine modeling one can obtain the quadrature parameters through a load rejection under an arbitrary reference, reducing the present difficulties. The proposed method is applied to a real machine.

Comparison of a multiplex-PCR assay with mycolic acids analysis and conventional methods for the identification of Mycobacteria

A fast, sensitive and cost-effective multiplex-PCR assay for Mycobacterium tuberculosis complex (MTC) and Mycobacterium avium (M. avium) identification for routine diagnosis was evaluated. A total of 158 isolates of mycobacteria from 448 clinical specimens from patients with symptoms of mycobacterial disease were analyzed. By conventional biochemical methods 151 isolates were identified as M. tuberculosis, five as M. avium and two as Mycobacterium chelonae (M. chelonae). Mycolic acid patterns confirmed these results. Multiplex-PCR detected only IS6110 in isolates identified as MTC, and IS1245 was found only in the M. avium isolates. The method applied to isolates from two patients, identified by conventional methods and mycolic acid analysis, one as M. avium and other as M. chelonae, resulted positive for IS6110, suggesting co-infection with M. tuberculosis. These patients were successfully submitted to tuberculosis treatment. The multiplex-PCR method may offer expeditious identification of MTC and M. avium, which may minimize risks for active transmission of these organisms and provide useful treatment information.

rDNA-RFLP identification of Candida species in immunocompromised and seriously diseased patients

PCR was used to amplify a targeted region of the ribosomal DNA of 76 Candida spp. isolates from immunocompromised and seriously diseased patients. Thirty-seven strains isolated from different anatomical sites of 11 patients infected with HIV (Vitória, ES, Brazil), 26 isolates from patients under treatment at Odilon Behrens Hospital and 13 isolates from skin and urine samples from São Marcos Clinical Analysis Laboratory (Belo Horizonte, Brazil) were scored. Fragments of rDNA were amplified using primer pairs ITS1-ITS4, for the amplification of ITS1 and ITS2 regions, including the gene for the 5.8s subunit. Amplification resulted in fragments ranging in size from 350 to 950 bp. Amplicons were digested with eight restriction enzymes. A pattern of species-specificity among the different medically important Candida species could be identified following restriction digestion of the PCR products. Candida albicans was the species most frequently observed, except for the group of newborns under treatment at the Odilon Behrens Hospital and for the isolates from the clinical analysis laboratory. C. parapsilosis was the species most frequently observed in these two groups.

Arbitrarily primed polymerase chain reaction for fingerprinting the genotype identification of mutans streptococci in children with Down syndrome

This study investigated the possible intrafamilial similarity of mutans streptococcal strains in some families with a child with Down syndrome using chromosomal DNA fingerprinting. The isolates were genotyped using arbitrarily primed polymerase chain reaction with the OPA 02 and OPA 03 primers. The results showed that five children with Down syndrome harbored mutans streptococci genotypes different from those of their mothers. A matching of genotypes was observed within the control pair (mother/child without Down syndrome). After six months, new samples were collected from all participants. Analysis showed that samples from children with Down syndrome were colonized by a new strain of Streptococcus mutans that did not match the previously collected one. The results suggest the S. mutans indigenous bacteria change more than once in children with Down syndrome.