A century of research: what have we learned about the interaction of Trypanosoma cruzi with host cells?

Since the discovery of Trypanosoma cruzi and the brilliant description of the then-referred to "new tripanosomiasis" by Carlos Chagas 100 years ago, a great deal of scientific effort and curiosity has been devoted to understanding how this parasite invades and colonises mammalian host cells. This is a key step in the survival of the parasite within the vertebrate host, and although much has been learned over this century, differences in strains or isolates used by different laboratories may have led to conclusions that are not as universal as originally interpreted. Molecular genotyping of the CL-Brener clone confirmed a genetic heterogeneity in the parasite that had been detected previously by other techniques, including zymodeme or schizodeme (kDNA) analysis. T. cruzi can be grouped into at least two major phylogenetic lineages: T. cruzi I, mostly associated with the sylvatic cycle and T. cruzi II, linked to human disease; however, a third lineage, T. cruziIII, has also been proposed. Hybrid isolates, such as the CL-Brener clone, which was chosen for sequencing the genome of the parasite (Elias et al. 2005, El Sayed et al. 2005a), have also been identified. The parasite must be able to invade cells in the mammalian host, and many studies have implicated the flagellated trypomastigotes as the main actor in this process. Several surface components of parasites and some of the host cell receptors with which they interact have been described. Herein, we have attempted to identify milestones in the history of understanding T. cruzi- host cell interactions. Different infective forms of T. cruzi have displayed unexpected requirements for the parasite to attach to the host cell, enter it, and translocate between the parasitophorous vacuole to its final cytoplasmic destination. It is noteworthy that some of the mechanisms originally proposed to be broad in function turned out not to be universal, and multiple interactions involving different repertoires of molecules seem to act in concert to give rise to a rather complex interplay of signalling cascades involving both parasite and cellular components.

Sesquiterpenes from the essential oil of Laurencia dendroidea (Ceramiales, Rhodophyta): isolation, biological activities and distribution among seaweeds

Two known sesquiterpenes (1R*,2S*,3R*,5S*,8S*,9R*)-2,3,5,9-tetramethyltricyclo[6.3.0.0(1,5)]undecan-2-ol and (1S*,2S*,3S*,5S*,8S*,9S*)-2,3,5,9-tetramethyltricyclo-[6.3.0.0(1,5)]undecan-2-ol were isolated for the first time from the essential oil of the red seaweed Laurencia dendroidea collected in the Brazilian coast. These compounds were not active against eight bacteria strains and the yeast Candida albicans, but showed some antioxidant activity. Both compounds were also found in other seaweed species showing that they are not exclusive taxonomic markers to the genus Laurencia.

Characterization of Vibrio Parahaemolyticus isolated from oysters and mussels in São Paulo, Brazil

Vibrio parahaemolyticus is a marine bacterium, responsible for gastroenteritis in humans. Most of the clinical isolates produce thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) encoded by tdh and trh genes respectively. In this study, twenty-three V. parahaemolyticus, previously isolated from oysters and mussels were analyzed by PCR using specific primers for the 16S rRNA and virulence genes (tdh, trh and tlh) and for resistance to different classes of antibiotics and PFGE. Nineteen isolates were confirmed by PCR as V. parahaemolyticus. The tlh gene was present in 100% of isolates, the tdh gene was identified in two (10.5%) isolates, whereas the gene trh was not detected. Each isolate was resistant to at least one of the nine antimicrobials tested. Additionally, all isolates possessed the blaTEM-116 gene. The presence of this gene in V. parahaemolyticus indicates the possibility of spreading this gene in the environment. Atypical strains of V. parahaemolyticus were also detected in this study.

Microbiological and histopathological aspects of canine pyometra

As pyometra is recognized as one of the main causes of disease and death in the bitch the purposes of this study were to evaluate microbiological and histopathological aspects of canine pyometra and to research the virulence factors of the E. coli isolates identifying possible risks to human health. The microbiological isolation from the intrauterine contents of 100 dogs with pyometra was carried out and the virulence factors in the E. coli strains were identified using PCR method. This study also consisted of the counting of microorganisms colonies forming units in samples of intrauterine content, tests of antimicrobial susceptibility of the E. coli isolates and the histological examination of the uterus. E. coli was the most prevalent microorganism isolated (76.6%) and 120 strains (79.5%) were positive for sfa, 86 (56.9%) were positive for cnf, 87 (57.6%) were positive for pap, 52 (34.4%) were positive for hly, 51 (33.8%) were positive for iuc and 5 (3.3%) were positive for afa genes. One observed more sensitivity of E. coli to norfloxacin, polimixin B, sulphazotrin, chloranfenicol and enrofloxacin. In 42% of the samples of uterine walls where microorganisms were isolated, the sizes of the areas of the inflammatory responses corresponded to 39-56%. Virulence factors were identified in 98.0% of the strains evaluated, demonstrating a high frequency of potentially pathogenic E. coli. It must be considered that dogs are animals that are living in close proximity to man for thousands of years and have an important role in the transmission of E. coli to other animals and to man.

Emergence and disappearance of a virulent clone of Haemophilus influenzae biogroup aegyptius, cause of Brazilian Purpuric Fever

Summary: In 1984, children presented to the emergency department of a hospital in the small town of Promissão, São Paulo State, Brazil, with an acute febrile illness that rapidly progressed to death. Local clinicians and public health officials recognized that these children had an unusual illness, which led to outbreak investigations conducted by Brazilian health officials in collaboration with the U.S. Centers for Disease Control and Prevention. The studies that followed are an excellent example of the coordinated and parallel studies that are used to investigate outbreaks of a new disease, which became known as Brazilian purpuric fever (BPF). In the first outbreak investigation, a case-control study confirmed an association between BPF and antecedent conjunctivitis but the etiology of the disease could not be determined. In a subsequent outbreak, children with BPF were found to have bacteremia caused by Haemophilus influenzae biogroup aegyptius (H. aegyptius), an organism previously known mainly to cause self-limited purulent conjunctivitis. Molecular characterization of blood and other isolates demonstrated the clonal nature of the H. aegyptius strains that caused BPF, which were genetically distant from the diverse strains that cause only conjunctivitis. This led to an intense effort to identify the factors causing the unusual invasiveness of the BPF clone, which has yet to definitively identify the virulence factor or factors involved. After a series of outbreaks and sporadic cases through 1993, no additional cases of BPF have been reported

Aeromonas detection and their toxins from drinking water form reservoirs and drinking fountains

Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. A total of 200 drinking water samples from domestic and public reservoirs and drinking fountains located in São Paulo (Brazil), were analyzed for the presence of Aeromonas. Samples were concentrated by membrane filtration and enriched in APW. ADA medium was used for Aeromonas isolation and colonies were confirmed by biochemical characterization. Strains isolated were tested for hemolysin and toxin production. Aeromonas was detected in 12 samples (6.0%). Aeromonas strains (96) were isolated and identified as: A. caviae (41.7%), A. hydrophila (15.7%), A.allosacharophila (10.4%), A. schubertii (1.0%) and Aeromonas spp. (31.2%).The results revealed that 70% of A. caviae, 66.7% of A. hydrophila, 80% of A. allosacharophila and 46.6% of Aeromonas spp. were hemolytic. The assay for checking production of toxins showed that 17.5% of A. caviae, 73.3% of A. hydrophila, 60% of A. allosacharophila, 100% of A. schubertii, and 33.3% of Aeromonas spp. were able to produce toxins. The results demonstrated the pathogenic potential of Aeromonas, indicating that the presence of this emerging pathogen in water systems is a public health concern

Extended-spectrum beta-lactamases among Enterobacteriaceae isolated in a public hospital in Brazil

Extended-spectrum beta-lactamases (ESBL) in enterobacteria are recognized worldwide as a great hospital problem. In this study, 127 ESBL-producing Enterobacteriaceae isolated in one year from inpatients and Outpatients at a public teaching hospital at Sao Paulo, Brazil, were Submitted to analysis by PCR with specific primers for bla(SHV), bla(TEM) and bla(CTX-M) genes. From the 127 isolates, 96 (75.6%) Klebsiella pneumoniae, 12 (9.3%) Escherichia coli, 8 (6.2%) Morganella morganii, 3 (2.3%) Proteus mirabilis, 2 (1.6%) Klebsiella oxytoca, 2 (1.6%) Providencia rettgeri, 2 (1.6%) Providencia stuartti, 1 (0.8%) Enterobacter aerogenes and 1 (0.8%) Enterobacter cloacae were identified as ESBL producers. Bla(SHV), bla(TEM), and bla(CTX-M) were detected in 63%, 17.3% and 33.9% strains, respectively. Pulsed field get eletrophoresis genotyping of K. pneumoniae revealed four main molecular patterns and 29 unrelated profiles. PCR results showed a high variety of ESBL groups among strains, in nine different species. The results Suggest the spread of resistance genes among genetically different strains of ESBL-producing K. pneumoniae in some hospital wards, and also that some strongly related strains were identified in different hospital wards, Suggesting clonal spread in the institutional environment

Modulatory effect of Byrsonima basiloba extracts on the mutagenicity of certain direct and indirect-acting mutagens in Salmonella typhimurium assays

Byrsonima basiloba A. Juss. species is a native arboreal type from the Brazilian ""cerrado"" (tropical American savanna), and the local population uses it to treat diseases, such as diarrhea and gastric ulcer. It belongs to the Malpighiaceae family, and it is commonly known as ""murici."" Considering the popular use of B. basiloba derivatives and the lack of pharmacological potential studies regarding this vegetal species, the mutagenic and antimutagenic effect of methanol (MeOH) and chloroform extracts were evaluated by the Ames test, using strains TA97a, TA98, TA100, and TA102 of Salmonella typhimurium. No mutagenic activity was observed in any of the extracts. To evaluate the antimutagenic potential, direct and indirect mutagenic agents were used: 4 nitro-o-phenylenediamine, sodium azide, mitomycin C, aflatoxin B(1), benzo[a] pyrene, and hydrogen peroxide. Both the extracts evaluated showed antimutagenic activity, but the highest value of inhibition level (89%) was obtained with the MeOH extract and strain TA100 in the presence of aflatoxin B(1). Phytochemical analysis of the extracts revealed the presence of n-alkanes, lupeol, ursolic and oleanolic acid, (+)-catechin, quercetin- 3-O-alpha-L-arabinopyranoside, gallic acid, methyl gallate, amentoflavone, quercetin, quercetin-3-O-(2 ''-O-galloyl)-beta-D-galactopyranoside, and quercetin-3-O-(2 ''-O-galloyl)-alpha-L-arabinopyranoside.

The full Bayesian significance test for mixture models: results in gene expression clustering

Gene clustering is a useful exploratory technique to group together genes with similar expression levels under distinct cell cycle phases or distinct conditions. It helps the biologist to identify potentially meaningful relationships between genes. In this study, we propose a clustering method based on multivariate normal mixture models, where the number of clusters is predicted via sequential hypothesis tests: at each step, the method considers a mixture model of m components (m = 2 in the first step) and tests if in fact it should be m - 1. If the hypothesis is rejected, m is increased and a new test is carried out. The method continues (increasing m) until the hypothesis is accepted. The theoretical core of the method is the full Bayesian significance test, an intuitive Bayesian approach, which needs no model complexity penalization nor positive probabilities for sharp hypotheses. Numerical experiments were based on a cDNA microarray dataset consisting of expression levels of 205 genes belonging to four functional categories, for 10 distinct strains of Saccharomyces cerevisiae. To analyze the method's sensitivity to data dimension, we performed principal components analysis on the original dataset and predicted the number of classes using 2 to 10 principal components. Compared to Mclust (model-based clustering), our method shows more consistent results.

Direct measurement of intrinsic atomic scale magnetostriction

Using differential x-ray absorption spectroscopy (DiffXAS) we have measured and quantified the intrinsic, atomic-scale magnetostriction of Fe(81)Ga(19). By exploiting the chemical selectivity of DiffXAS, the Fe and Ga local environments have been assessed individually. The enhanced magnetostriction induced by the addition of Ga to Fe was found to originate from the Ga environment, where lambda(gamma,2)(approximate to (3/2)lambda(100)) is 390 +/- 40 ppm. In this environment, < 001 > Ga-Ga pair defects were found to exist, which mediate the magnetostriction by inducing large strains in the surrounding Ga-Fe bonds. For the first time, intrinsic, chemically selective magnetostrictive strain has been measured and quantified at the atomic level, allowing true comparison with theory.

Environmental isolation of black yeast-like fungi involved in human infection

The present study focuses on potential agents of chromoblastomycosis and other endemic diseases in the state of Parana, Southern Brazil. Using a highly selective protocol for chaetothyrialean black yeasts and relatives, environmental samples from the living area of symptomatic patients were analysed. Additional strains were isolated from creosote-treated wood and hydrocarbon-polluted environments, as such polluted sites have been supposed to enhance black yeast prevalence. Isolates showed morphologies compatible with the traditional etiological agents of chromoblastomycosis, e.g. Fonsecaea pedrosoi and Phialophora verrucosa, and of agents of subcutaneous or systemic infections like Cladophialophora bantiana and Exophiala jeanselmei. Some agents of mild disease were indeed encountered. However, molecular analysis proved that most environmental strains differed from known etiologic agents of pronounced disease syndromes: they belonged to the same order, but mostly were undescribed species. Agents of chromoblastomycosis and systemic disease thus far are prevalent on the human host. The hydrocarbon-polluted environments yielded yet another spectrum of chaetothyrialean fungi. These observations are of great relevance because they allow us to distinguish between categories of opportunists, indicating possible differences in pathogenicity and virulence.

Enzymatic differences between the endophyte Guignardia mangiferae (Botryosphaeriaceae) and the citrus pathogen G. citricarpa

The endophyte Guignardia mangiferae is closely related to G. citricarpa, the causal agent of citrus black spot; for many years these species had been confused with each other. The development of molecular analytical methods has allowed differentiation of the pathogen G. citricarpa from the endophyte G. mangiferae, but the physiological traits associated with pathogenicity were not described. We examined genetic and enzymatic characteristics of Guignardia spp strains; G. citricarpa produces significantly greater amounts of amylases, endoglucanases and pectinases, compared to G. mangiferae, suggesting that these enzymes could be key in the development of citrus black spot. Principal component analysis revealed pectinase production as the main enzymatic characteristic that distinguishes these Guignardia species. We quantified the activities of pectin lyase, pectin methylesterase and endopolygalacturonase; G. citricarpa and G. mangiferae were found to have significantly different pectin lyase and endopolygalacturonase activities. The pathogen G. citricarpa is more effective in pectin degradation. We concluded that there are significant physiological differences between the species G. citricarpa and G. mangiferae that could be associated with differences in pathogenicity for citrus plants.

Origins of the Xylella fastidiosa Prophage-Like Regions and Their Impact in Genome Differentiation

Xylella fastidiosa is a Gram negative plant pathogen causing many economically important diseases, and analyses of completely sequenced X. fastidiosa genome strains allowed the identification of many prophage-like elements and possibly phage remnants, accounting for up to 15% of the genome composition. To better evaluate the recent evolution of the X. fastidiosa chromosome backbone among distinct pathovars, the number and location of prophage-like regions on two finished genomes (9a5c and Temecula1), and in two candidate molecules (Ann1 and Dixon) were assessed. Based on comparative best bidirectional hit analyses, the majority (51%) of the predicted genes in the X. fastidiosa prophage-like regions are related to structural phage genes belonging to the Siphoviridae family. Electron micrograph reveals the existence of putative viral particles with similar morphology to lambda phages in the bacterial cell in planta. Moreover, analysis of microarray data indicates that 9a5c strain cultivated under stress conditions presents enhanced expression of phage anti-repressor genes, suggesting switches from lysogenic to lytic cycle of phages under stress-induced situations. Furthermore, virulence-associated proteins and toxins are found within these prophage-like elements, thus suggesting an important role in host adaptation. Finally, clustering analyses of phage integrase genes based on multiple alignment patterns reveal they group in five lineages, all possessing a tyrosine recombinase catalytic domain, and phylogenetically close to other integrases found in phages that are genetic mosaics and able to perform generalized and specialized transduction. Integration sites and tRNA association is also evidenced. In summary, we present comparative and experimental evidence supporting the association and contribution of phage activity on the differentiation of Xylella genomes.

Behavior and viability of spontaneous oxidative stress-resistant Lactococcus lactis mutants in experimental fermented milk processing

Previously, we isolated two strains of spontaneous oxidative (SpOx2 and SpOx3) stress mutants of Lactococcus lactis subsp cremoris. Herein, we compared these mutants to a parental wild- type strain (J60011) and a commercial starter in experimental fermented milk production. Total solid contents of milk and fermentation temperature both affected the acidification profile of the spontaneous oxidative stress- resistant L. lactis mutants during fermented milk production. Fermentation times to pH 4.7 ranged from 6.40 h (J60011) to 9.36 h (SpOx2); V(max) values were inversely proportional to fermentation time. Bacterial counts increased to above 8.50 log(10) cfu/mL. The counts of viable SpOx3 mutants were higher than those of the parental wild strain in all treatments. All fermented milk products showed post-fermentation acidification after 24 h of storage at 4 degrees C; they remained stable after one week of storage.

Long Terminal Repeat Sequence Analysis of HTLV-2 Molecular Variants Identified in Southern Brazil

In Brazil, human T-lymphotropic virus type 2 (HTLV-2) is endemic in Amerindians and epidemic in intravenous drug users (IDUs). The long terminal repeat (LTR) is the most divergent genomic region of HTLV-2, therefore useful to characterize subtypes. Nucleotide sequence and restriction fragment length polymorphism (RFLP) analysis of LTR genomic segments of fourteen HTLV-2 strains isolated from HIV-infected patients of Londrina, Southern Brazil, were carried out. Molecular analysis disclosed that all HTLV-2 strains belonged to 2a subtype, and RFLP detected the presence of the a4, a5, and a6 subgroups according to Switzer's nomenclature. RFLP correlated with nucleotide sequence, and phylogenetic analysis clustered HTLV-2 sequences of IDUs into subgroups a5 and a6. HTLV-2 sequences from individuals of sexual risk factor clustered into the a4 subgroup. These results extend the knowledge of the genetic diversity of HTLV-2 circulating in Brazil and provide insights into HTLV-2 transmission and virus movement in this geographic area.