Morphological and molecular characterization of cyanobacteria from a Brazilian facultative wastewater stabilization pond and evaluation of microcystin production

The cyanobacterial population in the Cajati waste stabilization pond system (WSP) from Sao Paulo State, Brazil was assessed by cell isolation and direct microscope counting techniques. Ten strains, belonging to five genera (Synechococcus, Merismopedia, Leptolyngbya, Limnothrix, and Nostoc), were isolated and identified by morphological and molecular analyses. Morphological identification of the isolated strains was congruent with their phylogenetic analyses based on 16S rDNA gene sequences. Six cyanobacterial genera (Synechocystis, Aphanocapsa, Merismopedia, Lyngbya, Phormidium, and Pseudanabaena) were identified by direct microscope inspection. Both techniques were complementary, since, of the six genera identified by direct microscopic inspection, only Merismopedia was isolated, and the four other isolated genera were not detected by direct inspection. Direct microscope counting of preserved cells showed that cyanobacteria were the dominant members (> 90%) of the phytoplankton community during both periods evaluated (summer and autumn). ELISA tests specific for hepatotoxicmicrocystins gave positive results for six strains (Synechococcus CENA108, Merismopedia CENA106, Leptolyngbya CENA103, Leptolyngbya CENA112, Limnothrix CENA109, and Limnothrix CENA110), and for wastewater samples collected from raw influent (3.70 mu g microcystins/l) and treated effluent (3.74 mu g microcystins/l) in summer. Our findings indicate that toxic cyanobacteria in WSP systems are of concern, since the treated effluent containing cyanotoxins will be discharged into rivers, irrigation channels, estuaries, or reservoirs, and can affect human and animal health.

Molecular identification of naturally occurring bacteriocinogenic and bacteriocinogenic-like lactic acid bacteria in raw milk and soft cheese

Lactic acid bacteria ( LAB) are currently used by food industries because of their ability to produce metabolites with antimicrobial activity against gram-positive pathogens and spoilage microorganisms. The objectives of this study were to identify naturally occurring bacteriocinogenic or bacteriocinogenic-like LAB in raw milk and soft cheese and to detect the presence of nisin-coding genes in cultures identified as Lactococcus lactis. Lactic acid bacteria cultures were isolated from 389 raw milk and soft cheese samples and were later characterized for the production of antimicrobial substances against Listeria monocytogenes. Of these, 58 (14.9%) LAB cultures were identified as antagonistic; the nature of this antagonistic activity was then characterized via enzymatic tests to confirm the proteinaceous nature of the antimicrobial substances. In addition, 20 of these antagonistic cultures were selected and submitted to genetic sequencing; they were identified as Lactobacillus plantarum (n = 2) and Lactococcus lactis ssp. lactis (n = 18). Nisin genes were identified by polymerase chain reaction in 7 of these cultures. The identified bacteriocinogenic and bacteriocinogenic-like cultures were highly variable concerning the production and activity of antimicrobial substances, even when they were genetically similar. The obtained results indicated the need for molecular and phenotypic methodologies to properly characterize bacteriocinogenic LAB, as well as the potential use of these cultures as tools to provide food safety.

Hydrothermal stable templated molecular sieve silica (TMSS) membranes for gas separation

In this work we compare the hydrothermal stability performance of a Templated Molecular Sieve Silica (TMSS) membrane against a standard, non-templated Molecular Sieve Silica (MSS) membrane. The tests were carried under dry and wet (steam) conditions for single gas (He, H2, CO and CO2) at 1-2 atm membrane pressure drop at 200oC. Single gas TMSS membrane H2, permeance and H2/CO permselectivity was found to be 2.05 x 10-8 mols.m-2.s-1.Pa-1 and 15, respectively. The MSS membrane showed similar selectivity, but increased overall flux. He permeance through membranes decayed at a rate of 4-5 x 10-10 mols.m-2.s-1.Pa-1 per day regardless of membrane ambience (dry or wet). Although H2/CO permselectivity of the TMSS membrane slightly improved from 15 to 18 after steam testing, the MSS membrane resulted in significant reduction from 16 to 8.3. In addition, membrane regeneration after more than 50 days resulted in the TMSS membrane reverting to its original permeation levels while no significant improvements were observed for the MSS membra ne. Results showed that the TMSS membrane had enhanced hydrothermal stability and regeneration ability.

Hemichordates and deuterostome evolution: robust molecular phylogenetic support for a hemichordate plus echinoderm clade

Hemichordates were traditionally allied to the chordates, but recent molecular analyses have suggested that hemichordates are a sister group to the echinoderms, a relationship that has important consequences for the interpretation of the evolution of deuterostome body plans. However, the molecular phylogenetic analyses to date have not provided robust support for the hemichordate + echinoderm clade. We use a maximum likelihood framework, including the parametric bootstrap, to reanalyze DNA data from complete mitochondrial genomes and nuclear 18S rRNA. This approach provides the first statistically significant support for the hemichordate + echinoderm clade from molecular data. This grouping implies that the ancestral deuterostome had features that included an adult with a pharynx and a dorsal nerve cord and an indirectly developing dipleurula-like larva.

Molecular evidence of human T-cell lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2) infections in HTLV seroindeterminate individuals from Sao Paulo, Brazil

Background: Using enzyme immunoassays and Western blot (Wb) tests, HTLV serodiagnosis yields indeterminate results in a significant number of cases. Objective: To determine the prevalence of HTLV infection among HTLV-seroindeterminate individuals. Study design: We studied peripheral blood mononuclear cells from 65 anti-HTLV Wb-seroindeterminate individuals by attempting to amplify proviral DNA sequences (tax and pot) to identify HTLV-I and HTLV-2 infections. Results: These 65 specimens exhibited predominantly (43%) anti-HTLV antibodies to gag-coded antigens in the absence of anti-p24 on Wb analysis. Tax proviral sequences were detected in 6 (9.2%) samples. According to restricted fragment polymorphism analysis (RFLP), we identified HTLV-1 proviral DNA in 4 samples. HTLV-2 in one and sequences from both in another. Nested PCR for the pot region was positive in 3 (4.6%) specimens, which were also positive for tax sequences. After hybridization HTLV-1 infection was confirmed in 2 samples (3.1%) and HTLV-2 in another (1.5%). Detection of a single HTLV DNA sequence may be due to infection by defective provirus, but its significance remains undefined. In this cohort, no Wb reactivity pattern was predictive of proviral detection. HTLV-I infection was demonstrated in an individual who had Wb reactivity to gag-coded antigens only. Conclusions: This emphasizes the importance of clinical and laboratory follow-up of HTLV-seroindeterminate individuals from endemic areas. (c) 2009 Elsevier B.V. All rights reserved.

Neither Molecular Diversity of the Envelope, Immunosuppression Status, Nor Proviral Load Causes Indeterminate HTLV Western Blot Profiles in Samples From Human T-Cell Lymphotropic Virus Type 2 (HTLV-2)-Infected Individuals

Although human T-cell lymphotropic virus type 2 (HTLV-2) is considered of low pathogenicity, serological diagnosis is important for counseling and monitoring. The confirmatory tests most used are Western blot (WB) and PCR. However, in high-risk populations, about 50% of the indeterminate WB were HTLV-2 positives by PCR. The insensitivity of the WB might be due to the use of recombinant proteins of strains that do not circulate in our country. Another possibility may be a high level of immunosuppression, which could lead to low production of virus, resulting in low stimulation of antibody. We found one mutation, proline to serine in the envelope region in the position 184, presented at least 1/3 of the samples, independent the indeterminate WB profile. In conclusion, we found no correlation of immune state, HTLV-2 proviral load, or env diversity in the K55 region and WB indeterminate results. We believe that the only WB kit available in the market is probably more accurate to detect HTLV-1 antibodies, and some improvement for HTLV-2 detection should be done in the future, especially among high-risk population. J. Med. Virol. 82:837-842,2010. (C) 2010 Wiley-Liss, Inc.

Evaluation of Adult Chronic Chagas` Heart Disease Diagnosis by Molecular and Serological Methods

Chagas` disease caused by Trypanosoma cruzi is endemic in Latin America. T. cruzi presents heterogeneous populations and comprises two main genetic lineages, named T. cruzi I and T. cruzi II. Diagnosis in the chronic phase is based on conventional serological tests, including indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA), and diagnosis in the acute phase based on parasitological methods, including hemoculture. The objective of this study was to evaluate the diagnostic procedures of Chagas` disease in adult patients in the chronic phase by using a PCR assay and conventional serological tests, including TESA-blot as the gold standard. Samples were obtained from 240 clinical chronic chagasic patients. The sensitivities, compared to that of TESA-blot, were 70% for PCR using the kinetoplast region, 75% for PCR using the nuclear repetitive region, 99% for IIF, and 95% for ELISA. According to the serological tests results, we recommend that researchers assess the reliability and sensitivity of the commercial kit Chagatest ELISA recombinant, version 3.0 (Chagatest Rec v3.0; Wiener Lab, Rosario, Argentina), due to the lack of sensitivity. Based on our analysis, we concluded that PCR cannot be validated as a conventional diagnostic technique for Chagas` disease. These data have been corroborated by low levels of concordance with serology test results. It is recommended that PCR be used only for alternative diagnostic support. Using the nuclear repetitive region of T. cruzi, PCR could also be applicable for monitoring patients receiving etiologic treatment.

Towards a molecular epidemiology of alcohol dependence analysing the interplay of genetic and environmental risk factors

Background Progress in identifying genetic factors protective against alcohol dependence (AlcD) requires a paradigm shift in psychiatric epidemiology. Aims To integrate analysis of research into the genetics of alcoholism. Method Data from prospective questionnaire and interview surveys of the Australian twin panel, and from a subsample who underwent alcohol challenge, were analysed. Results In men, effects of alcohol dehydrogenase ADH2*1/*2 genotype or high alcohol sensitivity (risk-decreasing), and of history of childhood conduct disorder, or having monozygotic co-twin or twin sister with AlcD (risk-increasing) were significant and comparable in magnitude. Religious affiliation (Anglican versus other) was associated with the ADH2 genotype, but did not explain the associations with AlcD symptoms. No protective effect of the ADH2*1/*2 genotype was observed in women. Conclusions The early onset and strong familial aggregation of AlcD, and opportunity for within-family tests of genetic association to avoid confounding effects, make epidemiological family studies of adolescents and young adults and their families a priority.

Phylogenetic revision of Agapophytinae subf.n. (Diptera : Therevidae) based on molecular and morphological evidence

Agapophytinae subf.n. is a highly diverse lineage of Australasian Therevidae, comprising eight described and two new genera: Agapophytus Guerin-Meneville, Acupalpa Krober, Acraspisa Krober, Belonalys Krober, Bonjeania Irwin & Lyneborg, Parapsilocephala Krober, Acatopygia Krober, Laxotela Winterton & Irwin, Pipinnipons gen.n. and Patanothrix gen.n. A genus-level cladistic analysis of the subfamily was undertaken using sixty-eight adult morphological characters and c. 1000 base pairs of the elongation factor-1 alpha (EF-1 alpha) protein coding gene. The morphological data partition produced three most parsimonious cladograms, whereas the molecular data partition gave a single most parsimonious cladogram, which did not match any of the cladograms found in the morphological analysis. The level of congruence between the data partitions was determined using the partition homogeneity test (HTF) and Wilcoxon signed ranks rest. Despite being significantly incongruent in at least one of the incongruence tests, the partitions were combined in a simultaneous analysis. The combined data yielded a single cladogram that was better supported than that of the individual partitions analysed separately. The relative contributions of the data partitions to support for individual nodes on the combined cladogram were investigated using Partitioned Bremer Support. The level of support for many nodes on the combined cladogram was non-additive and often greater than the sum of support for the respective nodes on individual partitions. This synergistic interaction between incongruent data partitions indicates a common phylogenetic signal in both partitions. It also suggests that criteria for partition combination based solely on incongruence may be misleading. The phylogenetic relationships of the genera are discussed using the combined data. A key to genera of Agapophytinae is presented, with genera diagnosed and figured. Two new genera are described: Patanothrix with a new species (Pat. skevingtoni) and Pat. wilsoni (Mann) transferred from Parapsilocephala, and Pipinnipons with a new species (Pip. kroeberi). Pipinnipons fascipennis (Krober) is transferred from Squamopygin Krober and Pip. imitans (Mann) is transferred from Agapophytus. Agapophytus bicolor (Krober) is transferred from Parapsilocephala. Agapophytus varipennis Mann is synonymised with Aga, queenslandi Krober and Aga. flavicornis Mann is synonymised with Aga. pallidicornis (Krober).

Resolution of natural groups using iterative assignment tests: an example from two species of Australian native rats (Rattus)

Sympatric individuals of Rattus fuscipes and Rattus leucopus, two Australian native rats from the tropical wet forests of north Queensland, are difficult to distinguish morphologically and are often confused in the field. When we started a study on fine-scale movements of these species, using microsatellite markers, we found that the species as identified in the field did not form coherent genetic groups. In this study, we examined the potential of an iterative process of genetic assignment to separate specimens from distinct (e.g. species, populations) natural groups. Five loci with extensive overlap in allele distributions between species were used for the iterative process. Samples were randomly distributed into two starting groups of equal size and then subjected to the test. At each iteration, misassigned samples switched groups, and the output groups from a given round of assignment formed the input groups for the next round. All samples were assigned correctly on the 10th iteration, in which two genetic groups were clearly separated. Mitochondrial DNA sequences were obtained from samples from each genetic group identified by assignment, together with those of museum voucher specimens, to assess which species corresponded to which genetic group. The iterative procedure was also used to resolve groups within species, adequately separating the genetically identified R. leucopus from our two sampling sites. These results show that the iterative assignment process can correctly differentiate samples into their appropriate natural groups when diagnostic genetic markers are not available, which allowed us to resolve accurately the two R. leucopus and R. fuscipes species. Our approach provides an analytical tool that may be applicable to a broad variety of situations where genetic groups need to be resolved.

Testing the link between the latitudinal gradient in species richness and rates of molecular evolution

Numerous hypotheses have been proposed to explain latitudinal gradients in species richness, but all are subject to ongoing debate. Here we examine Rohde's (1978, 1992) hypothesis, which proposes that climatic conditions at low latitudes lead to elevated rates of speciation. This hypothesis predicts that rates of molecular evolution should increase towards lower latitudes, but this prediction has never been tested. We discuss potential links between rates of molecular evolution and latitudinal diversity gradients, and present the first test of latitudinal variation in rates of molecular evolution. Using 45 phylogenetically independent, latitudinally separated pairs of bird species and higher taxa, we compare rates of evolution of two mitochondrial genes and DNA-DNA hybridization distances. We find no support for an effect of latitude on rate of molecular evolution. This result casts doubt on the generality of a key component of Rohde's hypothesis linking climate and speciation.

Caracterização molecular de papilomavírus humano (HPV) e vírus adeno-associado (AAV) em lesões intraepiteliais de colo uterino: um estudo de seguimento

O câncer de colo uterino (CCU), cujo agente etiológico é o papilomavírus humano (HPV), é um dos tipos de câncer mais frequentes em mulheres em todo o mundo, não só em incidência como também em mortalidade. Alguns genótipos de HPV, denominados de alto risco (HR-HPV), e suas variantes gênicas, estão mais associados à indução de lesões malignas, sendo HPV16 e 18 os mais frequentes. Algumas infecções do trato genital podem atuar como cofatores da progressão carcinogênica do CCU, porém a infecção por vírus adeno-associado (AAV) parece estar inversamente relacionada, o que pode refletir em um papel protetor no desenvolvimento do CCU induzido pelo HPV. Portanto, este estudo objetivou investigar o papel da infecção mista AAV-HPV e das variantes oncogênicas de HPV na progressão das lesões intraepiteliais de colo de útero e acompanhar a eliminação / persistência viral em relação à progressão / regressão das lesões cervicouterinas. Exames citológicos foram realizados em amostras de espécime cervical, coletadas em dois momentos, de mulheres atendidas no Hospital Universitário Cassiano Antonio Moraes – HUCAM e seguiram para tratamento conforme preconizado. DNA foi extraído pelo kit comercial QIAamp® DNA Mini Kit, seguindo instruções do fabricante. DNA de AAV foi investigado por PCR e nPCR e, de HPV, por PCR e Captura Híbrida® (CH). Genotipagem de AAV e HPV foram realizadas por RFLP e RLB, respectivamente. Dos casos encaminhados ao ambulatório de colposcopia, 57,3% tiveram citologia normal, 23,1% lesões de baixo grau e 19,6% lesões de alto grau. Dos casos com citologia normal, 78% permaneceram normais, enquanto 22% progrediram à lesão; dos casos com lesão de baixo grau, 74% regrediram para citologia normal, enquanto 78,6% dos casos com lesão de alto grau apresentaram lesão de baixo grau ou citologia normal na segunda coleta. Foram positivas para HPV, 56% e 36,5% das amostras da primeira e segunda coletas, respectivamente. Foi observada boa correlação (kappa= 0,66) entre os testes de PCR e CH para detecção de HPV. Os HR-HPV foram detectados em mais de 90% das amostras de ambas as coletas, sendo os mais frequentes os HPV16, 58, 51, 52 e 53. Variante não-europeia esteve associada ao desenvolvimento de lesão cervical de alto grau, enquanto a presença de AAV foi inversamente relacionada à progressão da lesão cervical induzida por HPV.

Polymer indentation with mesoscopic molecular dynamics

Indentation tests are used to determine the hardness of a material, e.g., Rockwell, Vickers, or Knoop. The indentation process is empirically observed in the laboratory during these tests; the mechanics of indentation is insufficiently understood. We have performed first molecular dynamics computer simulations of indentation resistance of polymers with a chain structure similar to that of high density polyethylene (HDPE). A coarse grain model of HDPE is used to simulate how the interconnected segments respond to an external force imposed by an indenter. Results include the time-dependent measurement of penetration depth, recovery depth, and recovery percentage, with respect to indenter force, indenter size, and indentation time parameters. The simulations provide results that are inaccessible experimentally, including continuous evolution of the pertinent tribological parameters during the entire indentation process.

Caracterização molecular da resistência a antimicrobianos em Escherichia coli produtoras de β-lactamases de espectro alargado isoladas em cães e estudo da partilha de clones bacterianos entre animais de companhia e coabitantes humanos

A pressão seletiva originada pelo uso excessivo de antimicrobianos na medicina humana e veterinária tem contribuído para a emergência de estirpes bacterianas multirresistentes, sendo os estudos mais escassos relativamente à sua presença nos animais de companhia. Porque os animais e os seus proprietários partilham o mesmo espaço habitacional, apresentando comportamentos de contacto próximo, existe uma hipótese elevada de transferência microbiana inter-espécie. Ante esta possibilidade é importante escrutinar o papel dos animais de companhia enquanto reservatórios de estirpes e de genes de resistência, bem como a sua envolvência na disseminação de estirpes bacterianas multirresistentes. Importa também, investigar o papel das superfícies e objetos domésticos partilhados por ambos, como potenciadores deste fenómeno. O objetivo deste trabalho foi, identificar o filogrupo e fazer a caracterização molecular dos genes que conferem resistência aos β-lactâmicos e às quinolonas, em quarenta isolados de Escherichia coli produtoras de β-lactamases de espectro alargado (ESBL), obtidas em zaragatoas fecais de cães consultados no Hospital Veterinário do ICBAS-UP. Complementarmente pretendeu-se inferir sobre a partilha de clones de Escherichia coli e Enterococcus spp. com elevadas resistências, em cinco agregados familiares (humanos e seus animais de companhia) assim como avaliar a potencial disseminação de estirpes multirresistentes no ambiente doméstico. Previamente foram recolhidas zaragatoas de fezes, pelo e mucosa oral dos animais e em alguns casos, dos seus proprietários, e ainda do ambiente doméstico. As zaragatoas foram processadas e as estirpes isoladas com base em meios seletivos. Foram realizados testes de suscetibilidade antimicrobiana de modo a estabelecer o fenótipo de resistência de cada isolado. O DNA foi extraído por varias metodologias e técnicas de PCR foram utilizadas para caracterização de filogrupos (Escherichia coli) e identificação da espécie (Enterococcus spp.). A avaliação da proximidade filogenética entre isolados foi efetuada por ERIC PCR e PFGE. No conjunto de quarenta isolados produtores de ESBL e/ou resistentes a quinolonas verificou-se que 47,5% pertenciam ao filogrupo A, havendo uma menor prevalência do filogrupo D (25,0%), B1 (17,5%), e B2 (10,0%).A frequência de resistência nestes isolados é factualmente elevada, sendo reveladora de uma elevada pressão seletiva. Com exceção de dois isolados, os fenótipos foram justificados pela presença de β-lactamases. A frequência da presença de genes foi: 47% blaTEM, 34% blaSHV, 24% blaOXA , 18% blaCTX-M-15, 8% blaCTX-M-2, 3% blaCTX-M-9. Nos isolados resistentes às quinolonas verificou-se maioritariamente a presença de mutações nos genes cromossomais gyrA e parC, e em alguns casos a presença de um determinante de resistência mediado por plasmídeo – qnrS. Nos cinco “agregados familiares” (humanos e animais) estudados foi observada uma partilha frequente de clones de E. coli e Enterococcus faecalis com múltiplas resistências, isolados em fezes e mucosa oral de cães e gatos e fezes e mãos dos respetivos proprietários, evidenciando-se assim uma possível transferência direta entre coabitantes (agregados A, C, D, E). Ficou também comprovado com percentagens de similaridade genotípica superiores a 94% que essa disseminação também ocorre para o ambiente doméstico, envolvendo objetos dos animais e de uso comum (agregados A, E). Os resultados obtidos reforçam a necessidade de um uso prudente dos antimicrobianos, pois elevados padrões de resistências terão um impacto não só na qualidade de vida dos animais mas também na saúde humana. Adicionalmente importa sensibilizar os proprietários para a necessidade de uma maior vigilância relativamente às formas de interação com os animais, bem como para a adoção de medidas higiénicas cautelares após essa mesma interação.

Conventional and molecular typing of Salmonella typhi strains from Brazil

Phenotypic and genotypic characteristics of Salmonella Typhi were studied in 30 strains, isolated in different years, from some areas in Brazil. Conventional typing methods were performed by biochemical tests, Vi phage-typing scheme, and antimicrobial susceptibility test. Molecular typing methods were performed by analysis of plasmid DNA and by random amplified polymorphic DNA (RAPD-PCR). For the latter, an optimization step was performed to ensure the reproducibility of the process in genetic characterization of S. Typhi. The predominance of 76.7% of biotype I (xylose +, arabinose -) was noticed in all studied areas. Three phage types were recognized, with prominence for the phage types A (73.3%) and I+IV (23.3%). All the strains were susceptible to the drugs used. However, 36.7% of the strains contained plasmids, with predominance of the 105 Kb plasmid. RAPD was capable of grouping the strains in 8 genotypic patterns using primer 784, in 6, using primer 787 and in 7, using primer 797. Conventional phenotypic typing methods, as well as the DNA plasmid analysis, presented nonsignificant discriminatory power; however, RAPD-PCR analysis showed discriminatory power, reproducibility, easy interpretation and performance, being considered as a promising alternative typing method for S. Typhi.