Besnoitia besnoiti protein disulfide isomerase (BbPDI): molecular characterization, expression and in silico modelling

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor an efficacious vaccine is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neosporacaninum and Toxoplasmagondii, which are closely related to B. besnoiti, PDI play an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we present the nucleotide sequence of the B. besnoiti PDI gene. BbPDI belongs to the thioredoxin-like superfamily (cluster 00388) and is included in the PDI_a family (cluster defined cd02961) and the PDI_a_PDI_a'_c subfamily (cd02995). A 3D theoretical model was built by comparative homology using Swiss-Model server, using as a template the crystallographic deduced model of Tapasin-ERp57 (PDB code 3F8U chain C). Analysis of the phylogenetic tree for PDI within the phylum apicomplexa reinforces the close relationship among B. besnoiti, N. caninum and T. gondii. When subjected to a PDI-assay based on the polymerisation of reduced insulin, recombinant BbPDI expressed in E. coli exhibited enzymatic activity, which was inhibited by bacitracin. Antiserum directed against recombinant BbPDI reacted with PDI in Western blots and by immunofluorescence with B. besnoiti tachyzoites and bradyzoites.

Culture-dependent characterization of cyanobacterial diversity in the intertidal zones of the Portuguese coast: A polyphasic study

Cyanobacteria are important primary producers, and many are able to fix atmospheric nitrogen playing a key role in the marine environment. However, not much is known about the diversity of cyanobacteria in Portuguese marine waters. This paper describes the diversity of 60 strains isolated from benthic habitats in 9 sites (intertidal zones) on the Portuguese South and West coasts. The strains were characterized by a morphological study (light and electron microscopy) and by a molecular characterization (partial 16S rRNA, nifH, nifK, mcyA, mcyE/ndaF, sxtI genes). The morphological analyses revealed 35 morphotypes (15 genera and 16 species) belonging to 4 cyanobacterial Orders/Subsections. The dominant groups among the isolates were the Oscillatoriales. There is a broad congruence between morphological and molecular assignments. The 16S rRNA gene sequences of 9 strains have less than 97% similarity compared to the sequences in the databases, revealing novel cyanobacterial diversity. Phylogenetic analysis, based on partial 16S rRNA gene sequences showed at least 12 clusters. One-third of the isolates are potential N2-fixers, as they exhibit heterocysts or the presence of nif genes was demonstrated by PCR. Additionally, no conventional freshwater toxins genes were detected by PCR screening.

Molecular analysis of the dengue virus type 1 and 2 in Brazil based on sequences of the genomic envelope-nonstructural protein 1 junction region

The genomic sequences of the Envelope-Non-Structural protein 1 junction region (E/NS1) of 84 DEN-1 and 22 DEN-2 isolates from Brazil were determined. Most of these strains were isolated in the period from 1995 to 2001 in endemic and regions of recent dengue transmission in São Paulo State. Sequence data for DEN-1 and DEN-2 utilized in phylogenetic and split decomposition analyses also include sequences deposited in GenBank from different regions of Brazil and of the world. Phylogenetic analyses were done using both maximum likelihood and Bayesian approaches. Results for both DEN-1 and DEN-2 data are ambiguous, and support for most tree bipartitions are generally poor, suggesting that E/NS1 region does not contain enough information for recovering phylogenetic relationships among DEN-1 and DEN-2 sequences used in this study. The network graph generated in the split decomposition analysis of DEN-1 does not show evidence of grouping sequences according to country, region and clades. While the network for DEN-2 also shows ambiguities among DEN-2 sequences, it suggests that Brazilian sequences may belong to distinct subtypes of genotype III.

Enzymatic and hemolytic activities of Candida dubliniensis strains

Candida dubliniensis is an opportunistic yeast that has been recovered from several body sites in many populations; it is most often recovered from the oral cavities of human immunodeficiency virus-infected patients. Although extensive studies on epidemiology and phylogeny of C. dubliniensis have been performed, little is known about virulence factors such as exoenzymatic and hemolytic activities. In this study we compared proteinase, hyaluronidase, chondroitin sulphatase and hemolytic activities in 18 C. dubliniensis and 30 C. albicans strains isolated from AIDS patients. C. albicans isolates produced higher amounts of proteinase than C. dubliniensis (p < 0.05). All the tested C. dubliniensis strains expressed hyaluronidase and chondroitin sulphatase activities, but none of them were significantly different from those observed with C. albicans (p > 0.05). Hemolytic activity was affected by CaCl2; when this component was absent, we did not notice any significant difference between C. albicans and C. dubliniensis hemolytic activities. On the contrary, when we added 2.5 g% CaCl2, the hemolytic activity was reduced on C. dubliniensis and stimulated on C. albicans tested strains (p < 0.05).

THE INFLUENCE OF HIV-1 SUBTYPES C, CRF31_BC AND B ON DISEASE PROGRESSION AND INITIAL VIROLOGIC RESPONSE TO HAART IN A SOUTHERN BRAZILIAN COHORT

Background: Although most HIV-1 infections in Brazil are due to subtype B, Southern Brazil has a high prevalence of subtype C and recombinant forms, such as CRF31_BC. This study assessed the impact of viral diversity on clinical progression in a cohort of newly diagnosed HIV-positive patients. Methods: From July/2004 to December/2005, 135 HIV-infected patients were recruited. The partial pol region was subtyped by phylogeny. A generalized estimating equation (GEE) model was used to examine the relationship between viral subtype, CD4+ T cell count and viral load levels before antiretroviral therapy. Hazard ratio (Cox regression) was used to evaluate factors associated with viral suppression (viral load < 50 copies/mL at six months). Results: Main HIV-1 subtypes included B (29.4%), C (28.2%), and CRF31_BC (23.5%). Subtypes B and C showed a similar trend in CD4+ T cell decline. Comparison of non-B (C and CRF31_BC) and B subtypes revealed no significant difference in the proportion of patients with viral suppression at six months (week 24). Higher CD4+ T cell count and lower viral load were independently associated with viral suppression. Conclusion: No significant differences were found between subtypes; however, lower viral load and higher CD4+ T cell count before therapy were associated with better response.

MOLECULAR CHARACTERIZATION AND SEQUENCE PHYLOGENETIC ANALYSIS OF SURFACE ANTIGEN 3 (SAG3) GENE OF LOCAL INDIAN ISOLATES (CHENNAI AND IZATNAGAR) OF Toxoplasma gondii

Context and objective:The molecular characterization of local isolates of Toxoplasma gondii is considered significant so as to assess the homologous variations between the different loci of various strains of parasites.Design and setting:The present communication deals with the molecular cloning and sequence analysis of the 1158 bp entire open reading frame (ORF) of surface antigen 3 (SAG3) of two Indian T. gondii isolates (Chennai and Izatnagar) being maintained as cryostock at the IVRI.Method:The surface antigen 3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences.Results:The sequence comparison analysis revealed 99.9% homology with the standard published RH strain sequence of T. gondii. The strains were also compared with other established published sequences and found to be most related to the P-Br strain and CEP strain (both 99.3%), and least with PRU strain (98.4%). However, the two Indian isolates had 100% homology between them.Conclusion:Finally, it was concluded that the Indian isolates were closer to the RH strain than to the P-Br strain (Brazilian strain), the CEP strain and the PRU strains (USA), with respect to nucleotide homology. The two Indian isolates used in the present study are known to vary between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as SAG3 locus is concerned. This could be attributed to the fact that this SAG3 might be a conserved locus and thereby, further detailed studies are thereby warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The findings are important from the point of view of molecular phylogeny.

Revealing fungal activity in the presence of ionic liquids

Dissertation presented to obtain the Ph.D degree in Biochemistry

Paracoccidioides brasiliensis AND Paracoccidioides lutzii, A SECRET LOVE AFFAIR

SUMMARYTo commemorate Prof. Carlos da Silva Lacaz's centennial anniversary, the authors have written a brief account of a few, out of hundreds, biological, ecological, molecular and phylogenetic studies that led to the arrival of Paracoccidioides lutzii, hidden for more than a century within Paracoccidioides brasiliensis. Lacaz's permanent interest in this fungus, and particularly his conviction on the benefits that research on paracoccidioidomycosis would bring to patients, were pivotal in the development of the field.

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.

Evolution of Diplodocid Sauropod dinosaurs with emphasis on specimens from Howe Ranch, Wyoming (USA)

Diplodocidae are among the best known sauropod dinosaurs. Several species were described in the late 1800s or early 1900s. Since then, numerous additional specimens were recovered in the USA, Tanzania, Portugal, as well as possibly Spain, England, and Asia. To date, the clade includes about 12 to 15 different species, some of them with questionable taxonomic status (e.g. ‘Diplodocus’ hayi or Dyslocosaurus polyonychius). However, intrageneric relationships of the multi-species, iconic genera Apatosaurus and Diplodocus are still poorly known. The way to resolve this issue is a specimen-based phylogenetic analysis, which was done for Apatosaurus, but is here performed for the first time for the entire clade of Diplodocidae. New material from different localities and stratigraphic levels on the Howe Ranch (Shell,Wyoming, USA) sheds additional light on the evolution of Diplodocidae. Three new specimens are described herein, considerably increasing our knowledge of the anatomy of the group. The new specimens (SMA 0004, SMA 0011, and SMA 0087) represent two, to possibly three new diplodocid species. They preserve material from all parts of the skeleton, including two nearly complete skulls, as well as fairly complete manus and pedes, material which is generally rare in diplodocids. Thereby, they considerably increase anatomical overlap between the sometimes fragmentary holotype specimens of the earlier described diplodocid species, allowing for significant results in a specimenbased phylogenetic analysis. Furthermore, clavicles and interclavicles are identified, the latter for the first time in dinosaurs. Their presence seems restricted to early sauropods, flagellicaudatans, and early Macronaria, and might thus be a retained plesiomorphy, with the loss of these bones being synapomorphic for Titanosauriformes and possibly Rebbachisauridae. The new material allows to test previous hypotheses of diplodocid phylogeny. In order to do so, any type specimen previously proposed to belong to Diplodocidae was included in the study, as are relatively complete referred specimens, in order to increase the degree of overlapping material. For specimens subsequently suggested to be non-diplodocid sauropods, their hypothesized sister taxa were included as outgroups. The current phylogenetic analysis thus includes 76 operational taxonomic units, 45 of which belong to Diplodocidae. The specimens were scored for 477 morphological characters, representing one of the most extensive phylogenetic analyses done within sauropod dinosaurs. The resulting cladogram recovers the classical arrangement of diplodocid relationships. Basing on a newly developed numerical approach to reduce subjectivity in the decision of specific or generic separation, species that have historically been included into well-known genera like Apatosaurus or Diplodocus, were detected to be actually generically different. Thereby, the famous genus Brontosaurus is resuscitated, and evidence further suggests that also Elosaurus parvus (previously referred to Apatosaurus) or ‘Diplodocus’ hayi represent unique genera. The study increases our knowledge about individual variation, and helps to decide how to score multi-species genera. Such a specimen-based phylogenetic analysis thus proves a valuable tool to validate historic species in sauropods, and in paleontology as a whole.

Hepatitis C: sexual or intrafamilial transmission? Epidemiological and phylogenetic analysis of hepatitis C virus in 24 infected couples

The role of sexual or intrafamilial transmission of hepatitis C is controversial. A phylogenetic analysis was performed on the non-structural region 5B of the hepatitis C virus (NS5B-HCV). High percentages of homology (mean of 98.3%) were shown between the couples. Twenty (83.3%) of the 24 men but only two of the women (8.3%) reported having had sexually transmitted diseases during their lives. The risk factors for HCV acquisition were blood transfusion (10 couples), use of illegal injected drugs (17), use of inhalants (15), acupuncture (5) and tattoos (5). The shared use of personal hygiene items included toothbrushes between six couples (25%), razor blades between 16 (66.7%), nail clippers between 21 (87.5%) and manicure pliers between 14 (58.3%). The high degree of similarity of the hepatitis C virus genome supports the hypothesis of hepatitis C virus transmission between these couples. The shared use of personal hygiene items suggests the possibility of intrafamilial transmission of infection.

Prevalence of Mansonella ozzardi among riverine communities in the municipality of Lábrea, State of Amazonas, Brazil

INTRODUCTION: Estimate the prevalence of Mansonella ozzardi infection and calculate the parasitic infection rate (PIR) in simuliid black flies in the municipality of Lábrea, State of Amazonas, Brazil. METHODS: Prevalence was measured using the thick blood smear method collected from the fingers and was related to age, sex and occupation. Simuliidae were collected with a suction apparatus, then stained with hematoxylin and dissected to verify the PIR. RESULTS: The average prevalence rate of M. ozzardi among the 694 individuals examined was 20.7%. Infection was higher in men (27.6%) than in women (14.3%) (p < 0.001) and occurred in most age groups, with the highest prevalence in the following age groups: 38-47 (40%), 48-57 (53.1%) and >58 (60.5%). The highest prevalence rates were observed in the retired (64%), followed by farm workers (47.1%). Infection by M. ozzardi was only identified in Cerqueirellum amazonicum (Simuliidae) with a PIR of 0.6%. CONCLUSIONS: This study showed a high prevalence of M. ozzardi in the riverine communities of Lábrea due to the lack of policies regarding the treatment of microfilaremic individuals in the region and an abundance of competent vectors for M. ozzardi.

Investigação da ocorrência da Mansonella ozzardi no Estado de Rondônia, Amazônia Ocidental

INTRODUÇÃO: Mansonella ozzardi é uma filária humana que tem como vetor dípteros simulídeos e é amplamente distribuída na Amazônia. Não há informações sobre a ocorrência de casos de mansonelose no Estado de Rondônia, e neste trabalho procurou-se investigar a situação epidemiológica desta parasitose em áreas urbanas e ribeirinhas na Amazônia Ocidental. MÉTODOS: Foram examinados 4.452 moradores às margens dos rios Madeira, Mamoré, Guaporé, Machado e Preto em Rondônia, através da técnica da gota espessa de sangue. Capturou-se às margens do rio Machado e Guaporé 2.112 simulídeos adultos das espécies Cerqueirellum pydanieli e Chirostilbia pertinax. RESULTADOS: Não foram encontradas pessoas infectadas com M. ozzardi e foram encontradas diferentes espécies de simulídeos com potencial de transmissão da mansonelose. CONCLUSÕES: Não foram encontrados indivíduos infectados com M. ozzardi neste estudo. Este fato pode ser explicado pela existência de pacientes com baixas microfilaremias, associado à técnica diagnóstica de baixa sensibilidade utilizada no estudo. Pode-se ainda aventar a possibilidade de ter ocorrido migração insuficiente de pacientes infectados de áreas endêmicas, impossibilitando a implantação da endemia. Estudos posteriores com técnicas diagnósticas mais sensíveis podem ajudar a esclarecer esta questão.

Seasonality, parity rates and transmission indices of Mansonella ozzardi (Manson) (Nematoda: Onchocercidae) by Cerqueirellum argentiscutum (Shelley & Luna Dias) (Diptera: Simulidae) in a lower Solimões River community, Amazonas, Brazil

Mansonella ozzardi is transmitted by two dipterian families, Ceratopogonidae (midges) and Simuliidae (black flies). In Brazil, black flies are vectors for this filariasis until now. In this paper, we determined the seasonality, parity capacity and parasitic infection rate of Cerqueirellum argentiscutum. The work was carried out in the Porto Japão community, Lower Solimões River, Amazonas, Brazil. Results show that the black flies were more abundant during the rainy season (from December to May). The number of parous flies was higher in every sampling during the course of year. Monthly Biting Rate (MBR1 123742.00, MBR2 86701.50) was high, although Parasitic Infection Rate (PIR1 0.06, PIR2 0.08) and Annual Transmission Potential (ATP 7.25) were low in numbers.

Epidemiological studies of Mansonella ozzardi (Nematoda, Onchocercidae) in indigenous communities of Pauini municipality, Amazonas, Brazil

The Mansonella ozzardi has a widespread distribution among the indigenous and riverine communities of Amazonas, Brazil. We estimated the prevalence of Mansonella ozzardi in indigenous communities of the Pauini municipality, Amazonas state, Brazil and the rate of parasitic infection in vectors. We collected thick blood smears from individuals from six Apurinã indigenous communities along the Purus River and its tributaries. Collections of simuliids were made and dissected, and the larval instars of M. ozzardi identified. The overall prevalence of M. ozzardi was 28.40%, with the highest incidence among males and agricultural workers. Among age groups, children 2-9 years of age had the lowest incidence, while individuals older than 58 exhibited the highest rates of infection. We found infected simuliids in three communities, with Parasitic Infection Rates (PIR) of 0.34-6.58%. The prevalence of M. ozzardi among the Apurinã people is high, possibly related to the diary activities of the riparian people, where a high abundance of the vectors exists.