Polymorphism in the flanking regions of the PbGP43 gene from the human pathogen Paracoccidioides brasiliensis: search for protein binding sequences and poly(A) cleavage sites

<p>Abstract</p> <p>Background</p> <p><it>Paracoccidioides brasiliensis </it>is a thermo-dimorphic fungus that causes paracoccidiodomycosis (PCM). Glycoprotein gp43 is the fungal main diagnostic antigen, which can also protect against murine PCM and interact with extracellular matrix proteins. It is structurally related to glucanases, however not active, and whose expression varies considerably. We have presently studied polymorphisms in the Pb<it>GP43 </it>flanking regions to help understand such variations.</p> <p>Results</p> <p>we tested the protein-binding capacity of oligonucleotides covering the Pb<it>GP43 </it>proximal 5' flanking region, including overlap and mutated probes. We used electrophoretic mobility shift assays and found DNA binding regions between positions -134 to -103 and -255 to -215. Only mutation at -230, characteristic of <it>P. brasiliensis </it>phylogenetic species PS2, altered binding affinity. Next, we cloned and sequenced the 5' intergenic region up to position -2,047 from <it>P. brasiliensis </it>Pb339 and observed that it is composed of three tandem repetitive regions of about 500 bp preceded upstream by 442 bp. Correspondent PCR fragments of about 2,000 bp were found in eight out of fourteen isolates; in PS2 samples they were 1,500-bp long due to the absence of one repetitive region, as detected in Pb3. We also compared fifty-six Pb<it>GP43 </it>3' UTR sequences from ten isolates and have not observed polymorphisms; however we detected two main poly(A) clusters (1,420 to 1,441 and 1,451 to 1,457) of multiple cleavage sites. In a single isolate we found one to seven sites.</p> <p>Conclusions</p> <p>We observed that the amount of Pb<it>GP43 </it>transcripts accumulated in <it>P. brasiliensis </it>Pb339 grown in defined medium was about 1,000-fold higher than in Pb18 and 120-fold higher than in Pb3. We have described a series of features in the gene flanking regions and differences among isolates, including DNA-binding sequences, which might impact gene regulation. Little is known about regulatory sequences in thermo-dimorphic fungi. The peculiar structure of tandem repetitive fragments in the 5' intergenic region of Pb<it>GP43</it>, their characteristic sequences, besides the presence of multiple poly(A) cleavage sites in the 3' UTR will certainly guide future studies.</p

Innes: Security Bonds

Risk in society is a pertinent concept of late modernity. Most elements of our social and interpersonal lives are in some way linked to concerns about safety, security and fear of harm. As a consequence, we spend a great deal of time engaged in emotional, physical and economic processes that facilitate our safety. Whether this be through purchasing anti-theft devices, or subscribing to self-defense training courses; participating in neighbourhood-watch schemes or altering our behavior to prevent susceptibility to victimization, all demonstrate an inherent pre-occupation with risk and perceived danger. The work presented in this paper offers an in-depth socio-criminological analysis focusing on the issue of citizens insecurity, and proposes an original interpretative paradigm emerging from findings on the INNES (Intimate Neighborhood Strengthening) European Project. A presentation of the idiographic and nomothetic motivations and conditions influencing and predicting social fears and insecurities over the last two decades is discussed, with the presentation of the new interpretative model, 'Social Cobweb Theory'. This model focuses on solidarity and on the strengthening of intimate neighborhood bonds and argues that these aforementioned concepts function as an effective approach in lowering citizens' perceptions of individual insecurities and risk

Inhibition of PbGP43 expression may suggest that gp43 is a virulence factor in Paracoccidioides brasiliensis

ABSTARCT: Glycoprotein gp43 is an immunodominant diagnostic antigen for paracoccidioidomycosis caused by Paracoccidioides brasiliensis. It is abundantly secreted in isolates such as Pb339. It is structurally related to beta-1,3-exoglucanases, however inactive. Its function in fungal biology is unknown, but it elicits humoral, innate and protective cellular immune responses; it binds to extracellular matrix-associated proteins. In this study we applied an antisense RNA (aRNA) technology and Agrobacterium tumefaciens-mediated transformation to generate mitotically stable PbGP43 mutants (PbGP43 aRNA) derived from wild type Pb339 to study its role in P. brasiliensis biology and during infection. Control PbEV was transformed with empty vector. Growth curve, cell vitality and morphology of PbGP43 aRNA mutants were indistinguishable from those of controls. PbGP43 expression was reduced 80-85% in mutants 1 and 2, as determined by real time PCR, correlating with a massive decrease in gp43 expression. This was shown by immunoblotting of culture supernatants revealed with anti-gp43 mouse monoclonal and rabbit polyclonal antibodies, and also by affinity-ligand assays of extracellular molecules with laminin and fibronectin. In vitro, there was significantly increased TNF-α production and reduced yeast recovery when PbGP43 aRNA1 was exposed to IFN-γ-stimulated macrophages, suggesting reduced binding/uptake and/or increased killing. In vivo, fungal burden in lungs of BALB/c mice infected with silenced mutant was negligible and associated with decreased lung ΙΛ-10 and IL-6. Therefore, our results correlated low gp43 expression with lower pathogenicity in mice, but that will be definitely proven when PbGP43 knockouts become available.

COLORECTAL CANCER IN PATIENTS WITH TYPE 2 DIABETES MELLITUS: PRELIMINARY RESULTS FROM AN ONGOING CASE-CONTROL STUDY

Background and Aim:Understanding the risk factors for colorectal cancer (CRC) is crucial to the development of effective strategies for its prevention. meta-analysis and epidemiological studies have already shown that type 2 diabetes mellitus (DM) is associated with an increased risk of CRC and have provided data to support a positive relationship between these diseases. Material and Methods: We retrospectively evaluated 741 consecutive caucasian patients with type 2 DM who underewnt colonoscopic screening cof CRC and followed in our tertiary referrral center in 200-208 for incidence of CRC. Patients were stratified based on gender, age, body mass index (MBI), alchool and NSAIDS assumption, family history for cancer blood glycated hemoglobin levels, hypertension, hypertrigliceridemia, age at diabetes onset and duration, treatment with insulin or other hypoglicemic drugs. A total of 257 consecutive control patients were selected from a cohort of patients followed as outpatients for thyroid diseases. Results: At a median follow-up of 132,5 months (range 33,3-175,7) 56 cases of cancer (prevalence 7,56%) occurred; among these, 14 cases of CRC were reported (prevalence 18,8%) among the diabetic patients, while only one case (prevalence 0,004%) occurred in the control group, although this difference is not statistically significant (chi-square 2,9, P=0,08). Median duration of DM to CRC diagnosis was 156 months (range 1-768). At the univariate analysis older age (p=0,001), and diabetes duration (p=0,001) were related to higher risk of cancer, while metformin seems to be protective towards cancer (p=0,058). in the subset of patients with CRC, older age (p=0,001) and diabetes duration (p=0,001) were related to higher risk of CRC, such as treatment with sulphonylureas (p=0,01). Conclusions: Our preliminbar data show that the prevalence of CRC in the cohort of patients with type 2 DM was higher compared to that from our control group, and to that from the National Tumor Register up 2010 (0,5%). Furthermore we could interestingly hypotize that sulphonylureas may play a role in CRC carcinogenesis altering the physiological insulin secretion

Empreendedorismo em turismo rural: o caso do Norte de Portugal

A presente dissertação tem como objectivo principal estudar o empreendedorismo em turismo rural: o caso do Norte de Portugal, recorrendo para isso, a uma análise quantitativa. A análise efectuada é baseada numa amostra formada por empresas pertencentes ao sector do turismo em Portugal do Norte de Portugal que fossem empresas activas e que tivessem registado na base de dados um contacto de e-mail. Foram apuradas 267 empresas a quem se enviou um questionário tendo-se obtido 41 respostas válidas, sendo esse o número de empresas da nossa amostra final. Além dos dados obtidos via questionário foram recolhidos dados económico-financeiros através da base de dados empresarial SABI (Sistema de Análise de Balanços Ibéricos). Os resultados obtidos indicam que um maior nível de internacionalização, bem como mais elevadas habilitações académicas dos gestores das empresas se associam a melhores níveis de desempenho organizacional.This dissertation´s main objective is to study entrepreneurship in rural tourism: the case of Northern Portugal, using a quantitative analysis. The analysis is based on a sample of companies belonging to the tourism sector in northern Portugal who were active companies and had registered an e-mail contact in the database. To a total of 267 companies a questionnaire was sent and 41 valid answers were obtained, which is the number of companies in our final sample. In addition to the data obtained through the questionnaire, economic and financial data was collected through the SABI (Iberian Balance Sheet Analysis System) business database. The results obtained indicate that a higher level of internationalization and higher academic qualifications of company managers are associated with a better level of organizational performance

Gene Copy-Number Variation in Haploid and Diploid Strains of the Yeast Saccharomyces cerevisiae

The increasing ability to sequence and compare multiple individual genomes within a species has highlighted the fact that copy-number variation (CNV) is a substantial and underappreciated source of genetic diversity. Chromosome-scale mutations occur at rates orders of magnitude higher than base substitutions, yet our understanding of the mechanisms leading to CNVs has been lagging. We examined CNV in a region of chromosome 5 (chr5) in haploid and diploid strains of Saccharomyces cerevisiae. We optimized a CNV detection assay based on a reporter cassette containing the SFA1 and CUP1 genes that confer gene dosage-dependent tolerance to formaldehyde and copper, respectively. This optimized reporter allowed the selection of low-order gene amplification events, going from one copy to two copies in haploids and from two to three copies in diploids. In haploid strains, most events involved tandem segmental duplications mediated by nonallelic homologous recombination between flanking direct repeats, primarily Ty1 elements. In diploids, most events involved the formation of a recurrent nonreciprocal translocation between a chr5 Ty1 element and another Ty1 repeat on chr13. In addition to amplification events, a subset of clones displaying elevated resistance to formaldehyde had point mutations within the SFA1 coding sequence. These mutations were all dominant and are proposed to result in hyperactive forms of the formaldehyde dehydrogenase enzyme

Highlights of the São Paulo ISEV workshop on extracellular vesicles in cross-kingdom communication

In the past years, extracellular vesicles (EVs) have become an important field of research since EVs have been found to play a central role in biological processes. In pathogens, EVs are involved in several events during the host–pathogen interaction, including invasion, immunomodulation, and pathology as well as parasite–parasite communication. In this report, we summarised the role of EVs in infections caused by viruses, bacteria, fungi, protozoa, and helminths based on the talks and discussions carried out during the International Society for Extracellular Vesicles (ISEV) workshop held in São Paulo (November, 2016), Brazil, entitled Cross-organism Communication by Extracellular Vesicles: Hosts, Microbes and Parasites. © 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.11Ysciescopu

Serological detection of antibodies against Paracoccidioides brasiliensis in dogs with leishmaniasis

The aim of this study was to detect antibodies against Paracoccidioides brasiliensis in dogs seropositive and seronegative for leishmaniasis. Sera from 836 dogs (449 positive and 387 negative to leishmaniasis) were analysed by ELISA and the immunodiffusion test using gp43 and exoantigen, respectively. The analysis of the 836 serum samples by ELISA and the immunodiffusion test showed a positivity of 67.8 % and 7.3%, respectively, for P. brasiliensis infection. The dogs positive to leishmaniasis showed a higher reactivity to gp43 (79.9%) and exoantigen (12.7%) than the negative ones (54.0% and 1.0%, respectively). The higher reactivity to P. brasiliensis antigens may be due to cross-reactivity or a co-infection of dogs by Leishmania and P. brasiliensis. The lower correlation (0.187) observed between reactivity to gp43 and Leishmania antigen reinforces the latter hypothesis