Toward a point-of-care diagnostic for specific detection of Mycobacterium tuberculosis from sputum samples.

This study reports the development of a new PCR-free device, using IS6110 gene as biomarker, for Tuberculosis (TB) diagnosis. An arginine film (ARGFILM) was used to prepare the biosensor platform. MT-probe was immobilized on this biosensor platform to identify IS6110 gene. This gene is an excellent biomarker for Mycobacterium tuberculosis (MT). Electrochemical analyses were carried out using differential pulse voltammetry method (DPV) by methylene blue (MB) reduction signal measurement before and after hybridization either between probe and synthetic target or extracted DNA from clinical sputum samples. The optimization study of MT-probe immobilization on modified-electrode surface showed that the best probe concentration was 15 µM. The analytical analysis of hybridization assays was performed using different concentrations of synthetic MT-target (15-500 nM). The linear response was between 15 and 100 nM and the detection limit was 4.4 nM. The biosensor performance was also investigated with extracted DNA from sputum samples (PCR-free). The results showed that the biosensor was able to detect the MT from samples, exhibiting a high sensitivity and satisfactory selectivity. Thus, these results allow for the possibility of developing a portable detection device for effective diagnosis of TB patients.

Clinical and molecular aspects of breast cancer: Targets and therapies.

Breast Cancer is a complex disease characterized by the occurrence of multiple molecular alterations. Currently, some molecular markers are in use for breast cancer diagnostic, prognostic, and predictive purposes. Thus, genetic signatures are available for improving the decision-making. The biomarkers are also essential as therapeutic approaches, but many questions remain due to the lack of efficacy on breast cancer treatment, mainly for triple-negative breast cancer subtype. Since the genetic profile of breast cancer can also be related to different ethnic groups and geographic areas, the reference populations of the genetic assays and clinical trials need to include a broader population beyond the European and North American patients. In this review, we analyzed the current and potential molecular markers that could help to improve the strategies for breast cancer therapy.

Molecular analysis and conventional cytology: association between HPV and bacterial vaginosis in the cervical abnormalities of a Brazilian population.

We investigated the association between bacterial vaginosis (BV) and human papillomavirus (HPV) infection in Papanicolaou smears in a Brazilian population. Cross-sectional analysis was performed on 673 samples collected from women attending public health centers in Olinda (PE, Brazil) by conventional cytology methodology and molecular analysis, PCR tests (GP5+/6+ and MY09/11). Cytological abnormalities, BV, and HPV-DNA were detected in 23 (3.4%) samples, 189 samples (28.1%), and 210 samples (31.2%), respectively. GP5+/6+ primers resulted in higher detection performance than MY09/11 primers, with 81% concordance between both primers (P < 0.0001). The occurrence of HPV-DNA and BV had ORs of 8.59 (P < 0.0001) and 2.91 (P = 0.0089) for abnormal cytology, respectively, whereas the concomitant presence of both infections showed an OR equal to 3.82 (P = 0.0054). Therefore, we observed an association between abnormal cervical cytology and HPV infection, BV, or both HPV infection and BV. These results highlight the necessity of monitoring patients presenting not only HPV, but also BV, as risk factors for cervical lesion development.

Hemocytes of Rhipicephalus sanguineus (Acari: Ixodidae): Characterization, Population Abundance, and Ultrastructural Changes Following Challenge with Leishmania infantum.

Few studies have examined the cellular immune response of ticks, and further research on the characterization of the hemocytes of ticks is required, particularly on those of Rhipicephalus sanguineus (Latreille) because of the medical and veterinary importance of this tick. The aims of this study were to characterize the morphology and the ultrastructure of the different types of hemocytes of adult R. sanguineus and to determine the population abundance and the ultrastructural changes in the hemocytes of ticks infected with Leishmania infantum. The hemocytes were characterized through light and transmission electron microscopy. Within the variability of circulating cells in the hemolymph of adult R. sanguineus, five cell types were identified, which were the prohemocytes, plasmatocytes, granulocytes, spherulocytes, and adipohemocytes. The prohemocytes were the smallest cells found in the hemolymph. The plasmatocytes had polymorphic morphology with vesicles and cytoplasmic projections. The granulocytes had an elliptical shape with the cytoplasm filled with granules of different sizes and electrodensities. The spherulocytes were characterized by several spherules of uniform shapes and sizes that filled the entire cytoplasm, whereas the adipohemocytes had an irregular shape with multiple lipid inclusions that occupied almost the entire cytoplasmic space. The total counts of the hemocyte population increased in the group that was infected with L. infantum. Among the different cell types, the numbers increased and the ultrastructural changes occurred in the granulocytes and the plasmatocytes in the infected group of ticks.

Persistence or clearance of human papillomavirus infections in women in Ouro Preto, Brazil.

Persistent high-risk (HR) human papillomavirus (HPV) infection is necessary for development of precursor lesions and cervical cancer. We investigate persistence and clearance of HPV infections and cofactors in unvaccinated women. Cervical samples of 569 women (18-75 years), received for routine evaluation in the Health Department of Ouro Preto, Brazil, were collected and subjected to PCR (MY09/11 or GP5+/6+ primers), followed by RFLP or sequencing. All women were interviewed to collect sociodemographic and behavioral information. Viral infection persistence or clearance was reevaluated after 24 months and was observed in 59.6% and 40.4% of women, respectively. HPVs 16, 33, 59, 66, 69, and 83 (HR) were the most persistent types whereas HPVs 31, 45, and 58 were less persistent. Clearance or persistence did not differ between groups infected by HPVs 18, 53, and 67. In low-risk (LR) types, HPV 6 infected samples were associated with clearance, while HPV 11, 61, 72, or 81 infected samples were persistent in the follow-up. No statistically significant association was detected between persistent HPV infections and sociodemographic and behavioral characteristics analyzed. To study persistence or clearance in HPV infection allows the identification of risk groups, cofactors, and strategies for prevention of cervical cancer.

Morphological and morphometric study of cercariae and adult worms of Schistosoma mansoni (SLM strain) isolated from infected mice.

In northeastern Brazil, the schistosomiasis is historically endemic and considered as a public health problem. The Schistosoma mansoni São Lourenço da Mata (SLM-PE, Brazil) strain was used in several paper already published; however, morphological and morphometric studies about this strain was never done. In this work, scanning electron microscopy (SEM) was used in morphological and morphometric analysis of cercariae and adult worms. Cercariae were obtained from Biomphalaria glabrata snails and adult worms from mice, both infected by the S. mansoni SLM strain, fixed and prepared for SEM. The results showed that cercariae of S. mansoni measures 254.9 µm of length. The bodies are covered by spines, with a ventral sucker, an oral sucker with sensory receivers, and a pair of penetration glands in the head. The area of tail and body and the distance between suckers were 3,011.77, 1,530.32, and 42.9 µm, respectively. Adult worms of S. mansoni were divided into three main regions: the anterior, medial, and posterior, besides the gynecophoral canal in males. The measure of adult worms of S. mansoni was 4 mm males and 5 mm females. The anterior region length of the male was 470 µm and of the female 271 µm. All the parameters were assayed in ten samples. The morphometric values found in the SLM strain were smaller than other S. mansoni strains described in the literature as well as other helminths. This is the first morphological and morphometric study with the SLM strain of S. mansoni being extremely important for improving control strategies and life quality of the local population.

Avaliação da microbiota bacteriana do queijo de coalho artesanal produzido na região agreste do estado de Pernambuco / Evaluation of the bacterial microbiota of the artisanal "Coalho" of cheese produced in agreste region of the state of Pernambuco

O objetivo desta pesquisa foi avaliar a qualidade microbiológica e o perfil ácido-láctico do queijo de coalho artesanal. Todas as amostras de queijo apresentaram coliformes totais, termotolerantes e presença de Escherichia coli, porém com os valores dentro dos padrões estabelecidos pela legislação vigente no país. O perfil ácido-láctico estudado mostrou uma microbiota heterogênea, constituída por lactobacilos, lactococos, estreptococos e enterococos, confirmadas as espécies Enterococcus faecalis, Enterococcus faecium, Streptococcus thermophilus e Lactococcus lactis.

The aim of this study was to evaluate the microbiological quality and lactic-acid profile of artisanal coalho cheese. All cheese samples analyzed showed total coliforms, were thermotolerant, and had Escherichia coli, but all the values were within the standards established by current legislation in the country, and could be considered a food fit for human consumption. The cheese showed a heterogeneous microbiota, being constituted of all tested genus, such as lactobacilli, lactococcus, streptococcus and enterococcus, and confirmed the species: Enterococcus faecalis, Enterococcus faecium, Streptococcus thermophilus and Lactococcus lactis.

Can artisanal "Coalho" cheese from Northeastern Brazil be used as a functional food?

Brazilian artisanal "Coalho" cheeses from six Northeast towns were investigated as a functional food based on their peptide profiles and antioxidant, zinc-binding and antimicrobial activities. The peptides (WSP) from "Coalho" cheese showed high antioxidant activity, the best value of TEAC being 2223±10.10µM, which means 91.1±0.43% oxidative inhibition and peptide concentration for IC(50) of 7mg/mL (21µg of peptides) for sample from the town of Correntes. The smallest TEAC value (1896±17µM), which means 75.9±0.7% oxidative inhibition and IC(50) of 10.5mg/mL (31.5µg of peptide), was obtained for samples from the town of São Bento do Una. The zinc-binding activities were: Arcoverde (72.21±0.24%) Cachoeirinha (75.02±0.02%), Capoeiras (61.78±0.65%), Correntes (75.47±0.5%), São Bento do Una (75.41±0.15%), and Venturosa (74.36±0.04%). The WSP extracts showed antimicrobial activity against Enterococcus faecalis, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. All the results obtained suggest that "Coalho" cheese has potential as a functional food.

Human papillomavirus infection in Brazilian women with normal cervical cytology.

We examined the prevalence of human papillomavirus (HPV) infection in a sample of Brazilian women presenting normal cervical cytology. Possible interactions between patient characteristics and HPV infection were analyzed in order to provide background data to improve cervical cancer screening and prophylaxis. Cervical samples of 399 women, received for routine evaluation in the Health Department of Ouro Preto, MG, Brazil, were subjected to HPV-DNA testing by PCR with MY09/11 primers. HPV-positive specimens were typed by RFLP. A structured epidemiological questionnaire was administered to each woman. HPV prevalence among these cytologically normal women was 11%. Twelve viral types were detected, the most common being HPV-16, -6, -61, -83, and -66. HPV was more prevalent in younger women; high-risk viral types were detected in 61% of the infected women and 27% of the infected women had multiple HPV infections. Significant associations of HPV infection were found with age, literacy, residence, marital status, lifetime number of sexual partners, and parity. We detected a great diversity of HPV types in women with normal cytology. This kind of information about local populations is useful for HPV prevention and vaccination strategies.

Association of MBL2 gene exon 1 variants with autoimmune thyroid disease in Brazilian patients.

We investigated the association between MBL2 gene exon 1 functional polymorphisms and autoimmune thyroid disease (AITD) in 163 Brazilian patients (87 with Hashimoto thyroiditis, HT; 76 with Graves' disease) and 214 healthy controls. Individuals carrying MBL2 O allele are at higher risk of developing AITD (OR = 1.58, 95% CI: 1.11-2.26; P-value = 0.009) and HT (OR = 1.67, 95% CI: 1.09-2.55; P-value = 0.013) as suggesting a possible role for mannose-binding lectin in influencing disease susceptibility.

Functional single-nucleotide polymorphisms in the DEFB1 gene are associated with systemic lupus erythematosus in Southern Brazilians.

Systemic lupus erythematosus (SLE) is an autoimmune disease that results in inflammation and tissue damage. The etiology of SLE remains unknown, but recent studies have shown that the innate immune system may have a role in SLE pathogenesis through the secretion of small cationic peptides named defensins. The aim of the study was to determine the possible involvement in SLE of three functional single nucleotide polymorphisms (SNPs) (c.-52G>A, c.-44C>G and c.-20G>A) in the 5'UTR region of DEFB1 gene, by analyzing them in a population of 139 SLE patients and 288 healthy controls. The c.-52G>A SNP showed significant differences in allele and genotype frequency distribution between SLE patients and controls (p = 0.01 and p = 0.02 respectively) indicating protection against SLE (A allele, OR = 0.68, AA genotype OR = 0.51). Significant differences were also observed for c.-44C>G SNP, the C/G genotype being associated with susceptibility to SLE (OR = 1.60, p = 0.04). Moreover, statistically significant differences between patients and controls were found for two DEFB1 haplotypes (GCA and GGG, p = 0.01 and p = 0.02 respectively). When considering DEFB1 SNPs and SLE clinical and laboratory manifestations, significant association was found with neuropsychiatric disorders, immunological alterations and anti-DNA antibodies. In conclusion, our results evidence a possible role for the c.-52G>A and c.-44C>G DEFB1 polymorphisms in SLE pathogenesis, that can be considered as possible risk factors for development of disease and disease-related clinical manifestations. Additional studies are needed, to corroborate these results as well as functional studies to understand the biological role of these SNPs in the pathogenesis of SLE.

Mannose-binding lectin (MBL2) polymorphisms and inflammation in hypertensive patients.

We investigated the possible role of Mannose binding lectin 2 (MBL2) functional polymorphisms in the prevalence of hypertension and hypertensive end-organ damage in 300 hypertensive patients and 313 normotensive individuals from Southern Brazil. Hypertensive subjects with MBL2 AO/OO genotypes presented lower C-reactive protein levels than AA individuals and consequently lower inflammatory status.

Extractive fermentation of clavulanic acid by Streptomyces DAUFPE 3060 using aqueous two-phase system.

The influence of four variables, specifically PEG molar mass (400, 1,000, and 8,000 g/mol), concentrations of PEG and phosphate salts (15, 20, and 25% for both), and agitation intensity (110, 150, and 200 rpm), on clavulanic acid (CA) extraction by extractive fermentation with PEG/phosphate salts aqueous two-phase system was investigated in shaken flasks using a 2(4-1) -fractional factorial design. After selection of the two most significant variables (agitation intensity and PEG molar mass), an optimization study conducted according to a 2(2) -central composite design revealed that 25% PEG 8,000 g/mol and phosphate salts at 240 rpm (run 6) were the best conditions for the extractive fermentation, leading to the best results in terms of partition coefficient (k = 8.2), yield of CA in the PEG-rich phase (η(T) = 93%) and productivity (P = 5.3 mg/Lh). As a first attempt to make a scale-up of these results, the effectiveness of the extractive fermentation was then checked in a bench-scale bioreactor under conditions as close as possible to the optimum ones determined in flasks. The highest CA concentration obtained in the PEG-rich phase (691 mg/L) was 30% higher than in flasks, thus demonstrating the potential of such a new process, integrating the production and extraction steps, as a promising, low-cost tool to obtain high yields of this and similar products.

Actinomicetos produtores de inibidores de beta-lactamases com atividade antimicrobiana frente a isolados de mastite bovina / Actinomycetes producers of beta-lactamases inhibitors with antimicrobial activity against microorganisms isolated from bovine mastitis

Avaliou-se a capacidade de 71 actinomicetos isolados de líquens da região amazônica em produzir inibidores de β-lactamases com atividade antimicrobiana sobre Staphylococcus aureus, resistentes à penicilina, isolados de mastite bovina do estado de Pernambuco. A seleção dos actinomicetos produtores de inibidores de β-lactamases foi realizada pela técnica de bloco de gelose contra Klebsiella pneumoniae ATCC 29665, e os actinomicetos selecionados foram testados frente a 17 linhagens de Staphylococcus aureus resistentes à penicilina. Os melhores produtores de inibidores de β-lactamases foram Streptomyces sp. DPUA 1542 e Nocardia sp. DPUA 1571, os quais foram submetidos ao cultivo submerso para determinação da curva de crescimento, pH e atividade antimicrobiana. Os maiores halos de inibição foram obtidos pelos metabólitos produzidos após 96 horas de cultivo tanto para Nocardia sp. - 13,5 e 12,0mm - como para Streptomyces sp. - 8,0 e 14,0mm - com os testes de difusão nos discos e poços, respectivamente. Os resultados permitiram concluir que os actinomicetos são fonte promissora de inibidores de β-lactamases, com potencial uso no tratamento de mastites bovinas.

The ability of 71 actinomycetes, isolated from the Amazon lichens, to produce β-lactamase inhibitors with antimicrobial activity was evaluated against penicillin-resistant Staphylococcus aureus, isolated from bovine mastitis in Pernambuco State. The selection of actinomycetes producers of β-lactamase inhibitors was performed using agar-plug method against Klebsiella pneumoniae ATCC 29665 and the selected actinomycetes were tested against 17 penicillin-resistant Staphylococcus aureus strains. The best producers of β-lactamase inhibitors were Streptomyces sp. DPUA 1542 and Nocardia sp. DPUA 1571. They were submitted to the submerged cultivation to determine the growth and pH curve, and antimicrobial activity. The highest inhibition halo zonewas obtained by metabolites produced after 96 hours of cultivation for both Nocardia sp. (13.5 and 12.0mm) and Streptomyces sp. (8.0 and 14.0mm) with discs and well diffusion tests, respectively. The results showed that the actinomycetes are a promising source of β-lactamase inhibitors, with potential for use in the bovine mastitis treatment.

The sound of silence: human beta-defensin-1 gene untranslated SNPs change the predicted mRNA secondary structure in a length-dependent manner.

This study investigated how 5'-UTR of DEFB1 gene (encoding for the human beta-defensin-1) affects mRNA secondary structure and its correlation with translation efficiency in the susceptibility of diseases. It was possible to determine DEFB1 mRNA folding under the influence of 5'-UTR SNPs haplotypes and putative alternative transcript lengths. Different DEFB1 mRNAs that fold in a pattern that is haplotype and length-dependent are potentially able to drive changes in peptide expression dynamics.

Covalent immobilization of invertase on polyurethane, plast-film and ferromagnetic Dacron.

Invertase was covalently immobilized on polyurethane (PU), inox plate covered with plast-film layer and ferromagnetic azide-Dacron. The immobilization processes, physico-chemical parameters and a model for coupling reactions were studied. The preliminary studies for selection of the support showed that the best activity was obtained for PU treated with HCl, polyethylenimine and glutaraldehyde (156.7+/-4.9 U/g support). All plast-film-invertase derivatives did not show activity and the Dacron-invertase derivative showed an activity of 105.39 U/g support. The invertase immobilized in presence of substrate (10% w/v sucrose) was the most efficient (832.74+/-1.48 U/g support). The optimal pH was shifted from 4.5 (free enzyme) to 5.0 (immobilized derivative) and optimal temperature was not affected. Activation energy values of free enzyme, Dacron-invertase and PU-invertase were 32.4+/-0.34 kJ/mol, 33.4+/-0.36 kJ/mol and 44.0+/-0.67 kJ/mol, respectively. The PU-invertase could be used over 2 months without considerable activity loss (68.5% activity retention) and retained 12.6% (287.97+/-27.9U/g support) of the activity after five cycles.

Extraction of ascorbate oxidase from Cucurbita maxima by continuous process in perforated rotating disc contactor using aqueous two-phase systems.

The ascorbate oxidase is the enzyme used to determine the content of ascorbic acid in the pharmaceutical and food industries and clinics analyses. The techniques currently used for the purification of this enzyme raise its production cost. Thus, the development of alternative processes and with the potential to reduce costs is interesting. The application of aqueous two-phase system is proposed as an alternative to purification because it enables good separation of biomolecules. The objective of this study was to determine the conditions to continuously pre-purify the enzyme ascorbate oxidase by an aqueous two-phase system (PEG/citrate) using rotating column provided with perforated discs. Under the best conditions (20,000 g/mol PEG molar mass, 10% PEG concentration, and 25% citrate concentration), the system showed satisfactory results (partition coefficient, 3.35; separation efficiency, 54.98%; and purification factor, 1.46) and proved suitable for the pre-purification of ascorbate oxidase in continuous process.

Screening of variables influencing the clavulanic acid production by Streptomyces DAUFPE 3060 strain.

Clavulanic acid (CA) is a beta-lactam antibiotic, which has a potent beta-lactamase inhibiting activity. The influence of five variables, namely pH (6.0, 6.4, and 6.8), temperature (28 degrees C, 30 degrees C, and 32 degrees C), agitation intensity (150, 200, and 250 rpm), glycerol concentration (5.0, 7.5, and 10 g/L) and soybean flour concentration (5.0, 12.5, and 20 g/L), on CA production by a new isolate of Streptomyces (DAUFPE 3060) was investigated in 250-mL Erlenmeyer flasks using a fractional factorial design. Temperature and soybean flour concentration were shown to be the two variables that exerted the most important effects on the production of CA at 95% confidence level. The highest CA concentration (494 mg/L) was obtained after 48 h at 150 rpm, 32 degrees C, pH 6.0, 5.0 g/L glycerol, and 20 g/L soybean flour concentrations. Under these conditions, the yields of biomass and product on consumed substrate were 0.26 g(X)/g(S) and 64.3 mg(P)/g(S), respectively. Fermentations performed in 3.0-L bench-scale fermenter allowed increasing the CA production by about 60%.

HPV31 and HPV33 incidence in cervical samples from women in Recife, Brazil.

Human papillomavirus (HPV) has been extensively studied concerning genomic structure, infection mechanisms, and diversity of types, as well as disease progression stages and development of vaccines. HPV type prevalence can differ in specific populations in different countries, according to ethnicity. This is the first report of an integrated project to evaluate the incidence of HPV types in different regions in Brazil in order to obtain data for vaccine development. Cervical samples were collected from women seen at a public hospital in Pernambuco, Northeast Brazil, for routine evaluation of genital alterations. Selection of the patients was random. There was a strong prevalence of HPV16 and a high incidence of HPV types 31 and 33. These data foster the discussion about the need to evaluate viral prevalence in each geographic region in order to develop targeted vaccine programs.

Functional polymorphisms of DEFB1 gene in type 1 diabetes Brazilian children.

We analyzed three functional 5' un-translated region beta-defensin 1 (DEFB1) single nucleotide polymorphism (SNPs) in a group of 170 type 1 diabetes (T1D) patients. In order to evaluate the SNPs influence on the disease onset and the development of other autoimmune disorder, such as celiac disease (CD) and autoimmune thyroid disease (AITD), patients were stratified according to the presence of AITD, CD, and both AITD and CD. As control group, we studied 191 healthy children and adolescent not presenting a familiar historic of T1D, CD or AITD. DEFB1 SNPs were in Hardy-Weinberg equilibrium both in healthy controls and T1D patients, as well in the T1D patients stratified according to the presence of other autoimmune disorder(s). Allele, genotype, and haplotype frequencies of T1D patients globally considered were comparable to healthy controls ones. No evidence of any association of DEFB1 SNPs with the onset of AIDT, CD, and both AITD and CD on T1D patients was evidenced. Only a minor trend was found for an increased frequency of the - 20 G allele in T1D patients only presenting AITD vs. T1D patients not presenting AITD or CD, as well as an increase of those haplotypes comprising the - 20 G allele when compared with the GCA haplotype. We also evaluated the influence of functional DEFB1 SNPs on the age of T1D onset: no significant statistical conclusion was achieved. Further studies are envisaged, in order to elucidate the possible role of functional DEFB1 polymorphisms in the onset of TD1 and other autoimmune-related disorders.