Metabolômica, uma abordagem otimizada para exploração da biodiversidade brasileira: estado da arte, perspectivas e desafios

Brazilian biodiversity is a colossal source of secondary metabolites with remarkable structural features, which are valuable in further biodiscovery studies. In order to fully understand the relations and interactions of a living system with its surroundings, efforts in natural product chemistry are directed toward the challenge of detecting and identifying all the molecular components present in complex samples. It is plausible that this endeavor was born out of recent technological sophistication in secondary metabolite identification with sensitive spectroscopic instruments (MS and NMR) and higher resolving power of chromatographic systems, which allow a decrease in the amount of required sample and time to acquire data. Nevertheless, the escalation of data acquired in these analyses must be sorted with statistical and multi-way tools in order to select key information. Chromatography is also of paramount importance, more so when selected compounds need to be isolated for further investigation. However, in the course of pursuing a "greener" environment, new policies, with an aim to decrease the use of energy and solvents, are being developed and incorporated into analytical methods. Metabolomics could be an effective tool to answer questions on how living organisms in our huge biodiversity work and interact with their surroundings while also being strategic to the development of high value bio-derived products, such as phytotherapeutics and nutraceuticals. The incorporation of proper phytotherapeutics in the so-called Brazilian Unified Health System is considered an important factor for the urgent improvement and expansion of the Brazilian national health system. Furthermore, this approach could have a positive impact on the international interest toward scientific research developed in Brazil as well as the development of high value bio-derived products, which appear as an interesting economic opportunity in national and global markets. Thus, this study attempts to highlight the recent advances in analytical tools used in detection of secondary metabolites, which can be useful as bioproducts. It also emphasizes the potential avenues to be explored in Brazilian biodiversity, known for its rich chemical diversity.

Liposomes as a gene delivery system

Gene therapy is an active field that has progressed rapidly into clinical trials in a relatively short time. The key to success for any gene therapy strategy is to design a vector able to serve as a safe and efficient gene delivery vehicle. This has encouraged the development of nonviral DNA-mediated gene transfer techniques such as liposomes. Many liposome-based DNA delivery systems have been described, including molecular components for targeting given cell surface receptors or for escaping from the lysosomal compartment. Another recent technology using cationic lipids has been evaluated and has generated substantial interest in this approach to gene transfer.

Boosting the suppressive effects of Ascaris suum components in IFN-γ-deficient mice

High molecular weight components from Ascaris suum extract suppress ovalbumin-specific immunity in mice. In IFN-γ-deficient mice, ovalbumin-specific delayed-type hypersensitivity reactions are more strongly downregulated by these suppressive components. Here, the cellularity of the delayed-type hypersensitivity reaction in IFN-γ-deficient mice and the increased downregulation induced by Ascaris suum components were analyzed. IL-12p40-dependent neutrophilic influx was predominant. Suboptimal doses of the suppressive fraction from this nematode completely inhibited the hypersensitivity reaction, thus indicating intensification of the immunosuppression under conditions of intense recruitment of IFN-γ-independent neutrophils.

Carbohydrate-rich high-molecular-mass antigens are strongly recognized during experimental Histoplasma capsulatum infection

INTRODUCTION: During histoplasmosis, Histoplasma capsulatum soluble antigens (CFAg) can be naturally released by yeast cells. Because CFAg can be specifically targeted during infection, in the present study we investigated CFAg release in experimental murine histoplasmosis, and evaluated the host humoral immune response against high-molecular-mass antigens (hMMAg. >150 kDa), the more immunogenic CFAg fraction. METHODS: Mice were infected with 2.2x10(4) H. capsulatum IMT/HC128 yeast cells. The soluble CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg circulating immune complexes (CIC) levels were determined by enzymelinked immunosorbent assay, at days 0, 7, 14, and 28 post-infection. RESULTS: We observed a progressive increase in circulating levels of CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg CIC after H. capsulatum infection. The hMMAg showed a high percentage of carbohydrates and at least two main immunogenic components. CONCLUSIONS: We verified for the first time that hMMAg from H. capsulatum IMT/HC128 strain induce humoral immune response and lead to CIC formation during experimental histoplasmosis.

Molecular characterization of Salmonella strains in individuals with acute diarrhea syndrome in the State of Sucre, Venezuela

INTRODUCTION:In Venezuela, acute diarrheic syndrome (ADS) is a primary cause of morbi-mortality, often involving the Salmonella genus. Salmonella infections are associated with acute gastroenteritis, one of the most common alimentary intoxications, and caused by the consumption of contaminated water and food, especially meat. METHODS: Conventional and molecular methods were used to detect Salmonella strains from 330 fecal samples from individuals of different ages and both sexes with ADS. Polymerase chain reaction (PCR) was used for the molecular characterization of Salmonella, using invA, sefA, and fliC genes for the identification of this genus and the serotypes Enteritidis and Typhimurium, respectively. RESULTS: The highest frequency of individuals with ADS was found in children 0-2 years old (39.4%), and the overall frequency of positive coprocultures was 76.9%. A total of 14 (4.2%) strains were biochemically and immunologically identified as Salmonella enterica subsp. enterica, of which 7 were classified as belonging to the Enteritidis serotype, 4 to the Typhimurium serotype, and 3 to other serotypes. The S. enterica strains were distributed more frequently in the age groups 3-4 and 9-10 years old. CONCLUSIONS: The molecular characterization method used proved to be highly specific for the typing of S. enterica strains using DNA extracted from both the isolated colonies and selective enrichment broths directly inoculated with fecal samples, thus representing a complementary tool for the detection and identification of ADS-causing bacteria.

Molecular approaches to malaria and Babesisosis diagnosis

The development of additional methods for detecting and identifuing Babesia and Plasmodium infections may be useful in disease monitoring, management and control efforts. To preliminarily evaluate sunthetic peptide-based serodiagnosis, a hydrophilic sequence (DDESEFDKEK)was selected from published BabR gene of B. bovis. Immunization of rabbits and cattle with the hemocyanin-conjugated peptide elicited antibody responses that specifically detected both P. falciparum and B. bovis antigens by immunofluorescence and Western blots. Using a dot-ELISA with this peptide, antisera from immunized and naturally-infected cattle, and immunized rodents, were specifically detected. Reactivity was weak and correlated with peptide immunization or infection. DNA-based detection using repetitive DNA was species-specific in dot-blot formats for B. bovis DNA, and in both dot-blot and in situ formats for P. falciparum; a streamlined enzymelinked synthetic DNA assay for P. falciparum detected 30 parasites/mm(cúbicos) from patient blood using either colorimetric (2-15 h color development) or chemiluminescent detection (0.5-6-min. exposures). Serodiagnostic and DNA hybridization methods may be complementary in the respective detection of both chronic and acute infections. However, recent improvements in the polymerase chain reaction (PCR) make feasible a more sensitive and uniform approach to the diagnosis of these and other infectious disease complexes, with appropriate primers and processing methods. An analysis of ribosomal DNA genes of Plasmodium and Toxoplasma identified Apicomplexa-conserved sequence regions. Specific and distinctive PCR profiles were obtained for primers spanning the internal transcribed spacer locus for each of several Plasmodium and Babesia species.

Malaria diagnosis from pooled blood samples: comparative analysis of real-time PCR, nested PCR and immunoassay as a platform for the molecular and serological diagnosis of malaria on a large-scale

Malaria diagnoses has traditionally been made using thick blood smears, but more sensitive and faster techniques are required to process large numbers of samples in clinical and epidemiological studies and in blood donor screening. Here, we evaluated molecular and serological tools to build a screening platform for pooled samples aimed at reducing both the time and the cost of these diagnoses. Positive and negative samples were analysed in individual and pooled experiments using real-time polymerase chain reaction (PCR), nested PCR and an immunochromatographic test. For the individual tests, 46/49 samples were positive by real-time PCR, 46/49 were positive by nested PCR and 32/46 were positive by immunochromatographic test. For the assays performed using pooled samples, 13/15 samples were positive by real-time PCR and nested PCR and 11/15 were positive by immunochromatographic test. These molecular methods demonstrated sensitivity and specificity for both the individual and pooled samples. Due to the advantages of the real-time PCR, such as the fast processing and the closed system, this method should be indicated as the first choice for use in large-scale diagnosis and the nested PCR should be used for species differentiation. However, additional field isolates should be tested to confirm the results achieved using cultured parasites and the serological test should only be adopted as a complementary method for malaria diagnosis.

Biogeographic analysis of Crocidiinae (Diptera, Bombyliidae): finding congruence among morphological, molecular, fossil and paleogeographical data

Biogeographic studies dealing with Bombyliidae are rare in the literature and no information is available on its origin and early diversification. In this study, we found evidence from molecular phylogeny and from fossil record supporting a Middle Jurassic origin of the Bombylioidea, taken as a starting point to discuss the biogeography and diversification of Crocidiinae. Based on a previously published phylogenetic hypothesis, we performed a Brooks Parsimony Analysis (BPA) to discuss the biogeographical history of Crocidiinae lineages. This subfamily is mostly distributed over arid areas of the early components of the Gondwanaland: Chile and southern Africa, but also in southwestern Palaearctic and southwestern Nearctic. The vicariant events affecting the Crocidiinae biogeography at the generic level seems to be related to the sequential separation of a Laurasian clade from a Gondwanan clade followed by the splitting of the latter into smaller components. This also leads to a hypothesis of origin of the Crocidiinae in the Middle Jurassic, the same period in which other bombyliid lineages are supposed to have arisen and irradiated.

Genetic diversity of irrigated barley based on molecular and quantitative data and on malting quality

The objective of this work was to quantify the genetic diversity of elite genotypes of irrigated barley in the Brazilian savanna. Thirty elite barley genotypes from Embrapa Cerrados' collection were evaluated using 160 RAPD markers, 12 agronomic traits related to yield components, and 10 malting quality parameters. The genetic dissimilarity matrices based on molecular markers, quantitative traits, and malting quality characters were calculated and a cluster analysis was performed using the unweighted pair-group method with arithmetic mean (UPGMA) as grouping criterion. High genetic diversity among accessions were observed. The estimated genetic dissimilarities were weakly correlated, showing the complementarity of the different character groups. Selection indices and graphical dispersion analysis allowed the selection of promising genotypes and the indication of suitable crosses for maximizing the heterotic effects in breeding programs for irrigated barley in the Brazilian savanna.

Modelagem Molecular: Uma Ferramenta para o Planejamento Racional de Fármacos em Química Medicinal

The molecular basis of modern therapeutics consist in the modulation of cell function by the interaction of microbioactive molecules as drug cells macromolecules structures. Molecular modeling is a computational technique developed to access the chemical structure. This methodology, by means of the molecular similarity and complementary paradigm, is the basis for the computer-assisted drug design universally employed in pharmaceutical research laboratories to obtain more efficient, more selective, and safer drugs. In this work, we discuss some methods for molecular modeling and some approaches to evaluate new bioactive structures in development by our research group.

Comparison of nuclear grade and immunohistochemical features in situ and invasive components of ductal carcinoma of breast

PURPOSE:To compare the prognostic and predictive features between in situ and invasive components of ductal breast carcinomas. METHODS:We selected 146 consecutive breast samples with ductal carcinoma in situ (DCIS) associated with adjacent invasive breast carcinoma (IBC). We evaluated nuclear grade and immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6 (CK5/6), and epidermal growth factor receptor (EGFR) in both components, in situ and invasive, and the Ki-67 percentage of cells in the invasive part. The DCIS and IBC were classified in molecular surrogate types determined by the immunohistochemical profile as luminal (RE/PR-positive/ HER2-negative), triple-positive (RE/RP/HER2-positive), HER2-enriched (ER/PR-negative/HER2-positive), and triple-negative (RE/RP/HER2-negative). Discrimination between luminal A and luminal B was not performed due to statistical purposes. Correlations between the categories in the two groups were made using the Spearman correlation method. RESULTS:There was a significant correlation between nuclear grade (p<0.0001), expression of RE/RP (p<0.0001), overexpression of HER2 (p<0.0001), expression of EGFR (p<0.0001), and molecular profile (p<0.0001) between components in situ and IBC. CK 5/6 showed different distribution in DCIS and IBC, presenting a significant association with the triple-negative phenotype in IBC, but a negative association among DCIS. CONCLUSIONS: Our results suggest that classical prognostic and predictive features of IBC are already determined in the preinvasive stage of the disease. However the role of CK5/6 in invasive carcinoma may be different from the precursor lesions.

Autoimmunity and molecular mimicry in tropical spastic paraparesis/human T-lymphotropic virus-associated myelopathy

Viruses share antigenic sites with normal host cell components, a phenomenon known as molecular mimicry. It has long been suggested that viral infections might trigger an autoimmune response by several mechanisms including molecular mimicry. More than 600 antiviral monoclonal antibodies generated against 11 different viruses have been reported to react with 3.5% of cells specific for uninfected mouse organs. The main pathological feature of tropical spastic paraparesis/human T-lymphotropic virus type I (HTLV-I)-associated myelopathy (TSP/HAM) is a chronic inflammation of the spinal cord characterized by perivascular cuffing of mononuclear cells accompanied by parenchymal lymphocytic infiltration. We detected the presence of autoantibodies against a 98- to 100-kDa protein of in vitro cultured human astrocytes and a 33- to 35-kDa protein from normal human brain in the serum of HTLV-I-seropositive individuals. The two cell proteins exhibited molecular mimicry with HTLV-I gag and tax proteins in TSP/HAM patients, respectively. Furthermore, the location of 33- to 35-kDa protein cross-reaction correlated with the anatomical spinal cord areas (in the rat model) in which axonal damage has been reported in several cases of TSP/HAM patients. Our experimental evidence strongly suggests that the demyelinating process occurring in TSP/HAM may be mediated by molecular mimicry between domains of some viral proteins and normal cellular targets of the spinal cord sections involved in the neurodegeneration.

Electron paramagnetic resonance study of lipid and protein membrane components of erythrocytes oxidized with hydrogen peroxide

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H2O2). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H2O2 (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H2O2 (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation.

Reversal of the anticoagulant and anti-hemostatic effect of low molecular weight heparin by direct prothrombin activation

Lopap, found in the bristles of Lonomia obliqua caterpillar, is the first exogenous prothrombin activator that shows serine protease-like activity, independent of prothrombinase components and unique lipocalin reported to interfere with hemostasis mechanisms. To assess the action of an exogenous prothrombin activator reversing the anticoagulant and antihemostatic effect induced by low molecular weight heparin (LMWH), male New Zealand rabbits (N = 20, weighing 3.8-4.0 kg) allocated to 4 groups were anticoagulated with 1800 IU/kg LMWH (iv) over 2 min, followed by iv administration of saline (SG) or recombinant Lopap (rLopap) at 1 µg/kg (LG1) or 10 µg/kg (LG10), 10 min after the injection of LMWH, in a blind manner. Control animals (CG) were treated only with saline. The action of rLopap was assessed in terms of activated partial thromboplastin time (aPTT), prothrombin fragment F1+2, fibrinogen, and ear puncture bleeding time (BT) at 5, 10, 15, 17, 20, 30, 40, 60, and 90 min after initiation of LMWH infusion. LG10 animals showed a decrease of aPTT in more than 50% and BT near to normal baseline. The level of prothrombin fragment F1+2 measured by ELISA had a 6-fold increase with rLopap treatment (10 µg/kg) and was inversely proportional to BT in LMWH-treated animals. Thus, Lopap, obtained in recombinant form using E. coli expression system, was useful in antagonizing the effect of LMWH through direct prothrombin activation, which can be a possible strategy for the reversal of bleeding and anticoagulant events.

Genética molecular da deficiência auditiva não-sindrômica

Aproximadamente 1/1000 recém-nascidos apresentam deficiência auditiva congênita, sendo 60% dessas de etiologia genética. Na maioria dos casos, a deficiência auditiva é uma doença multifatorial causada por ambos os fatores, genéticos e ambientais. A genética molecular da deficiência auditiva tem apresentado grandes avanços na última década, pois os genes responsáveis pela deficiência auditiva hereditária vêm sendo progressivamente mapeados e clonados. Esta revisão enfatiza a deficiência auditiva não-sindrômica, uma vez que, os genes envolvidos nesse tipo de deficiência foram identificados recentemente.