Barnacle Allergy: Allergen Characterization and Cross-Reactivity with Mites

Background: Barnacles are a type of seafood with worldwide distribution and abundant along the shores of temperate seas. They are particularly appreciated and regularly consumed in Portugal as well as in Spain, France and South America, but barnacle allergy is a rare condition of which there is only one reference in the indexed literature. The molecular allergens and possible cross-reactivity phenomena implicated (namely with mites) have not been established. Objective: To demonstrate the IgE-mediated allergy to barnacle and to identify the proteins implicated as well as possible cross-reactivity phenomena with mites. Methods: We report the clinical and laboratory data of five patients with documented IgE-mediated allergy to barnacle. The diagnosis was based on a suggestive clinical history combined with positive skin prick tests (SPT) to barnacle – prick to prick method. Two barnacle extracts were prepared (raw and cooked barnacle) and sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and IgE-immunoblotting were performed. An immunoblotting inhibition assay with Dermatophagoides pteronyssinus was also done in order to evaluate cross-reactivity. Results: All patients had mite-related asthma and the allergic rhinoconjunctivitis; they all experienced mucocutaneous symptoms. All of them had positive SPT to barnacle, and the immunoblotting showed several allergenic fractions with a wide molecular weight range (19 – 94 kDa). The D. pteronyssinus extract inhibited several IgE-binding protein fractions in the barnacle extract. Conclusions: We describe five patients with IgE-mediated barnacle allergy. We also describe a group of IgEbinding+ proteins between 30 and 75 kDa as the allergenic fractions of this type of Crustacea. Cross-reactivity with D. pteronyssinus was demonstrated in two cases.

Exposure of Lycopersicon Esculentum to Microcystin-LR: Effects in the Leaf Proteome and Toxin Translocation from Water to Leaves and Fruits

Natural toxins such as those produced by freshwater cyanobacteria have been regarded as an emergent environmental threat. However, the impact of these water contaminants in agriculture is not yet fully understood. The aim of this work was to investigate microcystin-LR (MC-LR) toxicity in Lycopersicon esculentum and the toxin accumulation in this horticultural crop. Adult plants (2 month-old) grown in a greenhouse environment were exposed for 2 weeks to either pure MC-LR (100 μg/L) or Microcystis aeruginosa crude extracts containing 100 μg/L MC-LR. Chlorophyll fluorescence was measured, leaf proteome investigated with two-dimensional gel electrophoresis and Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF)/TOF, and toxin bioaccumulation assessed by liquid chromatography-mass spectrometry (LC-MS)/MS. Variations in several protein markers (ATP synthase subunits, Cytochrome b6-f complex iron-sulfur, oxygen-evolving enhancer proteins) highlight the decrease of the capacity of plants to synthesize ATP and to perform photosynthesis, whereas variations in other proteins (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and ribose-5-phosphate isomerase) suggest an increase of carbon fixation and decrease of carbohydrate metabolism reactions in plants exposed to pure MC-LR and cyanobacterial extracts, respectively. MC-LR was found in roots (1635.21 μg/kg fw), green tomatoes (5.15–5.41 μg/kg fw), mature tomatoes (10.52–10.83 μg/kg fw), and leaves (12,298.18 μg/kg fw). The results raise concerns relative to food safety and point to the necessity of monitoring the bioaccumulation of water toxins in agricultural systems affected by cyanotoxin contamination.

Echovirus 4 associated to hand, foot and mouth disease

Hand, foot and mouth disease (HFMD) is a contagious enteroviral infection occurring primarily in children and characterized by vesicular palmoplantar eruptions and erosive stomatitis. Echovirus 4 (EV-4) has been commonly associated with aseptic meningitis. The association of HFMD with EV-4 has not been reported previously. Two samples of a 14-month child who presented mild fever, sores in the mouth, rash with blisters on the palm of hands and soles of feet were sent to Enteric Viruses Laboratory of Adolfo Lutz Institute. Clinical samples were inoculated in three different cell lines, and those which presented cytopathic effect (CPE), were submitted to Indirect Immunofluorescence Assay (IFA) and "one step" RT-PCR. Agarose gel electrophoresis from RT-PCR product, showed a product with 437 bp, which is characteristic of Enterovirus group. Echovirus 4 was identified by IFA. Although HFMD is a viral infection associated mainly with Enterovirus 71 (HEV-71) and Coxsackievirus A16 (CV-A16), our results demonstrate a diversity of serotype related to HFMD and stress the importance of epidemiological surveillance to this disease and its complications.

Neurotoxic activity and ultrastructural changes in muscles caused by the brown widow spider Latrodectus geometricus venom

Brown widow spider (Latrodectus geometricus) venom (BrWSV) produces few local lesions and intense systemic reactions such as cramps, harsh muscle pains, nausea, vomiting and hypertension. Approximately 16 protein bands under reducing conditions and ~ 14 bands under non-reducing conditions on a 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis were observed. Neurotoxic clinical manifestations were confirmed in vivo, while proteolytic activity was demonstrated on gelatine film. Severe ultrastructural damages in mice skeletal muscles were observed at 3, 6, 12 and 24 h postinjection with at total of 45 µg of venom protein. Infiltration of eosinophils and ruptures of the cellular membranes were observed in the muscles along with swelling of the nuclear cover and interruption of the collagen periodicity. Altered mitochondrias and autophage vacuoles, nuclear indentation and mitochondria without cristae, slight increment of intermyofibrillar and subsarcolemic spaces and myelinic figures formation were also observed. In the capillary, endothelial membrane unfolding into the lumen was noticed; along with myelinic figures compatible with a toxic myopathy. Swollen sarcotubular systems with lysis of membrane, intense mitochondria autophagia and areas without pinocytic vesicles were observed. Swollen mitochondria surrounded by necrotic areas, myofibrillar disorganization and big vacuolas of the sarcotubular system, degenerated mitochondrium with formation of myelinic figure was seen. Glycogenosomes with small particulate, muscle type glycogen was noticed. Autophagic vacuole (autophagolysosomes) and necrotic areas were also noticed. These damages may be due to interactive effects of the multifactorial action of venom components. However, Latrodectus geometricus venom molecules may also be utilized as neuro therapeutic tools, as they affect neuronal activities with high affinity and selectivity. To our knowledge, the present study is the first ultrastructural report in the literature of muscle injuries and neurological and proteolytic activities caused by BrWSV.

EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES

Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.

Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica

Biochemistry, 2011, 50 (20), pp 4251–4262 DOI: 10.1021/bi101605p

Candida albicans PROTEIN PROFILE CHANGES IN RESPONSE TO THE BUTANOLIC EXTRACT OF Sapindus saponariaL.

Candida albicans is an opportunistic human pathogen that is capable of causing superficial and systemic infections in immunocompromised patients. Extracts of Sapindus saponaria have been used as antimicrobial agents against various organisms. In the present study, we used a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify the changes in protein abundance of C. albicans after exposure to the minimal inhibitory concentration (MIC) and sub-minimal inhibitory concentration (sub-MIC) of the butanolic extract (BUTE) of S. saponaria and also to fluconazole. A total of six different proteins with greater than 1.5 fold induction or repression relative to the untreated control cells were identified among the three treatments. In general, proteins/enzymes involved with the glycolysis (GPM1, ENO1, FBA1), amino acid metabolism (ILV5, PDC11) and protein synthesis (ASC1) pathways were detected. In conclusion, our findings reveal antifungal-induced changes in protein abundance of C. albicans. By using the previously identified components of the BUTE of S. saponaria(e.g., saponins and sesquiterpene oligoglycosides), it will be possible to compare the behavior of compounds with unknown mechanisms of action, and this knowledge will help to focus the subsequent biochemical work aimed at defining the effects of these compounds.

Development of ion jelly thin films for electrochemical devices

Dissertação para obtenção do Grau de Doutor em Química Sustentável

Microbial contribution to biofuels production

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica

Enrichment of a PHA producing microbial community in a continuous bioreactor setup

Polyhydroxyalkanoates (PHAs) are biosynthetic polyesters, biodegradable and biocompatible making them of great interest for industrial purposes. The use of low value substrates with mixed microbial communities (MMC) is a strategy currently used to decrease the elevated PHA production costs. PHA production process requires an important step for selection and enrichment of PHA-storing microorganisms which is usually carried out in a Sequencing Batch Reactor (SBR). The aim of this study was to optimize the PHA accumulating culture selection stage using a 2-stage Continuous Stirrer Tank Reactor (CSTR) system. The system was composed by two separate feast and famine bioreactors operated continuously, mimicking the feast and famine phases in a SBR system. Acetate was used as carbon source and biomass seed was highly enriched in Plasticicumulans acidivorans obtained from activated sludge. The system was operated under two different sets of conditions (setup 1 and 2), maintaining a system total retention time of 12 hours and an OLR of 2.25 Cmmol/L.h-1. An average PHB-content of 3.3 % wt was obtained in setup 1 and 4.8% wt in setup 2. Several other experiments were performed in order to better understand the continuous system behaviour, using biomass from the continuous system. With the fed-batch experiment a maximum of 8.1% PHB was stored and the maximum substrate uptake and specific growth rates obtained in the growth experiment (1.15 Cmol Cmol-1.h-1 and 0.53 Cmol Cmol-1.h-1) were close to the ones from continuous system (1.12 Cmol Cmol-1.h-1 and 0.59 Cmol Cmol-1.h-1). The microbial community was characterized trough microscopic visualization, Denaturing Gradient Gel Electrophoresis (DGGE) analysis and Fluorescent in situ hybridization (FISH). The last studied performed mimicked the continuous system by building up a SBR system with all the same operational conditions while adding an extra acetate dosage during the 12 h cycle, simulating the substrate passing from the feast to the famine reactors under continuous operation. It was shown that possibly the continuous system was not able to efficiently select for PHB storing organisms under the operational conditions imposed, although the selected culture was capable of consuming the substrate and grow fast. This main conclusion might have resulted from two major factors affecting the system performance: the ammonium concentration in the Feast reactor and the amount of substrate leaching from the Feast to the Famine reactor.

Gastroenteric virus detection in fecal samples from women in Goiânia, State of Goiás, Brazil

INTRODUCTION: This was a prospective study that included women seen in the obstetrics and gynecology sector of Hospital das Clínicas, Federal University of Goiás, in Goiânia, State of Goiás, with the aim of detecting rotaviruses, adenoviruses, caliciviruses and astroviruses. Eighty-four women participated in the study and from these, 314 fecal samples were collected. Out of all of the women, 29 were seropositive for HIV and 55 were seronegative, and 45 and 39 were pregnant and non-pregnant, respectively. METHODS: Fecal samples were collected from each woman once every two months over the period from July 2006 to June 2007, and they were screened for rotaviruses by means of polyacrylamide gel electrophoresis and immunoenzymatic assays, for caliciviruses and astroviruses by means of RT-PCR and for adenovirus by means of immunoenzymatic assays. The astroviruses were genotyped using nested PCR. RESULTS: Among the 84 patients, 19 (22.6%) were positive for either calicivirus (14/19) or astrovirus (6/19), while one women was positive for both viruses in fecal samples collected on different occasions. Most of the positive samples were collected during the months of July and August (astrovirus) and September and October (calicivirus). None of the samples analyzed was positive for rotavirus or adenovirus. Gastroenteric viruses were detected in 13/19 (68.4%) of the pregnant women, whether HIV-seropositive or not. CONCLUSIONS: The results from the present study showed that neither pregnancy nor HIV-seropositive status among the women increased the risk of infection by any of the gastroenteric viruses studied. This study presents data on gastroenteric virus detection among pregnant and/or HIV-positive women.

High molecular mass fraction in clinical isolates of Paracoccidioides brasiliensis

INTRODUCTION: Different serum levels of the IgG/IgE for Paracoccidioides brasiliensis high mass molecular (hMM) fraction (~366kDa) in the acute and chronic forms of the disease have been reported. Considering the nonexistence of hMM fraction investigation involving clinical isolates of P. brasiliensis, the present study aimed to investigate the presence of the hMM fraction (~366kDa) in cell free antigens (CFA) from P. brasiliensis clinical isolates. METHODS: CFA from 10 clinical isolates and a reference strain (Pb18) were submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by gel image capturing and densitometer analysis. Additionally, CFA from 20 isolates and Pb18 were analyzed by capture ELISA (cELISA) using polyclonal (polAb) or monoclonal (mAb) antibodies to the hMM fraction. RESULTS: The presence of the hMM component was observed in CFA of all samples analyzed by SDS-PAGE/densitometry and by cELISA. In addition, Pearson's correlation test demonstrated stronger coefficients between hMM fraction levels using pAb and mAb (R = 0.853) in cELISA. CONCLUSIONS: The soluble hMM fraction was present in all the P. brasiliensis clinical isolates analyzed and the reference strain Pb18, which could be used as a source of this antigen. The work also introduces for first time, the cELISA method for P. brasiliensis hMM fraction detection. Analysis also suggests that detection is viable using polAb or mAb and this methodology may be useful for future investigation of the soluble hMM fraction (~366kDa) in sera from PCM patients.

Rotavirus genotyping in gastroenteritis cases of an infantile population from Western Brazilian Amazonia

INTRODUCTION: During the period from 2000 to 2002, 79 rotavirus-positive stool samples were collected from children presenting diarrhea in the Western Brazilian Amazon. METHODS: Molecular characterization of the G and P genotypes was performed using RT-PCR and electropherotyping analysis by polyacrylamide gel electrophoresis. RESULTS: A total of 59 samples were confirmed as group A rotavirus. A long electrophoretic profile was exhibited by the G1P[8], G3P[8], and G4P[8] genotypes. The G1P[8] genotype was found in greater proportion. The short electropherotype was exhibited only by G2 genotype strains. CONCLUSIONS: The proportion of the rotavirus genotypes observed was not different from that in other areas of Brazil. This study is the first genotyping of rotavirus in the Western Brazilian Amazon.

Analysis of the chemical components of hydatid fluid from Echinococcus granulosus

Introduction The aim of this study was to explore the environment of Echinococcus granulosus (E. granulosus) protoscolices and their relationship with their host. Methods Proteins from the hydatid-cyst fluid (HCF) from E. granulosus were identified by proteomics. An inductively coupled plasma atomic emission spectrometer (ICP-AES) was used to determine the elements, an automatic biochemical analyzer was used to detect the types and levels of biochemical indices, and an automatic amino acid analyzer was used to detect the types and levels of amino acids in the E. granulosus HCF. Results I) Approximately 30 protein spots and 21 peptide mass fingerprints (PMF) were acquired in the two-dimensional gel electrophoresis (2-DE) pattern of hydatid fluid; II) We detected 10 chemical elements in the cyst fluid, including sodium, potassium, calcium, magnesium, copper, and zinc; III) We measured 19 biochemical metabolites in the cyst fluid, and the amount of most of these metabolites was lower than that in normal human serum; IV) We detected 17 free amino acids and measured some of these, including alanine, glycine, and valine. Conclusions We identified and measured many chemical components of the cyst fluid, providing a theoretical basis for developing new drugs to prevent and treat hydatid disease by inhibiting or blocking nutrition, metabolism, and other functions of the pathogen.

Evaluation of arboviruses of public health interest in free-living non-human primates (Alouatta spp., Callithrix spp., Sapajus spp.) in Brazil

INTRODUCTION: The aim of the present study was to evaluate the presence of arboviruses from the Flavivirus genus in asymptomatic free-living non-human primates (NHPs) living in close contact with humans and vectors in the States of Paraná and Mato Grosso do Sul, Brazil. METHODS: NHP sera samples (total n = 80, Alouatta spp. n = 07, Callithrix spp. n = 29 and Sapajus spp. n = 44) were screened for the presence of viral genomes using reverse transcription polymerase chain reaction and 10% polyacrylamide gel electrophoresis techniques. RESULTS: All of the samples were negative for the Flavivirus genome following the 10% polyacrylamide gel electrophoresis analysis. CONCLUSIONS: These negative results indicate that the analyzed animals were not infected with arboviruses from the Flavivirus genus and did not represent a risk for viral transmission through vectors during the period in which the samples were collected.