Characterization and regulation of a bacterial sugar phosphatase of the haloalkanoate dehalogenase superfamily, AraL, from Bacillus subtilis

FEBS journal, Volume 278, Issue 14, pages 2511-2524, July 2011

Diagnosis of hepatitis C virus in Brazilian blood donors using a reverse transcriptase nested polymerase chain reaction: comparison with enzyme immunoassay and recombinant protein immunoblot assay

Screening blood donations for anti-HCV antibodies and alanine aminotransferase (ALT) serum levels generally prevents the transmission of hepatitis C virus (HCV) by transfusion. The aim of the present study was to evaluate the efficiency of the enzyme immunoassay (EIA) screening policy in identifying potentially infectious blood donors capable to transmit hepatitis C through blood transfusion. We have used a reverse transcriptase (RT)-nested polymerase chain reaction (PCR) to investigate the presence of HCV-RNA in blood donors. The prevalence of HCV-RNA positive individuals was compared with the recombinant immunoblot assay (RIBA-2) results in order to assess the usefulness of both tests as confirmatory assays. Both tests results were also compared with the EIA-2 OD/C ratio (optical densities of the samples divided by the cut off value). ALT results were expressed as the ALT quotient (qALT), calculated dividing the ALT value of the samples by the maximum normal value (53UI/l) for the method. Donors (n=178) were divided into five groups according to their EIA anti-HCV status and qALT: group A (EIA > or = 3, ALT<1), group B (EIA > or = 3, ALT>1), group C (1<=EIA<3, ALT<1), group D (1<=EIA<3, ALT>1) and group E (EIA<=0.7). HCV sequences were detected by RT-nested PCR, using primers for the most conserved region of viral genome. RIBA-2 was applied to the same samples. In group A (n=6), all samples were positive by RT-nested PCR and RIBA-2. Among 124 samples in group B, 120 (96.8%) were RIBA-2 positive and 4 (3.2%) were RIBA-2 indeterminate but were seropositive for antigen c22.3. In group B, 109 (87.9%) of the RIBA-2 positive samples were also RT-nested PCR positive, as well as were all RIBA-2 indeterminate samples. In group C, all samples (n=9) were RT-nested PCR negative: 4 (44.4%) were also RIBA-2 negative, 4 (44.4%) were RIBA-2 positive and 1 (11.1%) was RIBA-2 indeterminate. HCV-RNA was detected by RT-nested PCR in 3 (37.5%) out of 8 samples in group D. Only one of them was also RIBA-2 positive, all the others were RIBA-2 indeterminate. All of the group E samples (controls) were RT- nested PCR and RIBA-2 negative. Our study suggests a strong relation between anti-HCV EIA-2 ratio > or = 3 and detectable HCV-RNA by RT-nested PCR. We have also noted that blood donors with RIBA-2 indeterminate presented a high degree of detectable HCV-RNA using RT-nested PCR (75%), especially when the c22.3 band was detected.

hsp65 PCR-restriction enzyme analysis (PRA) for identification of mycobacteria in the clinical laboratory

More than 70 species of mycobacteria have been defined, and some can cause disease in humans, especially in immunocompromised patients. Species identification in most clinical laboratories is based on phenotypic characteristics and biochemical tests and final results are obtained only after two to four weeks. Quick identification methods, by reducing time for diagnosis, could expedite institution of specific treatment, increasing chances of success. PCR restriction-enzyme analysis (PRA) of the hsp65 gene was used as a rapid method for identification of 103 clinical isolates. Band patterns were interpreted by comparison with published tables and patterns available at an Internet site (http://www.hospvd.ch:8005). Concordant results of PRA and biochemical identification were obtained in 76 out of 83 isolates (91.5%). Results from 20 isolates could not be compared due to inconclusive PRA or biochemical identification. The results of this work showed that PRA could improve identification of mycobacteria in a routine setting because it is accurate, fast, and cheaper than conventional phenotypic identification.

Descending pain modulation in a Kaolin-induced hydrocephalus rat model

Pain transmission at the spinal cord is modulated by descending actions that arise from supraspinal areas which collectively form the endogenous pain control system. Two key areas involved of the endogenous pain control system have a circunventricular location, namely the periaqueductal grey (PAG) and the locus coeruleus (LC). The PAG plays a crucial role in descending pain modulation as it conveys the input from higher brain centers to the spinal cord. As to the LC, it is involved in descending pain inhibition by direct noradrenergic projections to the spinal cord. In the context of neurological defects, several diseases may affect the structure and function of the brain. Hydrocephalus is a congenital or acquired disease characterized by an enlargement of the ventricles which leads to a distortion of the adjacent tissues, including the PAG and LC. Usually, patients suffering from hydrocephalus present dysfunctions in learning and memory and also motor deficits. It remains to be evaluated if lesions of the periventricular brain areas involved in pain control during hydrocephalus may affect descending pain control and, herein, affect pain responses. The studies included in the present thesis used an experimental model of hydrocephalus (the rat injected in the cisterna magna with kaolin) to study descending modulation of pain, focusing on the two circumventricular regions referred above (the PAG and the LC). In order to evaluate the effects of kaolin injection into the cisterna magna, we measured the degree of ventricular dilatation in sections encompassing the PAG by standard cytoarquitectonic stanings (thionin staining). For the LC, immunodetection of the noradrenaline-synthetizing enzyme tyrosine hydroxylase (TH) was performed, due to the noradrenergic nature of the LC neurons. In general, rats with kaolin-induced hydrocephalus presented a higher dilatation of the 4th ventricle, along with a tendency to a higher area of the PAG. Due to the validated role of detection the c-fos protooncogene as a marker of neuronal activation, we also studied neuronal activation in the several subnuclei which compose the PAG, namely the dorsomedial, dorsolateral, lateral and ventrolateral (VLPAG) parts. A decrease in the numbers of neurons immunoreactive for Fos protein (the product of activation of the c-fos protooncogene) was detected in rats injected with kaolin, whereas the remaining PAG subnuclei did not present changes in Fos-immunoreactive nuclei. Increases in the levels of TH in the LC, namely at the rostral parts of the nucleus, were detected in hydrocephalic animals. The following pain-related parameters were measured, namely 1) pain behavioural responses in a validated pain inflammatory test (the formalin test) and 2) the nociceptive activation of spinal cord neurons. A decrease in behavioral responses was detected in rats with kaolin-induced hydrocephalus was detected, namely in the second phase of the test (inflammatory phase). This is the phase of the formalin test in which the motor behaviour is less important, which is important since a semi-quantitative analysis of the motor performance of rats injected with kaolin indicates that these animals may present some motor impairments. Collectively, the results of the behavioral studies indicate that rats with kaolin-induced hydrocephalus exhibit hypoalgesia. A decrease in Fos expression was detected at the superficial dorsal layers of the spinal cord in rats with kaolin-induced hydrocephalus, further indicating that hydrocephalus decreases nociceptive responses. It remains to be ascertained if this is due to alterations in the PAG and LC in the rats with kaolin-induced hydrocephalus, which may affect descending pain modulation. It remains to be evaluated what are the mechanisms underlying the increased pain inhibition at the spinal dorsal horn in the hydrocephalus rats. Regarding the VLPAG, the decrease in neuronal activity may impair descending modulation. Since the LC has higher levels of TH in rats with kaolininduced hydrocephalus, which also appears to increase the noradrenergic innervation in the spinal dorsal horn, it is possible that an increase in the release of noradrenaline at the spinal cord accounts for pain inhibition. Our studies also determine the need to study in detail patients with hydrocephalus namely in what concerns their thresholds to pain and to perform imaging studies focused on the structure and function of pain control areas in the brain.

Crystallographic studies on molybdopterin-dependent enzymes

Dissertação para obtenção do Grau de Doutor em Bioquímica, Especialidade Bioquímica Estrutural

Backside-surface imprinting as a new strategy to generate specific plastic antibody materials

A backside protein-surface imprinting process is presented herein as a novel way to generate specific synthetic antibody materials. The template is covalently bonded to a carboxylated-PVC supporting film previously cast on gold, let to interact with charged monomers and surrounded next by another thick polymer. This polymer is then covalently attached to a transducing element and the backside of this structure (supporting film plus template) is removed as a regular “tape”. The new sensing layer is exposed after the full template removal, showing a high density of re-binding positions, as evidenced by SEM. To ensure that the templates have been efficiently removed, this re-binding layer was cleaned further with a proteolytic enzyme and solution washout. The final material was named MAPS, as in the back-side reading of SPAM, because it acts as a back-side imprinting of this recent approach. It was able to generate, for the first time, a specific response to a complex biomolecule from a synthetic material. Non-imprinted materials (NIMs) were also produced as blank and were used as a control of the imprinting process. All chemical modifications were followed by electrochemical techniques. This was done on a supporting film and transducing element of both MAPS and NIM. Only the MAPS-based device responded to oxLDL and the sensing layer was insensitive to other serum proteins, such as myoglobin and haemoglobin. Linear behaviour between log(C, μg mL−1) versus charged tranfer resistance (RCT, Ω) was observed by electrochemical impedance spectroscopy (EIS). Calibrations made in Fetal Calf Serum (FCS) were linear from 2.5 to 12.5 μg mL−1 (RCT = 946.12 × log C + 1590.7) with an R-squared of 0.9966. Overall, these were promising results towards the design of materials acting close to the natural antibodies and applied to practical use of clinical interest.

Measuring the cytochrome c nitrite reductase activity—practical considerations on the enzyme assays

Hindawi Publishing Corporation Bioinorganic Chemistry and Applications Volume 2010, Article ID 634597, 8 pages

Carbon key-properties for microcystin adsorption in drinking water treatment: structure or surface chemistry?

Dissertação para Obtenção de Grau de Mestre em Engenharia Química e Bioquímica

Update on the population structure of MRSA causing infection in a central hospital and in healthcare centers in Portugal

RESUMO:Staphylococcus aureus é um dos principais agentes patogénicos humanos, sendo frequentemente associado a infecções nosocomiais e infecções na comunidade. A prevalência de S. aureus resistentes à meticilina (MRSA) em hospitais portugueses é uma das mais elevadas da Europa e tem sido caracterizada extensivamente; contrariamente, a prevalência e epidemiologia de MRSA na comunidade em Portugal não tem sido devidamente seguida. Com o objectivo de compreender as causas possíveis do aumento na frequência de MRSA num dos maiores hospitais centrais portugueses (HSM) ao longo de 17 anos, isolados de MRSA recolhidos em 1993 (n=54) e 2010 (n=180) de pus, sangue e urina foram analisados por PFGE, MLST, tipagem do spa e tipagem de SCCmec. Os resultados mostraram que ocorreu uma mudança global nos tipos clonais predominantes, onde o clone ST22-IVh substituiu os clones, ST239-IIIvar e ST247-I, representando mais de 70% da população actual. Além disso, entre 1993 e 2010 verificou-se um aumento na diversidade genética dos tipos clonais de MRSA. Para determinar a frequência e a natureza clonal de MRSA e S. aureus sensíveis à meticilina (MSSA) isolados de infecções de pele e tecidos moles (SSTI) em pessoas que frequentam centros de saúde em Portugal, 73 amostras foram recolhidas em nove centros de saúde (Rede Médicos Sentinela). Isolou-se um total de 40 S. aureus (55%), dos quais 17,5% eram MRSA. Os isolados de MRSA pertenciam aos clones ST22-IVh (n=4), ST5-IVc (n=2) e ST105-II (n=1), que foram descritos neste estudo como sendo clones de origem hospitalar. Os nossos resultados sugerem que o aumento da frequência de MRSA no HSM pode estar associado à emergência de um clone de MRSA com maior capacidade epidémica. Além disso, verificámos que a principal causa de SSTI em pessoas que frequentam centros de saúde em Portugal são MRSA de origem hospitalar e não MRSA associados à comunidade.------ABSTRACT: Staphylococcus aureus is one of the most important human pathogens, being a major cause of infections worldwide both in the hospital and in the community. In Portugal, the prevalence of methicillin resistant S. aureus (MRSA) in hospitals is one of the highest in Europe and has been characterized extensively; contrarily the prevalence and epidemiology of MRSA in the community has not been followed in a meaningful way. To understand the epidemiological events that could explain a steep increase in MRSA frequency in a major Portuguese central hospital (HSM) within a 17 year period, two MRSA collections recovered in 1993 (n=54) and 2010 (n=180) from pus, blood and urine were analyzed by PFGE, MLST, spa and SCCmec typing. The results showed that a major clonal shift occurred, wherein ST22-IVh clone has replaced the previous ST239-IIIvar and ST247-I clones and accounts for more than 70% of the present population. Moreover, an increase in genetic diversity of MRSA clonal types was observed between the two study periods. With the aim of determining the frequency and clonal nature of MRSA and methicillin-susceptible S. aureus (MSSA) causing skin and soft tissue infections (SSTI) in patients attending healthcare centers in Portugal, 73 samples were collected from nine healthcare centers (Medicos Sentinela Network). A total of 40 S. aureus were isolated, accounting for 55% of the SSTI, of which 17.5% were MRSA. MRSA isolates belonged to ST22-IVh (n=4), ST5-IVc (n=2) and ST105-II (n=1) that have also been described in the hospital in an equivalent period. Our results suggest that the increase in MRSA frequency in HSM may be associated to the emergence of a MRSA clone with higher epidemic potential. Moreover, we propose that the spillover of MRSA from the hospital rather than community-associated-MRSA was the main cause of SSTI in persons attending healthcare centers in Portugal.

Insights into human carboxylesterase 2 stability and activity in vivo and in vitro

Dissertation to obtain a Master Degree in Biotechnology

APPLICATION OF 6-NITROCOUMARIN AS A SUBSTRATE FOR THE FLUORESCENT DETECTION OF NITROREDUCTASE ACTIVITY IN Sporothrix schenckii

Introduction Sporothrix schenckii is a thermal dimorphic pathogenic fungus causing a subcutaneous mycosis, sporotrichosis. Nitrocoumarin represents a fluorogenic substrate class where the microbial nitroreductase activity produces several derivatives, already used in several other enzyme assays. The objective of this study was the analysis of 6-nitrocoumarin (6-NC) as a substrate to study the nitroreductase activity in Sporothrix schenckii. Methods Thirty-five samples of S. schenckii were cultivated for seven, 14 and 21 days at 35 °C in a microculture containing 6-nitrocoumarin or 6-aminocoumarin (6-AC) dissolved in dimethyl sulfoxide or dimethyl sulfoxide as a negative control, for posterior examination under an epifluorescence microscope. The organic layer of the seven, 14 and 21-day cultures was analyzed by means of direct illumination with 365 nm UV light and by means of elution on G silica gel plate with hexane:ethyl acetate 1:4 unveiled with UV light. Results All of the strains showed the presence of 6-AC (yellow fluorescence) and 6-hydroxylaminocoumarin (blue fluorescence) in thin layer chromatography, which explains the green fluorescence observed in the fungus structure. Conclusion The nitroreductase activity is widely distributed in the S. schenckii complex and 6-NC is a fluorogenic substrate of easy access and applicability for the nitroreductase activity detection.

Chromosomal structure: a selectable trait for evolution

Dissertation presented to obtain the Ph.D degree in Evolutionary Biology

The electron transfer complex between nitrous oxide reductase and its electron donors

J Biol Inorg Chem (2011) 16:1241–1254 DOI 10.1007/s00775-011-0812-9

The tetranuclear copper active site of nitrous oxide reductase: the CuZ center

J Biol Inorg Chem (2011) 16:183–194 DOI 10.1007/s00775-011-0753-3

Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo-C Interaction in a Biological System

J. Am. Chem. Soc., 2009, 131 (23), pp 7990–7998 DOI: 10.1021/ja809448r