Sulphate-reducing bacteria associated with biocorrosion: a review

Biocorrosion means any process of corrosion in wich microorganisms are somehow involved. As far as the petroleum industry is concerned, the anaerobic type is the more important, with Sulphate-Reducing Bacteria (SRB) accouting for half of the described processes. SRB are obligate anaerobs that use sulphur, sulphate or other oxidized sulphur compounds as oxidizing agents when decomposing organic material. A typical product of SRB metabolism, hydrogen sulphide -H2S-, is extremely toxic. In the present work we review the literature on mechanisms underlying biocorrosive process in wich SRB are involved and summarize some of the ultrastructural and eletrochemical work developed using SRB obtained from water injection flow in wells located on PETROBRAS offshore marine plataforms, sampled directly in the field over metallic probes, or cultured under laboratory conditions. Biofilms develop when SRB adhere to inert surfaces. A high diversity of morphological types is found inside these biofilms. Their extracellular matrix is highly hydrated and mainly anionic, as shown by its avid reaction with cationic compounds like ruthenium red. We have noted that variations in iron contet lead to interesting changes in the ultrastructure of the bacterial cell coat and also in the rate of corrosion induced in metallic test cupons. Since routine methods to prevent and treat SRB contamination and biodeterioration involve the use of biocides that are toxic and always have some environmental impact, an accurate diagnosis of biocorrosion is always required prior to a treatment decision. We developed a method that detects and semi-quantifies the presence of living or dead SRB by using free silver potentials as an indicator of corrosive action by SRB-associated sulphides. We found a correlation between sulphide levels (determined either by spectrophotometry, or using a silver electrode -E(Ag)- that measured changes in free potentials induced by the presence of exogeneously added sulphide) and SRB concentration (enumerated by a culturing method). E (Ag) was characterized under a variety of conditions andwas found to be relatively immune to possible interference resulting from aeration of media or from the psence of iron corrosion products. The method offers a simple, rapid, and effective means of diagnosing biocorrosive processes prior to their control.

Evaluation of some organic compounds on bloodstream forms of Trypanosoma cruzi

Accidental transmission of Chagas' disease to man by blood transfusion is a serious problem in Latin-America. This paper describes the testing of several synthetic, semi-synthetic, and natural compounds for their activity against blood trypomastigotes in vitro at 4-C. The compounds embody several types of chemical structures: benzoquinone, naphthoquinone, anthracenequinone, phenanthrenequinone, imidazole, piperazine, quinoline, xanthene, and simple benzenic and naphthalenic derivates. Some of them are for the first time tested against Trypanosoma cruzi. The toxic effect these compounds on this parasite was done by two quite distinct sets of experiments. In one set, the compounds were added to infected blood as ethanolic solution. In this situation the most active one was a furan-1, 2-naphthoquinone, in the same range as gentian violet, a new fact to be considered in the assessment of structure-activity relationships in this class of compounds. In other set, we tentatively evaluated the biological activity of water insoluble compounds by adding them in a pure form without solvent into infected blood. In this way some appear to be very active and it was postulated that the effectiveness of such compounds must result from interactions between them and specific blood components.

Simultaneous Evaluation of Intact Glucosinolates and Phenolic Compounds by UPLC-DAD-MS/MS in Brassica oleracea L. var. botrytis

A method for the simultaneous determination of intact glucosinolates and main phenolic compounds (flavonoids and sinapic acid derivatives) in Brassica oleracea L. var. botrytis was proposed. A simplified sample extraction procedure and a UPLC separation were carried out to reduce the total time of analysis. Brassica oleracea samples were added with internal standards (glucotropaeolin and rutin), and extracted with boiling methanol. Crude extracts were evaporated under nitrogen, redissolved in mobile phase and analyzed by UPLC with double detection (ESI--MRM for glucosinolates and flavonoids, and DAD for main sinapic acid derivatives). The proposed method allowed a satisfactory quantification of main native sinapic acid derivatives, flavonoids and glucosinolates with a reduced time of analysis.

Development of an optimized methodology for tensile testing of carbon steels in hydrogen environment

The study was performed at OCAS, the Steel Research Centre of ArcelorMittal for the Industry market. The major aim of this research was to obtain an optimized tensile testing methodology with in-situ H-charging to reveal the hydrogen embrittlement in various high strength steels. The second aim of this study has been the mechanical characterization of the hydrogen effect on hight strength carbon steels with varying microstructure, i.e. ferrite-martensite and ferrite-bainite grades. The optimal parameters for H-charging - which influence the tensile test results (sample geometry type of electrolyte, charging methods effect of steel type, etc.) - were defined and applied to Slow Strain Rate testing, Incremental Step Loading and Constant Load Testing. To better understand the initiation and propagation of cracks during tensile testing with in-situ H-charging, and to make the correlation with crystallographic orientation, some materials have been analyzed in the SEM in combination with the EBSD technique. The introduction of a notch on the tensile samples permits to reach a significantly improved reproducibility of the results. Comparing the various steel grades reveals that Dual Phase (ferrite-martensite) steels are more sensitive to hydrogen induced cracking than the FB (ferritic-bainitic) ones. This higher sensitivity to hydrogen was found back in the reduced failure times, increased creep rates and enhanced crack initiation (SEM) for the Dual Phase steels in comparison with the FB steels.

Mutation analysis of the different tfd genes for degradation of chloroaromatic compounds in Ralstonia eutropha JMP134.

Ralstonia eutropha JMP134 possesses two sets of similar genes for degradation of chloroaromatic compounds, tfdCDEFB (in short: tfdI cluster) and tfdDII CII EII FII BII (tfdII cluster). The significance of two sets of tfd genes for the organism has long been elusive. Here, each of the tfd genes in the two clusters on the original plasmid pJP4 was replaced by double recombination with a gene fragment in which a kanamycin resistance gene was inserted into the respective tfd gene's reading frame. The insertion mutants were all tested for growth on 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), and 3-chlorobenzoate (3-CBA). None of the tfdDII CII EII FII BII genes appeared to be essential for growth on 2,4-D or on 3-CBA. Mutations in tfdC, tfdD and tfdF also did not abolish but only retarded growth on 2,4-D, indicating that they were redundant to some extent as well. Of all tfd genes tested, only tfdE and tfdB were absolutely essential, and interruption of those two reading frames abolished growth on 2,4-D, 3-CBA ( tfdE only), and MCPA completely. Interestingly, strains with insertion mutations in the tfdI cluster and those in tfdDII, tfdCII, tfdEII and tfdBII were severely effected in their growth on MCPA, compared to the wild-type. This indicated that not only the tfdI cluster but also the tfdII cluster has an essential function for R. eutropha during growth on MCPA. In contrast, insertion mutation of tfdDII resulted in better growth of R. eutropha JMP134 on 3-CBA, which is most likely due to the prevention of toxic metabolite production in the absence of TfdDII activity.

Adding diversity to ruthenium (II)-arene anticancer (RAPTA) compounds via click chemistry: the influence of hydrophobic chains

The application of click chemistry to develop libraries of organometallic ruthenium-arene complexes with potential anticancer properties has been investigated. A series of ruthenium-imidazole-triazole complexes, with hydrophobic tails, were prepared from a common precursor via click chemistry. The tail could be attached to the ligand prior to coordination to the ruthenium complex were screened for cytotoxicity in tumourigenic and non-tumourigenic cell lines, and while the compounds were only moderately cytotoxic, good selectivity for tumourigenic cells were abserved.

Increased Pro-inflammatory Cytokines (TNF-a and IL-6) and Anti-inflammatory Compounds (sTNFRp55 and sTNFRp75) in Brazilian Patients during Exanthematic Dengue Fever

Pro-inflammatory cytokines, tumor necrosis factor (TNF-a), interleukin-6 (IL-6) and interleukin-1b (IL-1b) as well as anti-inflammatory compounds, soluble TNF-Receptor p55 (sTNFRp55), sTNFRp75 and IL-1 receptor antagonist (sIL-1Ra), were investigated in 34 Brazilian cases of dengue fever (DF) originated from a study of exanthematic virosis. The presence of pro-inflammatory cytokines was detected in sera from these patients by ELISA. TNF-a and IL-6 levels were significantly higher than control subjects in 32% and 52% patients, respectively. To our knowledge this was the first time a receptor antagonist and soluble receptors for cytokines were detected in sera obtained during exanthematic DF without hemorrhagic manifestations. Both sTNFRp55 and sTNFRp75 were consistently elevated in 42% and 84% patients, respectively. Most patients had IL-1b levels not different from those of normal subjects, except for one case. Only 16% patients had altered levels of IL-1Ra. Previous studies in dengue hemorrhagic fever patients demonstrated production of these soluble factors; here we observed that they are found in absence of hemorrhagic manifestations. The possible role of these anti-inflammatory compounds in immune cell activation and in regulating cytokine-mediated pathogenesis during dengue infection is discussed.

Transcriptional control of the hydrogen cyanide biosynthetic genes hcnABC by the anaerobic regulator ANR and the quorum-sensing regulators LasR and RhlR in Pseudomonas aeruginosa.

Virulence factors of Pseudomonas aeruginosa include hydrogen cyanide (HCN). This secondary metabolite is maximally produced at low oxygen tension and high cell densities during the transition from exponential to stationary growth phase. The hcnABC genes encoding HCN synthase were identified on a genomic fragment complementing an HCN-deficient mutant of P. aeruginosa PAO1. The hcnA promoter was found to be controlled by the FNR-like anaerobic regulator ANR and by the quorum-sensing regulators LasR and RhlR. Primer extension analysis revealed two transcription starts, T1 and T2, separated by 29 bp. Their function was confirmed by transcriptional lacZ fusions. The promoter sequence displayed an FNR/ANR box at -42.5 bp upstream of T2 and a lux box centered around -42.5 bp upstream of T1. Expression of the hcn genes was completely abolished when this lux box was deleted or inactivated by two point mutations in conserved nucleotides. The lux box was recognized by both LasR [activated by N-(oxododecanoyl)-homoserine lactone] and RhlR (activated by N-butanoyl-homoserine lactone), as shown by expression experiments performed in quorum-sensing-defective P. aeruginosa mutants and in the N-acyl-homoserine lactone-negative heterologous host P. fluorescens CHA0. A second, less conserved lux box lying 160 bp upstream of T1 seems to account for enhanced quorum-sensing-dependent expression. Without LasR and RhlR, ANR could not activate the hcn promoter. Together, these data indicate that expression of the hcn promoter from T1 can occur under quorum-sensing control alone. Enhanced expression from T2 appears to rely on a synergistic action between LasR, RhlR, and ANR.

Development of a novel headspace sorptive extraction method to study the aging of volatile compounds in spent handgun cartridges

Estimating the time since the last discharge of firearms and/or spent cartridges may be a useful piece of information in forensic firearm-related cases. The current approach consists of studying the diffusion of selected volatile organic compounds (such as naphthalene) released during the shooting using solid phase micro-extraction (SPME). However, this technique works poorly on handgun car-tridges because the extracted quantities quickly fall below the limit of detection. In order to find more effective solutions and further investigate the aging of organic gunshot residue after the discharge of handgun cartridges, an extensive study was carried out in this work using a novel approach based on high capacity headspace sorptive extraction (HSSE). By adopting this technique, for the first time 51 gunshot residue (GSR) volatile organic compounds could be simultaneously detected from fired handgun cartridge cases. Application to aged specimens showed that many of those compounds presented significant and complementary aging profiles. Compound-to-compound ratios were also tested and proved to be beneficial both in reducing the variability of the aging curves and in enlarging the time window useful in a forensic casework perspective. The obtained results were thus particularly promising for the development of a new complete forensic dating methodology.

Regulation of the Gac/Rsm pathway in "Pseudomonas fluorescens" CHA0

SUMMARY : Two-component systems are key mediators implicated in the response of numerous bacteria to a wide range of signals and stimuli. The two-component system comprised of the sensor kinase GacS and the response regulator GacA is broadly distributed among γ-proteobacteria bacteria and fulfils diverse functions such as regulation of carbon storage and expression of virulence. In Pseudomonas fluorescens, a soil bacterium which protects plants from root-pathogenic fungi and nematodes, the GacS/GacA two-component system has been shown to be essential for the production of secondary metabolites and exoenzymes required for the biocontrol activity of the bacterium. The regulatory cascade initiated by GacS/GacA consists of two translational repressor proteins, RsmA and RsmE, as well as three GacAcontrolled small regulatory RNAs RsmX, RsmY and RsmZ, which titrate RsmA and RsmE to allow the expression of biocontrol factors. Genetic analysis revealed that two additional sensor kinases termed RetS and Lads were involved as negative and positive control elements, respectively, in the Gac/Rsm pathway in P. fluoresens CHAO. Furthermore, it could be proposed that RetS and Lads interact with GacS, thereby modulating the expression of antibiotic compounds and hydrogen cyanide, as well as the rpoS gene encoding the stress and stationary phase sigma factor σ. Temperature was found to be an important environmental cue that influences the Gac/Rsm network. Indeed, the production of antibiotic compounds and hydrogen cyanide was reduced at 35°C, by comparison with the production at 30°C. RetS was identified to be involved in this temperature control. The small RNA RsmY was confirmed to be positively regulated by GacA and RsmA/RsmE. Two essential regions were identified in the rsmY promoter by mutational analysis, the upstream activating sequence (UAS) and the linker sequence. Although direct experimental evidence is still missing, several observations suggest that GacA may bind to the UAS, whereas the linker region would be recognized by intermediate RsmA/RsmEdependent repressors and/or activators. In conclusion, this work has revealed new elements contributing to the function of the signal transduction mechanisms in the Gac/Rsm pathway. RESUME : Les systèmes ä deux composants sont des mécanismes d'une importance notoire que beaucoup de bactéries utilisent pour faire face et répondre aux stimuli environnementaux. Le système à deux composants comprenant le senseur GacS et le régulateur de réponse GacA est très répandu chez les γ-protéobactéries et remplit des fonctions aussi diverses que la régulation du stockage de carbone ou l'expression de la virulence. Chez Pseudomonas fluorescens CHAO, une bactérie du sol qui protège les racines des plantes contre des attaques de champignons et nématodes pathogènes, le système à deux composants GacS/GacA est essentiel à la production de métabolites secondaires et d'exoenzymes requis pour l'activité de biocontrôle de la bactérie. La cascade régulatrice initiée pas GacS/GacA fait intervenir deux protéines répresseur de traduction, RsmA et RsmE, ainsi que trois petits ARNs RsmX, RsmY et RsmZ, dont la production est contrôlée par GacA. Ces petits ARNs ont pour rôle de contrecarrer l'action des protéines répressseur de la traduction, ce qui permet l'expression de facteurs de biocontrôle. Des analyses génétiques ont révélé la présence de deux senseurs supplémentaires, appelés Rets et Lads, qui interviennent dans la cascade Gac/Rsm de P. fluorescens. L'impact de ces senseurs est, respectivement, négatif et positif. Ces interactions ont apparenunent lieu au niveau de GacS et permettent une modulation de l'expression des antibiotiques et de l'acide cyanhydrique, ainsi que du gène rpoS codant pour le facteur sigma du stress. La température s'est révélée être un facteur environnemental important qui influence la cascade Gac/Rsm. Il s'avère en effet que la production d'antibiotiques ainsi que d'acide cyanhydrique est moins importante à 35°C qu'à 30°C. L'implication du senseur Rets dans ce contrôle par la température a pu être démontrée. La régulation positive du petit ARN RsmY par GacA et RsmA/RsmE a pu être confirmée; par le biais d'une analyse mutationelle, deux régions essentielles ont pu être mises en évidence dans la région promotrice de rsmY. Malgré le manque de preuves expérimentales directes, certains indices suggèrent que GacA puisse directement se fixer sur une des deux régions (appelée UAS), tandis que la deuxième région (appelée linker) serait plutôt reconnue par des facteurs intermédiaires (activateurs ou répresseurs) dépendant de RsmA/RsmE. En conclusion, ce travail a dévoilé de nouveaux éléments permettant d'éclairer les mécanismes de transduction des signaux dans la cascade Gac/Rsm.

Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake

The evaluation of new antimalarial agents using older methods of monitoring sensitivity to antimalarial drugs are laborious and poorly suited to discriminate stage-specific activity. We used flow cytometry to study the effect of established antimalarial compounds, cysteine protease inhibitors, and a quinolone against asexual stages of Plasmodium falciparum. Cultured P. falciparum parasites were treated for 48 h with different drug concentrations and the parasitemia was determined by flow cytometry methods after DNA staining with propidium iodide. P. falciparum erythrocytic life cycle stages were readily distinguished by flow cytometry. Activities of established and new antimalarial compounds measured by flow cytometry were equivalent to results obtained with microscopy and metabolite uptake assays. The antimalarial activity of all compounds was higher against P. falciparum trophozoite stages. Advantages of flow cytometry analysis over traditional assays included higher throughput for data collection, insight into the stage-specificity of antimalarial activity avoiding use of radioactive isotopes.

Molluscicidal effect of nicotinanilide and its intermediate compounds against a freshwater snail Lymnaea luteola, the vector of animal schistosomiasis

The molluscicidal effect of nicotinanilide was evaluated and compared with niclosamide (2',5-dichloro-4'-nitrosalicylanilide, ethanolamide salt) against different stages of the freshwater snail Lymnaea luteola i.e., eggs, immature, young mature, and adults. Calculated values of lethal concentrations (LC50 and LC90 ) showed that both nicotinanilide and niclosamide as toxic against eggs, immature, and adults. The young mature stage of the snails was comparatively more tolerant to both molluscicides than the other stages. The toxicity of the intermediate compounds of nicotinanilide against the young mature stage of the snails showed them as ineffective. The mortality pattern of the snails exposed to LC90 concentration of these molluscicides showed niclosamide to kill faster (within 8 to 9 h) than nicotinanilide (26 to 28 h). In view of the above studies it may be concluded that both molluscicides are toxic against all the stages of the L. luteola snails.

Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHA0.

The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis in P. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2. The hcnA promoter was mapped by primer extension; the -40 sequence (TTGGC ... ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT ... ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion. Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.

Brazilian flora extracts as source of novel antileishmanial and antifungal compounds

Natural products have long been providing important drug leads for infectious diseases. Leishmaniasis is a protozoan parasitic disease found mainly in developing countries, and it has toxic therapies with few alternatives. Fungal infections have been the main cause of death in immunocompromised patients and new drugs are urgently needed. In this work, a total of 16 plant species belonging to 11 families, selected on an ethnopharmacological basis, were analyzed in vitro against Leishmania (L.) chagasi, Leishmania (L.) amazonensis, Candida krusei, and C. parapsilosis. Of these plant species, seven showed antifungal activity against C. krusei, five showed antileishmanial activity against L. chagasi and four against L. amazonensis, among them species of genus Plectranthus. Our findings confirm the traditional therapeutic use of these plants in the treatment of infectious and inflammatory disorders and also offer insights into the isolation of active and novel drug prototypes, especially those used against neglected diseases as Leishmaniasis.