Clinical outcomes after tricuspid surgery : The role of previous cardiac surgery.

BACKGROUND: The aim of our study was to assess the outcomes of surgical treatment for severe tricuspid regurgitation according to whether cardiac surgery had been performed before the tricuspid valve intervention. METHODS: Between 1996 and 2013, 201 consecutive patients with severe tricuspid regurgitation underwent tricuspid surgery at our center. Patients were classified according to whether or not they had undergone previous cardiac surgery, which 33% of the sample had. Perioperative as well as long-term morbidity and mortality were analyzed. RESULTS: Mean patient age was 62.3 years. 32.8% underwent suture annuloplasty, 41.3% underwent ring annuloplasty, 15.4% received a bioprosthesis, and 10.4% received a mechanical prosthesis. There were no significant differences in perioperative mortality between the group that had not undergone previous cardiac surgery and the group that had (12.7% vs. 17.9%, respectively; p = 0.32). The long-term mortality rate (median follow-up time: 53 months) was 43.3%. Long-term survival curves showed no significant differences between the two groups (p = 0.884), and previous cardiac surgery was not a predictive factor for long-term mortality (hazard ratio = 1.211; p = 0.521). CONCLUSION: In a series of patients who underwent tricuspid valve surgery, no significant differences were observed in perioperative mortality or in long-term survival according to whether or not subjects had undergone previous cardiac surgery.

Relationship between pathogenic, clinical, and virulence factors of Staphylococcus aureus in infective endocarditis versus uncomplicated bacteremia: a case-control study.

Pathogenic factors of Staphylococcus aureus (SA) in the development of infective endocarditis (IE) have not been sufficiently investigated. The purpose of this study was to analyze the pathogenesis and virulence factors of SA in patients with IE as compared to patients with uncomplicated bacteremia (un-BAC). This is a retrospective case-control study (2002-2014) performed at a tertiary hospital in Spain. Clinical and epidemiological factors were analyzed. We assessed the presence of toxin genes [toxic shock syndrome toxin 1 (tst-1) and enterotoxins A (etA), B (etB), and D (etD)] and the potential relationship between accessory gene regulator (agr) groups and the development of IE confirmed by polymerase chain reaction (PCR). Twenty-nine patients with IE were compared with 58 patients with uncomplicated S. aureus bacteremia (SAB). As many as 75.9 % of patients had community-acquired IE (p < 0.005). Multivariate analysis revealed that there is a significant relationship between community-acquired infection and severe sepsis or septic shock and IE. Also, a minimum inhibitory concentration (MIC) of vancomycin ≥1.5 µg/ml was found to be associated with IE. The agr group I was prevalent (55.2 % vs. 31.0 %; p = 0.030). No association was observed between toxin genes (tst-1, etA, etB, and etD) and IE. The superantigen (SAg) most frequently found in SA isolates was tst-1 (12.6 %). We found no association between toxin genes and IE, probably due to the small sample size. However, a direct relationship was found between agr I and the development of IE, which suggests that agr I strains may have more potential to cause IE.

Involvement of cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improved tolerance against drought stress.

In order to understand the role of cytosolic antioxidant enzymes in drought stress protection, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants overexpressing cytosolic Cu/Zn-superoxide dismutase (cytsod) (EC 1.15.1.1) or ascorbate peroxidase (cytapx) (EC 1.11.1.1) alone, or in combination, were produced and tested for tolerance against mild water stress. The results showed that the simultaneous overexpression of Cu/Znsod and apx or at least apx in the cytosol of transgenic tobacco plants alleviates, to some extent, the damage produced by water stress conditions. This was correlated with higher water use efficiency and better photosynthetic rates. In general, oxidative stress parameters, such as lipid peroxidation, electrolyte leakage, and H(2)O(2) levels, were higher in non-transformed plants than in transgenic lines, suggesting that, at the least, overexpression of cytapx protects tobacco membranes from water stress. In these conditions, the activity of other antioxidant enzymes was induced in transgenic lines at the subcellular level. Moreover, an increase in the activity of some antioxidant enzymes was also observed in the chloroplast of transgenic plants overexpressing cytsod and/or cytapx. These results suggest the positive influence of cytosolic antioxidant metabolism on the chloroplast and underline the complexity of the regulation network of plant antioxidant defences during drought stress.

Interaction between hydrogen peroxide and plant hormones during germination and the early growth of pea seedlings.

Hydrogen peroxide (H(2)O(2)) increased the germination percentage of pea seeds, as well as the growth of seedlings in a concentration-dependent manner. The effect of H(2)O(2) on seedling growth was removed by incubation with 10 microm ABA. The H(2)O(2)-pretreatment produced an increase in ascorbate peroxidase (APX), peroxidase (POX) and ascorbate oxidase (AAO). The increases in these ascorbate-oxidizing enzymes correlated with the increase in the growth of the pea seedlings as well as with the decrease in the redox state of ascorbate. Moreover, the increase in APX activity was due to increases in the transcript levels of cytosolic and stromal APX (cytAPX, stAPX). The proteomic analysis showed that H(2)O(2) induced proteins related to plant signalling and development, cell elongation and division, and cell cycle control. A strong correlation between the effect of H(2)O(2) on plant growth and the decreases in ABA and zeatin riboside (ZR) was observed. The results suggest an interaction among the redox state and plant hormones, orchestrated by H(2)O(2), in the induction of proteins related to plant signalling and development during the early growth of pea seedlings.

Two-stage fermentation process for alginate production by Azotobacter vinelandii mutant altered in poly-beta-hydroxybutyrate (PHB) synthesis.

AIMS: A two-stage fermentation strategy, based on batch cultures conducted first under non-oxygen-limited conditions, and later under oxygen-limited conditions, was used to improve alginate production by Azotobacter vinelandii (AT6), a strain impaired in poly-beta-hydroxybutyrate (PHB) production. METHODS AND RESULTS: The use of sucrose as carbon source, as well as a high oxygen concentration (10%), allowed to obtain a maximum biomass concentration of 7.5 g l(-1) in the first stage of cultivation. In the second stage, the cultures were limited by oxygen (oxygen close to 0%) and fed with a sucrose solution at high concentration. Under those conditions, the growth rate decreased considerably and the cells used the carbon source mainly for alginate biosynthesis, obtaining a maximum concentration of 9.5 g l(-1), after 50 h of cultivation. CONCLUSION: Alginate concentration obtained from the AT6 strain was two times higher than that obtained using the wild-type strain (ATCC 9046) and was the highest reported in the literature. However, the mean molecular mass of the alginate produced in the second stage of the process by the mutant AT6 was lower (400 kDa) than the polymer molecular mass obtained from the cultures developed with the parental strain (950 kDa). SIGNIFICANCE AND IMPACT OF THE STUDY: The use of a mutant of A. vinelandii impaired in the PHB production in combination with a two-stage fermentation process could be a feasible strategy for the production of alginate at industrial level.

RpoS controls the expression and the transport of the AlgE1-7 epimerases in Azotobacter vinelandii.

Azotobacter vinelandii produces differentiated cells, called cysts, surrounded by two alginate layers, which are necessary for their desiccation resistance. This alginate contains variable proportions of guluronate residues, resulting from the activity of seven extracytoplasmic epimerases, AlgE1-7. These enzymes are exported by a system secretion encoded by the eexDEF operon; mutants lacking the AlgE1-7 epimerases, the EexDEF or the RpoS sigma factor produce alginate, but are unable to form desiccation resistant cysts. Herein, we found that RpoS was required for full transcription of the algE1-7 and eexDEF genes. We found that the AlgE1-7 protein levels were diminished in the rpoS mutant strain. In addition, the alginate produced in the absence of RpoS was more viscous in the presence of proteases, a phenotype similar to that of the eexD mutant. Primer extension analysis located two promoters for the eexDEF operon, one of them was RpoS-dependent. Thus, during encysting conditions, RpoS coordinates the expression of both the AlgE1-7 epimerases and the EexDEF protein complex responsible for their transport.

A pragmatic approach for mortality prediction after surgery in infective endocarditis: optimizing and refining EuroSCORE.

OBJECTIVE: To simplify and optimize the ability of EuroSCORE I and II to predict early mortality after surgery for infective endocarditis (IE). METHODS: Multicentre retrospective study (n = 775). Simplified scores, eliminating irrelevant variables, and new specific scores, adding specific IE variables, were created. The performance of the original, recalibrated and specific EuroSCOREs was assessed by Brier score, C-statistic and calibration plot in bootstrap samples. The Net Reclassification Index was quantified. RESULTS: Recalibrated scores including age, previous cardiac surgery, critical preoperative state, New York Heart Association >I, and emergent surgery (EuroSCORE I and II); renal failure and pulmonary hypertension (EuroSCORE I); and urgent surgery (EuroSCORE II) performed better than the original EuroSCOREs (Brier original and recalibrated: EuroSCORE I: 0.1770 and 0.1667; EuroSCORE II: 0.2307 and 0.1680). Performance improved with the addition of fistula, staphylococci and mitral location (EuroSCORE I and II) (Brier specific: EuroSCORE I 0.1587, EuroSCORE II 0.1592). Discrimination improved in specific models (C-statistic original, recalibrated and specific: EuroSCORE I: 0.7340, 0.7471 and 0.7728; EuroSCORE II: 0.7442, 0.7423 and 0.7700). Calibration improved in both EuroSCORE I models (intercept 0.295, slope 0.829 (original); intercept -0.094, slope 0.888 (recalibrated); intercept -0.059, slope 0.925 (specific)) but only in specific EuroSCORE II model (intercept 2.554, slope 1.114 (original); intercept -0.260, slope 0.703 (recalibrated); intercept -0.053, slope 0.930 (specific)). Net Reclassification Index was 5.1% and 20.3% for the specific EuroSCORE I and II. CONCLUSIONS: The use of simplified EuroSCORE I and EuroSCORE II models in IE with the addition of specific variables may lead to simpler and more accurate models.

Beta-ketothiolase genes in Azotobacter vinelandii.

Azotobacter vinelandii is proposed to contain a single beta-ketothiolase activity participating in the formation of acetoacetyl-CoA, a precursor for poly-beta-hydroxybutyrate (PHB) synthesis, and in beta-oxidation (Manchak, J., Page, W.J., 1994. Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD. Microbiology 140, 953-963). We designed a degenerate oligonucleotide from a highly conserved region among bacterial beta-ketothiolases and used it to identify bktA, a gene with a deduced protein product with a high similarity to beta-ketothiolases. Immediately downstream of bktA, we identified a gene called hbdH, which encodes a protein exhibiting similarity to beta-hydroxyacyl-CoA and beta-hydroxybutyryl-CoA dehydrogenases. Two regions with homology to bktA were also observed. One of these was cloned and allowed the identification of the phbA gene, encoding a second beta-ketothiolase. Strains EV132, EV133, and GM1 carrying bktA, hbdH and phbA mutations, respectively, as well as strain EG1 carrying both bktA and phbA mutations, were constructed. The hbdH mutation had no effect on beta-hydroxybutyryl-CoA dehydrogenase activity or on fatty acid assimilation. The bktA mutation had no effect on beta-ketothiolase activity, PHB synthesis or fatty acid assimilation, whereas the phbA mutation significantly reduced beta-ketothiolase activity and PHB accumulation, showing that this is the beta-ketothiolase involved in PHB biosynthesis. Strain EG1 was found to grow under beta-oxidation conditions and to possess beta-ketothiolase activity. Taken together, these results demonstrate the presence of three genes coding for beta-ketothiolases in A. vinelandii.

Transcriptional organization of the Azotobacter vinelandii algGXLVIFA genes: characterization of algF mutants.

Azotobacter vinelandii forms desiccation-resistant cysts which contain a high proportion of the exopolysaccharide alginate in their envelope. We have previously shown that the A. vinelandii alginate biosynthetic genes algA and algL are transcribed from a promoter located somewhere upstream of algL. In this study we sequenced the A. vinelandii algX, algL, algV, algI and algF genes located between algG and algA. We carried out primer extension analysis of the algG, algX and algL genes and detected transcription start sites upstream algG but not upstream algX or algL, implying that algG and algX form part of the previously identified algL-A operon. A promoter upstream algA was also detected; however, transcription of algA exclusively from this promoter is not sufficient for the AlgA levels required for alginate production. An algF mutant (AJ34) was constructed by insertion of the Omega-tetracycline cassette in the non-polar orientation. As expected, AJ34 produced unacetylated alginate. Viability of 35day old cysts formed by strain AJ34, but not of those formed by the wild type, was reduced, indicating that acetylation of alginate plays a role in cyst resistance to desiccation.

The Rhizobium leguminosarum biovar phaseoli glnT gene, encoding glutamine synthetase III.

Plasmid pGE203 contains the Rhizobium leguminosarum biovar phaseoli glnT locus. Glutamine synthetase III (GSIII) was purified from a glutamine auxotrophic strain of Klebsiella pneumoniae carrying this plasmid. Sequencing of a 2.4-kb fragment containing the glnT locus reveals an open reading frame of 435 amino acids (aa), whose first eight aa are identical to those determined from pure GSIII by direct aa sequencing, thus confirming that glnT indeed codes for GSIII activity. The comparison of the GSIII aa sequence with the reported sequence of GSs from other organisms shows a significant degree of homology. Since the three-dimensional structure of GS from Salmonella typhimurium is known, a three-dimensional model of GSIII was built by homology.

The Azotobacter vinelandii alg8 and alg44 genes are essential for alginate synthesis and can be transcribed from an algD-independent promoter.

A 2.8-kb DNA region, located immediately downstream of algD, contains the A. vinelandii alg8 and alg44 genes, whose sequences are highly homologous to those of the corresponding Pseudomonas aeruginosa genes. These genes occur on a transcript that does not include algD, and are transcribed from a promoter different from that transcribing algD; this is the fourth promoter described within the alginate biosynthetic gene cluster. alg8 and alg44 mutants were constructed and shown to be completely impaired in alginate production. Alg8 shares 28.20% identity and 38.09% similarity to Azorhizobium caulinodans NodC, a glycosyl transferase catalyzing the formation of beta-1,4 linkages. A topological model is predicted, which supports the idea of Alg8 being the polymerase responsible for alginate synthesis.

Phenotype of a Rhizobium leguminosarum ntrC mutant.

A Tn5 insertion mutant, strain CFN2012, of Rhizobium leguminosarum biovar phaseoli devoid of glutamine synthetase II (GSII) activity was analysed. It was shown to contain Tn5 within an 11-kb BamHI DNA fragment, which was isolated (pSM261) from the wild-type strain and, when introduced into strain CFN2012, was shown to complement the absence of GSII activity. The DNA sequence of the corresponding region from the wild-type allele revealed the presence of an ntrC regulatory gene, and restriction analysis indicated that the mutant allele carried the Tn5 insertion within it. Further analysis of strain CFN2012 indicated that this mutant has reduced levels of the PII regulatory protein and that, in contrast to ntrC mutants of other Rhizobiaceae, it grows on nitrate as the sole nitrogen source.

Isolation and characterization of an Azotobacter vinelandii algK mutant.

Random Tn5 mutagenesis over Azotobacter vinelandii mucoid strain ATCC 9046 produced strain LA21, a non-mucoid, non-encysting mutant, carrying the Tn5 insertion within a gene homologous to algK from Pseudomonas aeruginosa encoding a periplasmic protein. algK, algJ and algG were shown to be transcribed as part of the palg8-alg44-algK-algJ-algG operon. A non-polar algK mutant was constructed and showed a non-mucoid phenotype, indicating that algK is essential for alginate production.

Salicylic acid negatively affects the response to salt stress in pea plants.

We studied the effect of salicylic acid (SA) treatment on the response of pea plants to salinity. Sodium chloride (NaCl)-induced damage to leaves was increased by SA, which was correlated with a reduction in plant growth. The content of reduced ascorbate and glutathione in leaves of salt-treated plants increased in response to SA, although accumulation of the respective oxidised forms occurred. An increase in hydrogen peroxide also occurred in leaves of salt-exposed plants treated with SA. In the absence of NaCl, SA increased ascorbate peroxidase (APX; 100 µm) and glutathione-S transferase (GST; 50 µm) activities and increased catalase (CAT) activity in a concentration-dependent manner. Salinity decreased glutathione reductase (GR) activity, but increased GST and CAT activity. In salt-stressed plants, SA also produced changes in antioxidative enzymes: 100 µm SA decreased APX but increased GST. Finally, a concentration-dependent increase in superoxide dismutase (SOD) activity was induced by SA treatment in salt-stressed plants. Induction of PR-1b was observed in NaCl-stressed plants treated with SA. The treatment with SA, as well as the interaction between salinity and SA treatment, had a significant effect on PsMAPK3 expression. The expression of PsMAPK3 was not altered by 70 mm NaCl, but was statistically higher in the absence than in the presence of SA. Overall, the results show that SA treatment negatively affected the response of pea plants to NaCl, and this response correlated with an imbalance in antioxidant metabolism. The data also show that SA treatment could enhance the resistance of salt-stressed plants to possible opportunistic pathogen attack, as suggested by increased PR-1b gene expression.

Benzothiadiazole and l-2-oxothiazolidine-4-carboxylic acid reduce the severity of Sharka symptoms in pea leaves: effect on antioxidative metabolism at the subcellular level.

The effect of treatment with benzothiadiazole (BTH) or l-2-oxothiazolidine-4-carboxylic acid (OTC), and their interaction with Plum pox virus (PPV) infection, on antioxidative metabolism of pea plants was studied at the subcellular level. PPV infection produced a 20% reduction in plant growth. Pre-treatment of pea plants with OTC or BTH afforded partial protection against PPV infection, measured as the percentage of leaves showing symptoms, but neither BTH nor OTC significantly reduced the virus content. PPV infection caused oxidative stress, as monitored by increases in lipid peroxidation and protein oxidation in soluble and chloroplastic fractions. In leaves of non-infected plants, OTC increased the content of reduced glutathione (GSH) and total glutathione; accordingly, an increase in the redox state of glutathione was observed. An increase in oxidized glutathione (GSSG) was found in symptomatic leaves from infected plants. A similar increase in GSSG was also observed in asymptomatic leaves from infected, untreated plants. However, no changes in GSSG occurred in asymptomatic leaves from infected plants treated with BTH and OTC and, accordingly, a higher redox state of GSH was recorded in those leaves, which could have had a role in the reduction of symptoms, as observed in asymptomatic leaves from infected plants treated with BTH or OTC. Treatment with BTH or OTC had some effect on antioxidant enzymes in soluble and chloroplastic fractions from infected pea leaves. An increase in antioxidative mechanisms, such as GSH-related enzymes (DHAR, GR and G6PDH), as well as APX and POX, at the subcellular level was observed, which could play a role in reducing the severity of cellular damage induced by Sharka in pea leaves.

Inactivation of pycA, encoding pyruvate carboxylase activity, increases poly-beta-hydroxybutyrate accumulation in Azotobacter vinelandii on solid medium.

Strain AJ1678, an Azotobacter vinelandii mutant overproducing the storage polymer poly-beta-hydroxybutyrate (PHB) in solid but not liquid complex medium with sucrose, was isolated after mini-Tn5 mutagenesis of strain UW136. Cloning and nucleotide sequencing of the affected locus led to identification of pycA, encoding a protein with high identity to the biotin carboxylase subunit of pyruvate carboxylase enzyme (PYC). A gene ( pycB) whose product is similar to the biotin-carrying subunit of PYC is present immediately downstream from pycA. An assay of pyruvate carboxylase activity and an avidin-blot analysis confirmed that pycA and pycB encode the two subunits of this enzyme. In many organisms, PYC catalyzes ATP-dependent carboxylation of pyruvate to generate oxaloacetate and is responsible for replenishing oxaloacetate for continued operation of the tricarboxylic acid cycle. We propose that the pycA mutation causes a slow-down in the TCA cycle activity due to a low oxaloacetate concentration, resulting in a higher availability of acetyl-CoA for the synthesis of poly-beta-hydroxybutyrate.

Mutational inactivation of a gene homologous to Escherichia coli ptsP affects poly-beta-hydroxybutyrate accumulation and nitrogen fixation in Azotobacter vinelandii.

Strain DS988, an Azotobacter vinelandii mutant with a reduced capacity to accumulate poly-beta-hydroxybutyrate, was isolated after mini-Tn5 mutagenesis of the UW136 strain. Cloning and nucleotide sequencing of the affected locus revealed a gene homologous to Escherichia coli ptsP which encodes enzyme INtr, a homologue of enzyme I of the phosphoenol pyruvate-sugar phosphotransferase system with an N-terminal domain similar to the N-terminal domain of some NifA proteins. Strain DS988 was unable to grow diazotrophically with 10 mM glucose as a carbon source. Diazotrophic growth on alternative carbon sources such as gluconate was only slightly affected. Glucose uptake, as well as glucose kinase and glucose-6-phosphate-dehydrogenase activities that lead to the synthesis of gluconate-6-phosphate, were not affected by the ptsP mutation. The inability of DS988 to grow diazotrophically in 10 mM glucose was overcome by supplying ammonium or other sources of fixed nitrogen. Acetylene reduction activity but not transcription of the nitrogenase structural gene nifH was shown to be impaired in strain DS988 when it was incubated in 10 mM glucose. The diazotrophic growth defect of DS988 was restored either by increasing the glucose concentration to above 20 mM or by lowering the oxygen concentration. These data suggest that a mutation in ptsP leads to a failure in poly-beta-hydroxybutyrate metabolism and in the respiratory protection of nitrogenase under carbon-limiting conditions.

Molecular genetics of the glutamine synthetases in Rhizobium species.

Soil bacteria of the genus Rhizobium and Bradyrhizobium establish symbiotic interactions with leguminous plants that result in the formation of specialized structures, the nodules, in which the bacteria differentiate into bacteroids and fix nitrogen. Rhizobial glutamine synthetase (GS) activity is very low in the nodule. The ammonia produced by the bacteroids is exported to the plant cell, where it is assimilated by the GS from the plant, whereas in the free-living state, Rhizobium and Bradyrhizobium species assimilate ammonia for growth. Another characteristic of these species is that they possess two glutamine synthetase isozymes, known as GSI and GSII. A third glutamine synthetase isozyme, called GSIII, has been found in R. meliloti and R. etli.

Glutamine metabolism in nitrogen-starved conidia of Neurospora crassa.

During nitrogen deprivation, de novo synthesis of glutamine synthetase was induced in non-growing conidia of Neurospora crassa. When ammonia or glutamine was added to conidia which had been deprived of nitrogen, glutamine and arginine accumulated at a higher rate than in condia not deprived of nitrogen. The degradation of exogenous glutamine to glutamate is apparently a necessary step in the accumulation of glutamine and arginine within the conidia. In non-growing conidia, a cycle probably operates in which glutamine is degraded and resynthesized. The advantages of such a cycle would be that the carbon and nitrogen could be used to synthesize amino acids in general, as well as for the synthesis and accumulation of arginine and/or glutamine in particular.

Azotobacter vinelandii mutants that overproduce poly-beta-hydroxybutyrate or alginate.

Azotobacter vinelandii produces two polymers of industrial importance, i.e. alginate and poly-beta-hydroxybutyrate (PHB). Alginate synthesis constitutes a waste of substrate when seeking to optimize PHB production and, conversely, synthesis of PHB is undesirable when optimizing alginate production. In this study we evaluated the effect of a mutation in algA, the gene encoding the enzyme that catalyzes the first step of the alginate biosynthetic pathway in the production of PHB. We also evaluated production of alginate in strain AT6 carrying a phbB mutation that impairs PHB synthesis. The algA mutation prevented alginate production and increased PHB accumulation up to 5-fold, determined in milligrams per milligram of protein. Similarly, the phbB mutation increased alginate production up to 4-fold.