Transcription profiles of hydrogenases related genes in the cyanobacterium Lyngbya majuscula CCAP 1446/4

<p>Abstract</p> <p>Background</p> <p><it>Lyngbya majuscula </it>CCAP 1446/4 is a N₂-fixing filamentous nonheterocystous strain that contains two NiFe-hydrogenases: an uptake (encoded by <it>hupSL</it>) and a bidirectional enzyme (encoded by <it>hoxEFUYH</it>). The biosynthesis/maturation of NiFe-hydrogenases is a complex process requiring several accessory proteins for e.g. for the incorporation of metals and ligands in the active center (large subunit), and the insertion of the FeS clusters (small subunit). The last step in the maturation of the large subunit is the cleavage of a C-terminal peptide from its precursor by a specific endopeptidase. Subsequently, the mature large and small subunits can assemble forming a functional enzyme.</p> <p>Results</p> <p>In this work we demonstrated that, in <it>L. majuscula</it>, the structural genes encoding the bidirectional hydrogenase are cotranscribed, and that <it>hoxW </it>(the gene encoding its putative specific endopeptidase) is in the same chromosomal region but transcribed from a different promoter. The gene encoding the putative specific uptake hydrogenase endopeptidase, <it>hupW</it>, can be cotranscribed with the structural genes but it has its own promoter. <it>hoxH</it>, <it>hupL</it>, <it>hoxW </it>and <it>hupW </it>transcription was followed in <it>L. majuscula </it>cells grown under N₂-fixing and non-N₂-fixing conditions over a 12 h light/12 h dark cycle. The transcription of <it>hoxH</it>, <it>hoxW </it>and <it>hupW </it>did not vary remarkably in the conditions tested, while the <it>hupL </it>transcript levels are significantly higher under N₂-fixing conditions with a peak occurring in the transition between the light and the dark phase. Furthermore, the putative endopeptidases transcript levels, in particular <it>hoxW</it>, are lower than those of the respective hydrogenase structural genes.</p> <p>Conclusion</p> <p>The data presented here indicate that in <it>L. majuscula </it>the genes encoding the putative hydrogenases specific endopeptidases, <it>hoxW </it>and <it>hupW</it>, are transcribed from their own promoters. Their transcript levels do not vary notably in the conditions tested, suggesting that HoxW and HupW are probably constantly present and available in the cells. These results, together with the fact that the putative endopeptidases transcript levels, in particular for <it>hoxW</it>, are lower than those of the structural genes, imply that the activity of the hydrogenases is mainly correlated to the transcription levels of the structural genes. The analysis of the promoter regions indicates that <it>hupL </it>and <it>hupW </it>might be under the control of different transcription factor(s), while both <it>hoxH </it>and <it>xisH </it>(<it>hoxW</it>) promoters could be under the control of LexA.</p

Análise da percepção ergonómica de postos de trabalho dotados de microscópio

A adequabilidade do dimensionamento dos postos e equipamentos de trabalho aos trabalhadores e às tarefas desenvolvidas é fundamental para a promoção de condições de conforto e prevenção do desenvolvimento de LMERT. A percepção dos trabalhadores é importante para a análise e intervenção ergonómica. Este estudo teve como objectivo analisar a percepção dos trabalhadores sobre a adequabilidade dos postos de trabalho que envolvam tarefas ao microscópio. Foi analisada a adequabilidade das dimensões dos elementos do mobiliário e do microscópio, e desenvolvido e aplicado o Questionário de Análise Ergonómica de Tarefas ao Microscópio (QAETM). O questionário contemplou questões associadas a características pessoais, actividades desenvolvidas, percepções de risco e sensações de dor e/ou desconforto. A análise das dimensões dos postos de trabalho mostrou que estas se apresentam em geral inadequadas às tarefas realizadas. Verificou-se ainda que alguns postos de trabalho tinham sofrido intervenções por parte dos profissionais. A maioria dos inquiridos considera as dimensões das cadeiras adequadas, contrariamente à das bancadas. No que respeita ao microscópio, foram sugeridas alterações ao nível dos parafusos de focagem, movimentação de platina e altura e inclinação das oculares. Este estudo permitiu verificar que alguns dos profissionais apresentam uma baixa percepção ergonómica, não sendo na sua maioria capazes de identificar a falta de adequabilidade das dimensões dos seus postos de trabalho. Apesar do referido, alguns revelaram preocupações ergonómicas, sugerindo e identificando, nomeadamente ao nível do microscópio, situações a alterar, tendo mesmo a iniciativa de intervir no seu posto de trabalho de modo a promover uma melhor postura.info:eu-repo/semantics/publishedVersio

Engineered biosynthesis of novel polyenes: a pimaricin derivative produced by targeted gene disruption in Streptomyces natalensis

AbstractBackground: The post-polyketide synthase biosynthetic tailoring of polyene macrolides usually involves oxidations catalysed by cytochrome P450 monooxygenases (P450s). Although members from this class of enzymes are common in macrolide biosynthetic gene clusters, their specificities vary considerably toward the substrates utilised and the positions of the hydroxyl functions introduced. In addition, some of them may yield epoxide groups. Therefore, the identification of novel macrolide monooxygenases with activities toward alternative substrates, particularly epoxidases, is a fundamental aspect of the growing field of combinatorial biosynthesis. The specific alteration of these activities should constitute a further source of novel analogues. We investigated this possibility by directed inactivation of one of the P450s belonging to the biosynthetic gene cluster of an archetype polyene, pimaricin.Results: A recombinant mutant of the pimaricin-producing actinomycete Streptomyces natalensis produced a novel pimaricin derivative, 4,5-deepoxypimaricin, as a major product. This biologically active product resulted from the phage-mediated targeted disruption of the gene pimD, which encodes the cytochrome P450 epoxidase that converts deepoxypimaricin into pimaricin. The 4,5-deepoxypimaricin has been identified by mass spectrometry and nuclear magnetic resonance following high-performance liquid chromatography purification.Conclusions: We have demonstrated that PimD is the epoxidase responsible for the conversion of 4,5-deepoxypimaricin to pimaricin in S. natalensis. The metabolite accumulated by the recombinant mutant, in which the epoxidase has been knocked out, constitutes the first designer polyene obtained by targeted manipulation of a polyene biosynthetic gene cluster. This novel epoxidase could prove to be valuable for the introduction of epoxy substituents into designer macrolides

Crosstalk between ROS Homeostasis and Secondary Metabolism in S. natalensis ATCC 27448: Modulation of Pimaricin Production by Intracellular ROS

Streptomyces secondary metabolism is strongly affected by oxygen availability. The increased culture aeration enhances pimaricin production in S. natalensis, however the excess of O2 consumption can lead to an intracellular ROS imbalance that is harmful to the cell. The adaptive physiological response of S. natalensis upon the addition of exogenous H2O2 suggested that the modulation of the intracellular ROS levels, through the activation of the H2O2 inducible catalase during the late exponential growth phase, can alter the production of pimaricin. With the construction of defective mutants on the H2O2 related enzymes SodF, AhpCD and KatA1, an effective and enduring modulation of intracellular ROS was achieved. Characterization of the knock-out strains revealed different behaviours regarding pimaricin production: whilst the superoxide dismutase defective mutant presented low levels of pimaricin production compared to the wild-type, the mutants defective on the H2O2-detoxifying enzymes displayed a pimaricin overproducer phenotype. Using physiological and molecular approaches we report a crosstalk between oxidative stress and secondary metabolism regulatory networks. Our results reveal that the redox-based regulation network triggered by an imbalance of the intracellular ROS homeostasis is also able to modulate the biosynthesis of pimaricin in S. natalensis

Mycobacterial OtsA Structures Unveil Substrate Preference Mechanism and Allosteric Regulation by 2-Oxoglutarate and 2-Phosphoglycerate.

Trehalose is an essential disaccharide for mycobacteria and a key constituent of several cell wall glycolipids with fundamental roles in pathogenesis. Mycobacteria possess two pathways for trehalose biosynthesis. However, only the OtsAB pathway was found to be essential in Mycobacterium tuberculosis, with marked growth and virulence defects of OtsA mutants and strict essentiality of OtsB2. Here, we report the first mycobacterial OtsA structures from Mycobacterium thermoresistibile in both apo and ligand-bound forms. Structural information reveals three key residues in the mechanism of substrate preference that were further confirmed by site-directed mutagenesis. Additionally, we identify 2-oxoglutarate and 2-phosphoglycerate as allosteric regulators of OtsA. The structural analysis in this work strongly contributed to define the mechanisms for feedback inhibition, show different conformational states of the enzyme, and map a new allosteric site.IMPORTANCE Mycobacterial infections are a significant source of mortality worldwide, causing millions of deaths annually. Trehalose is a multipurpose disaccharide that plays a fundamental structural role in these organisms as a component of mycolic acids, a molecular hallmark of the cell envelope of mycobacteria. Here, we describe the first mycobacterial OtsA structures. We show mechanisms of substrate preference and show that OtsA is regulated allosterically by 2-oxoglutarate and 2-phosphoglycerate at an interfacial site. These results identify a new allosteric site and provide insight on the regulation of trehalose synthesis through the OtsAB pathway in mycobacteria

Death ideation in cancer patients: contributing factors

Advances in cancer research and therapy have improved prognosis and the quality of life of many patients. However, previous epidemiological studies in oncologic patients have shown an increased risk of suicide. Suicidal thoughts, relatively well known in those terminally ill, may be just as important for cancer patients who are survivors or are living with the disease. Nonetheless, there is a relative paucity of data about suicidality in this setting. The authors conducted a prospective observational study to identify death thoughts and to explore the factors associated with suicidal ideation in cancer patients. A sample of 130 patients referred for psychiatric consultation was obtained following informed consent and authorization from the local ethics committee. A semistructured interview assessed sociodemographic data, psychosocial support, and information regarding the cancer process and its treatment. Psychometric instruments were used to evaluate psychopathology, namely the Hospital Anxiety and Depression Scale, the Beck Hopelessness Scale, and the Beck Scale for Suicide Ideation. Psychiatric diagnoses were obtained through the application of the Mini International Neuropsychiatric Interview. Death ideation was identified in 34.6% of patients, yet only 10% had active suicidal thoughts. Risk of suicide was associated with female gender, a psychiatric diagnosis (major depressive disorder, panic disorder, or dysthymia), difficult interpersonal relationships, associated pain, high hopelessness, and depressive and anxiety symptoms. Although suicidal thoughts are frequent in cancer patients at different stages of disease, most are transitory. Risk factors for suicidal ideation have been identified, such as depression, hopelessness, uncontrolled pain, and difficult interpersonal relationships. Further assessment is necessary to identify those at higher risk of attempting suicide, and underlying psychiatric disorders should be vigorously treated

Impact of safety-related dose reductions or discontinuations on sustained virologic response in HCV-infected patients: Results from the GUARD-C Cohort

BACKGROUND: Despite the introduction of direct-acting antiviral agents for chronic hepatitis C virus (HCV) infection, peginterferon alfa/ribavirin remains relevant in many resource-constrained settings. The non-randomized GUARD-C cohort investigated baseline predictors of safety-related dose reductions or discontinuations (sr-RD) and their impact on sustained virologic response (SVR) in patients receiving peginterferon alfa/ribavirin in routine practice. METHODS: A total of 3181 HCV-mono-infected treatment-naive patients were assigned to 24 or 48 weeks of peginterferon alfa/ribavirin by their physician. Patients were categorized by time-to-first sr-RD (Week 4/12). Detailed analyses of the impact of sr-RD on SVR24 (HCV RNA <50 IU/mL) were conducted in 951 Caucasian, noncirrhotic genotype (G)1 patients assigned to peginterferon alfa-2a/ribavirin for 48 weeks. The probability of SVR24 was identified by a baseline scoring system (range: 0-9 points) on which scores of 5 to 9 and <5 represent high and low probability of SVR24, respectively. RESULTS: SVR24 rates were 46.1% (754/1634), 77.1% (279/362), 68.0% (514/756), and 51.3% (203/396), respectively, in G1, 2, 3, and 4 patients. Overall, 16.9% and 21.8% patients experienced 651 sr-RD for peginterferon alfa and ribavirin, respectively. Among Caucasian noncirrhotic G1 patients: female sex, lower body mass index, pre-existing cardiovascular/pulmonary disease, and low hematological indices were prognostic factors of sr-RD; SVR24 was lower in patients with 651 vs. no sr-RD by Week 4 (37.9% vs. 54.4%; P = 0.0046) and Week 12 (41.7% vs. 55.3%; P = 0.0016); sr-RD by Week 4/12 significantly reduced SVR24 in patients with scores <5 but not 655. CONCLUSIONS: In conclusion, sr-RD to peginterferon alfa-2a/ribavirin significantly impacts on SVR24 rates in treatment-naive G1 noncirrhotic Caucasian patients. Baseline characteristics can help select patients with a high probability of SVR24 and a low probability of sr-RD with peginterferon alfa-2a/ribavirin

Clinical oxidative stress during leprosy multidrug therapy:impact of dapsone oxidation

This study aims to assess the oxidative stress in leprosy patients under multidrug therapy (MDT; dapsone, clofazimine and rifampicin), evaluating the nitric oxide (NO) concentration, catalase (CAT) and superoxide dismutase (SOD) activities, glutathione (GSH) levels, total antioxidant capacity, lipid peroxidation, and methemoglobin formation. For this, we analyzed 23 leprosy patients and 20 healthy individuals from the Amazon region, Brazil, aged between 20 and 45 years. Blood sampling enabled the evaluation of leprosy patients prior to starting multidrug therapy (called MDT 0) and until the third month of multidrug therapy (MDT 3). With regard to dapsone (DDS) plasma levels, we showed that there was no statistical difference in drug plasma levels between multibacillary (0.518±0.029 μg/mL) and paucibacillary (0.662±0.123 μg/mL) patients. The methemoglobin levels and numbers of Heinz bodies were significantly enhanced after the third MDTsupervised dose, but this treatment did not significantly change the lipid peroxidation and NO levels in these leprosy patients. In addition, CAT activity was significantly reduced in MDT-treated leprosy patients, while GSH content was increased in these patients. However, SOD and Trolox equivalent antioxidant capacity levels were similar in patients with and without treatment. These data suggest that MDT can reduce the activity of some antioxidant enzyme and influence ROS accumulation, which may induce hematological changes, such as methemoglobinemia in patients with leprosy. We also explored some redox mechanisms associated with DDS and its main oxidative metabolite DDS-NHOH and we explored the possible binding of DDS to the active site of CYP2C19 with the aid of molecular modeling software