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Lipases produced by solid-state fermentation were used directly as biocatalysts for continuous synthesis of ethyl oleate in a continuously stirred tank reactor. The effect of biocatalyst reutilisation, molar ratio of substrates, agitation rate and feed rate on the esterification of oleic acid with ethanol were investigated. The catalyst maintained 90% conversion for four batch cycles with a 1:2 molar ratio (oleic acid:ethanol). Mechanical agitation at 200 and 300 rpm during 12 h of continuous reaction did not affect the biocatalytic conversion, allowing substrate conversions greater than 90% that were obtained with 50 mM oleic acid at a molar ratio of 1:2 during 14 h reaction. In contrast, substrate conversion was 70% with 100 mM oleic acid at a flow rate of 2 mL/min during 25 h of reaction. These results are promising and offer a technical alternative for the development of accessible biocatalysts that can be used in continuous operations. [ABSTRACT FROM AUTHOR]

Summary Aging is associated with progressive white adipose tissue (WAT) enlargement initiated early in life, but the molecular mechanisms involved remain unknown. Here we show that mitochondrial complex IV (CIV) activity and assembly are already repressed in white adipocytes of middle-aged mice and involve a HIF1A-dependent decline of essential CIV components such as COX5B. At the molecular level, HIF1A binds to the Cox5b proximal promoter and represses its expression. Silencing of Cox5b decreased fatty acid oxidation and promoted intracellular lipid accumulation. Moreover, local in vivo Cox5b silencing in WAT of young mice increased the size of adipocytes, whereas restoration of COX5B expression in aging mice counteracted adipocyte enlargement. An age-dependent reduction in COX5B gene expression was also found in human visceral adipose tissue. Collectively, our findings establish a pivotal role for CIV dysfunction in progressive white adipocyte enlargement during aging, which can be restored to alleviate age-dependent WAT expansion. [ABSTRACT FROM AUTHOR]

Summary: The fine regulation of mitochondrial function has proved to be an essential metabolic adaptation to fluctuations in oxygen availability. During hypoxia, cells activate an anaerobic switch that favors glycolysis and attenuates the mitochondrial activity. This switch involves the hypoxia-inducible transcription factor-1 (HIF-1). We have identified a HIF-1 target gene, the mitochondrial NDUFA4L2 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2). Our results, obtained employing NDUFA4L2-silenced cells and NDUFA4L2 knockout murine embryonic fibroblasts, indicate that hypoxia-induced NDUFA4L2 attenuates mitochondrial oxygen consumption involving inhibition of Complex I activity, which limits the intracellular ROS production under low-oxygen conditions. Thus, reducing mitochondrial Complex I activity via NDUFA4L2 appears to be an essential element in the mitochondrial reprogramming induced by HIF-1. [Copyright &y& Elsevier]

Abstract: Although nitric oxide (NO) was identified more than 150years ago and its effects were clinically tested in the form of nitroglycerine, it was not until the decades of 1970–1990 that it was described as a gaseous signal transducer. Since then, a canonical pathway linked to cyclic GMP (cGMP) as its quintessential effector has been established, but other modes of action have emerged and are now part of the common body of knowledge within the field. Classical (or canonical) signaling involves the selective activation of soluble guanylate cyclase, the generation of cGMP, and the activation of specific kinases (cGMP-dependent protein kinases) by this cyclic nucleotide. Nonclassical signaling alludes to the formation of NO-induced posttranslational modifications (PTMs), especially S-nitrosylation, S-glutathionylation, and tyrosine nitration. These PTMs are governed by specific biochemical mechanisms as well as by enzymatic systems. In addition, a less classical but equally important pathway is related to the interaction between NO and mitochondrial cytochrome c oxidase, which might have important implications for cell respiration and intermediary metabolism. Cross talk trespassing these necessarily artificial conceptual boundaries is progressively being identified and hence an integrated systems biology approach to the comprehension of NO function will probably emerge in the near future. [Copyright &y& Elsevier]

Certain patients suffering from chronic diseases such as AIDS or cancer experience a constant cellular secretion of tumor necrosis factor α and other pro-inflammatory cytokines that results in a continuous release of nitric oxide (·NO) to the blood- stream. One immediate consequence of the deleterious action of ·NO is weight loss and the progressive destruction of muscular mass in a process known as cachexia. We have previously reported that caveolin-3, a specific marker of muscle cells, becomes down-regulated by the action of ·NO on muscular myotubes. We describe herein that the changes observed in caveolin-3 levels are due to the alteration of the DNA binding activity of the muscular transcription factor myogenin. In the presence of ·NO, the binding of transcription factors from cell nuclear extracts of muscular tissues to the E boxes present in the caveolin-3 promoter become substantially reduced. When we purified recombinant myogenin and treated it with ·NO donors, we could detect its S-nitrosylation by three independent methods, suggesting that very likely one of the cysteine residues of the molecule is being modified. Given the role of myogenin as a regulatory protein that determines the level of multiple muscle genes expressed during late myogenesis, our results might represent a novel mode of regulation of muscle development under conditions of nitric oxide-mediated toxicity. [ABSTRACT FROM AUTHOR]

Summary Cellular aspartate drives cancer cell proliferation, but signaling pathways that rewire aspartate biosynthesis to control cell growth remain largely unknown. Hypoxia-inducible factor-1α (HIF1α) can suppress tumor cell proliferation. Here, we discovered that HIF1α acts as a direct repressor of aspartate biosynthesis involving the suppression of several key aspartate-producing proteins, including cytosolic glutamic-oxaloacetic transaminase-1 (GOT1) and mitochondrial GOT2. Accordingly, HIF1α suppresses aspartate production from both glutamine oxidation as well as the glutamine reductive pathway. Strikingly, the addition of aspartate to the culture medium is sufficient to relieve HIF1α-dependent repression of tumor cell proliferation. Furthermore, these key aspartate-producing players are specifically repressed in VHL-deficient human renal carcinomas, a paradigmatic tumor type in which HIF1α acts as a tumor suppressor, highlighting the in vivo relevance of these findings. In conclusion, we show that HIF1α inhibits cytosolic and mitochondrial aspartate biosynthesis and that this mechanism is the molecular basis for HIF1α tumor suppressor activity. Graphical Abstract Highlights • HIF1α reduces intracellular aspartate levels • HIF1α impairs oxidative and reductive aspartate biosynthesis • The aspartate-generating GOT1 and GOT2 enzymes are repressed by HIF1α • Aspartate supplementation counteracts the antiproliferative influence of HIF1α Meléndez-Rodríguez et al. show that HIF1α impairs oxidative and reductive aspartate biogenesis, which consequently drives HIF1α-dependent suppression of tumor cell proliferation. Mechanistically, HIF1α represses the aspartate-producing enzymes GOT1 and GOT2 in several biological settings, including human VHL-deficient renal cell carcinoma, in which HIF1α can act as a tumor suppressor. [ABSTRACT FROM AUTHOR]

Like big human cities, cells are crowded environments with a lot of inhabitants, where a myriad of signals try to orchestrate how each component can do its best among the crowd, and how the whole system respond to changes in the environment. Some signals are robust and stable, so they can be easily studied by life science researchers. However, free radical researchers know well that other molecular signals are short-lived and unstable. I have been involved in several studies that can be seen as adventures searching for those subtle signals, overcoming the difficulties that appear in the way. One is the reliable proteomic identification of protein S-nitrosylation (or S-nitrosation), and of other reversible oxidations of cysteines, in physiological scenarios where they can act as signals (not just in nitrosative, oxidative or similar stresses). Different techniques have been used for that purpose (called “thiol redox proteomics”), with technical developments still ongoing. A related adventure is the study of the functional relevance of these modifications in individual proteins, and of the particularities of these modes of signalling. Another adventure deals with the role of reactive oxygen species production in hypoxia sensing and adaptation, on which a debate has been maintained for a long time. We have measured superoxide production in acute hypoxia, showing there is a fast burst at the first minutes of acute hypoxia, and we are currently studying its molecular mechanism, implying mitochondrial complex I and the sodium/calcium exchanger. This can be a common signal to different cell types that produces diverse effects depending on the signalling cascades and effectors of each cell type. Thiol redox proteomics can help in identifying the specific protein cysteine residues that can be affected by reversible oxidation and take part in those signals. [ABSTRACT FROM AUTHOR]

An abstract of the article "Detection and Identification of S-Nitrosylated Proteins in Endothelial Cells," by Antonio Martínez-Ruiz and Santiago Lamas is presented.

Although there is scientific evidence of the presence of immunometabolic alterations in major depression, not all patients present them. Recent studies point to the association between an inflammatory phenotype and certain clinical symptoms in patients with depression. The objective of our study was to classify major depression disorder patients using supervised learning algorithms or machine learning, based on immunometabolic and oxidative stress biomarkers and lifestyle habits. Taking into account a series of inflammatory and oxidative stress biomarkers (C-reactive protein (CRP), tumor necrosis factor (TNF), 4-hydroxynonenal (HNE) and glutathione), metabolic risk markers (blood pressure, waist circumference and glucose, triglyceride and cholesterol levels) and lifestyle habits of the participants (physical activity, smoking and alcohol consumption), a study was carried out using machine learning in a sample of 171 participants, 91 patients with depression (71.42% women, mean age = 50.64) and 80 healthy subjects (67.50% women, mean age = 49.12). The algorithm used was the support vector machine, performing cross validation, by which the subdivision of the sample in training (70%) and test (30%) was carried out in order to estimate the precision of the model. The prediction of belonging to the patient group (MDD patients versus control subjects), melancholic type (melancholic versus non-melancholic patients) or resistant depression group (treatment-resistant versus non-treatment-resistant) was based on the importance of each of the immunometabolic and lifestyle variables. With the application of the algorithm, controls versus patients, such as patients with melancholic symptoms versus non-melancholic symptoms, and resistant versus non-resistant symptoms in the test phase were optimally classified. The variables that showed greater importance, according to the results of the area under the ROC curve, for the discrimination between healthy subjects and patients with depression were current alcohol consumption (AUC = 0.62), TNF-α levels (AUC = 0.61), glutathione redox status (AUC = 0.60) and the performance of both moderate (AUC = 0.59) and vigorous physical exercise (AUC = 0.58). On the other hand, the most important variables for classifying melancholic patients in relation to lifestyle habits were past (AUC = 0.65) and current (AUC = 0.60) tobacco habit, as well as walking routinely (AUC = 0.59) and in relation to immunometabolic markers were the levels of CRP (AUC = 0.62) and glucose (AUC = 0.58). In the analysis of the importance of the variables for the classification of treatment-resistant patients versus non-resistant patients, the systolic blood pressure (SBP) variable was shown to be the most relevant (AUC = 0.67). Other immunometabolic variables were also among the most important such as TNF-α (AUC = 0.65) and waist circumference (AUC = 0.64). In this case, sex (AUC = 0.59) was also relevant along with alcohol (AUC = 0.58) and tobacco (AUC = 0.56) consumption. The results obtained in our study show that it is possible to predict the diagnosis of depression and its clinical typology from immunometabolic markers and lifestyle habits, using machine learning techniques. The use of this type of methodology could facilitate the identification of patients at risk of presenting depression and could be very useful for managing clinical heterogeneity. • Differences in inflammatory and oxidative stress biomarkers are found in some patients with major depressive disorder. • With the application of supervised machine learning algorithms, major depressive patients were optimally classified. • The study of immunometabolic biomarkers and lifestyle variables can help to disentangle the clinical heterogeneity of major depression. [ABSTRACT FROM AUTHOR]

Abstract: Oxygen is a key molecule for cell metabolism. Eukaryotic cells sense the reduction in oxygen availability (hypoxia) and trigger a series of cellular and systemic responses to adapt to hypoxia, including the optimization of oxygen consumption. Many of these responses are mediated by a genetic program induced by the hypoxia-inducible transcription factors (HIFs), regulated by a family of prolyl hydroxylases (PHD or EGLN) that use oxygen as a substrate producing HIF hydroxylation. In parallel to these oxygen sensors modulating gene expression within hours, acute modulation of protein function in response to hypoxia is known to occur within minutes. Free radicals acting as second messengers, and oxidative posttranslational modifications, have been implied in both groups of responses. Localization and speciation of the paradoxical increase in reactive oxygen species production in hypoxia remain debatable. We have observed that several cell types respond to acute hypoxia with a transient increase in superoxide production for about 10min, probably originating in the mitochondria. This may explain in part the apparently divergent results found by various groups that have not taken into account the time frame of hypoxic ROS production. We propose that this acute and transient hypoxia-induced superoxide burst may be translated into oxidative signals contributing to hypoxic adaptation and preconditioning. [Copyright &y& Elsevier]

Abstract: Glutathione oxidation and protein glutathionylation are considered hallmarks of oxidative stress in cells because they reflect thiol redox status in proteins. Our aims were to analyze the redox status of thiols and to identify mixed disulfides and targets of redox signaling in pancreas in experimental acute pancreatitis as a model of acute inflammation associated with glutathione depletion. Glutathione depletion in pancreas in acute pancreatitis is not associated with any increase in oxidized glutathione levels or protein glutathionylation. Cystine and homocystine levels as well as protein cysteinylation and γ-glutamyl cysteinylation markedly rose in pancreas after induction of pancreatitis. Protein cysteinylation was undetectable in pancreas under basal conditions. Targets of disulfide stress were identified by Western blotting, diagonal electrophoresis, and proteomic methods. Cysteinylated albumin was detected. Redox-sensitive PP2A and tyrosine protein phosphatase activities diminished in pancreatitis and this loss was abrogated by N-acetylcysteine. According to our findings, disulfide stress may be considered a specific type of oxidative stress in acute inflammation associated with protein cysteinylation and γ-glutamylcysteinylation and oxidation of the pair cysteine/cystine, but without glutathione oxidation or changes in protein glutathionylation. Two types of targets of disulfide stress were identified: redox buffers, such as ribonuclease inhibitor or albumin, and redox-signaling thiols, which include thioredoxin 1, APE1/Ref1, Keap1, tyrosine and serine/threonine phosphatases, and protein disulfide isomerase. These targets exhibit great relevance in DNA repair, cell proliferation, apoptosis, endoplasmic reticulum stress, and inflammatory response. Disulfide stress would be a specific mechanism of redox signaling independent of glutathione redox status involved in inflammation. [Copyright &y& Elsevier]

Alterations in cognitive performance have been described in patients with major depressive disorder (MDD). However, the specific risk factors of these changes are not yet known. This study aimed to explore whether inmunometabolic parameters are related to cognitive performance in MDD in comparison to healthy controls (HC) Sample consisted of 84 MDD patients and 78 HC. Both groups were compared on the results of cognitive performance measured with the Cambridge Neuropsychological Test Automated Battery (CANTAB), the presence of metabolic syndrome (MetS) and an inflammatory/oxidative index calculated by a principal component analysis of peripheral biomarkers (tumor necrosis factor, C-reactive protein and 4-hydroxynonenal). A multiple linear regression was carried out, to study the relationship between inmunometabolic variables and the global cognitive performance, being the latter the dependent variable. Significant differences were obtained in the inflammatory/oxidative index between both groups (F (1157) = 12.93; p <.001), also in cognitive performance (F (1157) = 56.75; p <.001). The inmunometabolic covariate regression model (i.e., condition (HC/MDD), sex, age and medication loading, MetS, inflammatory/oxidative index and the interaction between MetS and inflammatory/oxidative index) was statistically significant (F (7157) = 11.24; p <.01) and explained 31% of variance. The condition, being either MDD or HD, (B=˗0.97; p <.001), age (B=˗0.28; p <.001) and the interaction between inflammatory/oxidative index and MetS (B=˗0.38; p =.02) were factors associated to cognitive performance. Sample size was relatively small. The cross-sectional design of the study limits the possibilities of analysis. Our results provide evidence on the conjoint influence of metabolic and inflammatory dysregulation on cognitive dysfunction in MDD patients. In this way, our study opens a line of research in immunometabolic agents to deal with cognitive decline associated with MDD. • MDD patients show dysregulation in an inflammatory index obtained from peripheral biomarkers. • Suffering from MDD, being older and having MetS with high inflammatory index are related to poorer cognitive performance. • Cognitive impairment in MDD is increased in patients in which metabolic and inflammatory dysregulation coexist. [ABSTRACT FROM AUTHOR]

Disulfide stress has been associated with inflammation and characterized by an increase in cystine levels and protein cysteinylation. Furthermore, it was recently discovered that thioredoxin-related protein of 14 kDa (TRP14, encoded by TXNDC17) exhibits efficient cystine reductase activity. The aim of our research was to elucidate if TRP14 is also able to reduce cysteinylated proteins in mammalian cells. Thus, protein cysteinylation was assessed in control and TRP14 knockdown cells in vitro through their pre-treatment with 25 µg/ml cycloheximide for 30 min and incubation with 250 µM biotinylated cysteine for 1 h. Moreover, such TRP14 knockdown cell lysates were tested as cysteinylated substrates for TRP14 activity through their incubation with 20 µM human TRP14, 1 uM thioredoxin reductase 1 (TrxR1) and 1 mM NADPH for 15 min at 25ºC. TRP14 knockdown cells showed more protein cysteinylation than control cells. Human TRP14, in a reaction together with TrxR1 and NADPH, could also reduce cysteinylated protein substrates in cell lysates. In conclusion, TRP14 is able to directly reduce protein cysteinylation motifs. The potential in vivo significance of this enzymatic activity awaits further clarification. [ABSTRACT FROM AUTHOR]

The main machinery responsible for cellular protein maintenance is the ubiquitin-proteasomal system. The main task of the system is a fast and efficient degradation of proteins not needed anymore in cellular metabolism. It is accepted that upon oxidative stress, the proteasome suffers a series of functional alterations, including a reversible, oxidation-triggered 26 S proteasome disassembly into its catalytic (CP) and regulatory (RP) particles. Formation of low fluxes of oxidants were stimulated with antimycin or palmitic acid in pancreatic MIN6 cells, inducing a partial increase of dimerization of mitochondrial and citosolic peroxiredoxins. Under this conditions, no significant increase of total protein thiol oxidation was detected. However, ATP-stimulation of 26 S proteasomes was inhibited while the particle remained fully assemblied. Discrete cysteine residues of both CP and RP were identified oxidized by a cysteine-targeted proteomics approach. Such results may rely on differential reactivity of cysteines in the subunits and suggest specific regulation of particles by discrete cysteine oxidation. Further studies will elucidate the type of modification on each cysteine residue and the impact. Our results suggest a novel mechanism of redox regulation of 26 S proteasome that precedes oxidation-driven 26 S particle disassembly. [ABSTRACT FROM AUTHOR]

Disulfide stress is a specific type of oxidative stress that is associated with protein cysteinylation. The aim of this research was to characterize experimental models of disulphide stress. Thus, the redox status of free thiols and protein cysteinylation was studied in acute pancreatitis as an in vivo model of inflammation and in cystinosis an in vitro model of cystine accumulation due to its dysfunctional lysosomal transport. Cystine and homocystine levels, and protein cysteinylation rose after taurocholate-induced acute pancreatitis. Oxidation of cysteines in mitochondrial sulfide quinone oxidoreductase and 60S ribosomal protein L7a was observed. Cysteinylated albumin was also detected. Serine/threonine protein phosphatase PP2Ac, and Keap-1 were identified as targets of disulfide stress in acute pancreatitis. PP2Ac exhibited an intramolecular disulfide that was detected by diagonal electrophoresis. Silencing of PP2Ac in 266 pancreatic cells triggered up-regulation of IL-6 under basal conditions and enhanced TNF-a-induced expression of Cxcl1 and Tnf-a. Silencing of lysosomal transporter, cystinosin, triggered an increase in cystine levels and cysteinylation in PANC-1 cells. In conclusion, disulfide stress triggers protein cysteinylation associated with oxidation of the cysteine/cysteine pair in acute pancreatitis and cystinosis. PP2A is a major target in acute inflammation. [ABSTRACT FROM AUTHOR]