NOX1 and PRDX6 synergistically support migration and invasiveness of hepatocellular carcinoma cells through enhanced NADPH oxidase activity.

The NADPH oxidase 1 (NOX1) complex formed by proteins NOX1, p22phox, NOXO1, NOXA1, and RAC1 plays an important role in the generation of superoxide and other reactive oxygen species (ROS) which are involved in normal and pathological cell functions due to their effects on diverse cell signaling pathways. Cell migration and invasiveness are at the origin of tumor metastasis during cancer progression which involves a process of cellular de-differentiation known as the epithelial-mesenchymal transition (EMT). During EMT cells lose their polarized epithelial phenotype and express mesenchymal marker proteins that enable cytoskeletal rearrangements promoting cell migration, expression and activation of matrix metalloproteinases (MMPs), tissue remodeling, and cell invasion during metastasis. In this work, we explored the importance of the peroxiredoxin 6 (PRDX6)-NOX1 enzyme interaction leading to NOXA1 protein stabilization and increased levels of superoxide produced by NOX in hepatocarcinoma cells. This increase was accompanied by higher levels of N-cadherin and MMP2, correlating with a greater capacity for cell migration and invasiveness of SNU475 hepatocarcinoma cells. The increase in superoxide and the associated downstream effects on cancer progression were suppressed when phospholipase A2 or peroxidase activities of PRDX6 were abolished by site-directed mutagenesis, reinforcing the importance of these catalytic activities in supporting NOX1-based superoxide generation. Overall, these results demonstrate a clear functional cooperation between NOX1 and PRDX6 catalytic activities which generate higher levels of ROS production, resulting in a more aggressive tumor phenotype.

Loss of PRDX6 Aborts Proliferative and Migratory Signaling in Hepatocarcinoma Cell Lines.

Peroxiredoxin 6 (PRDX6), the only mammalian 1-Cys member of the peroxiredoxin family, has peroxidase, phospholipase A2 (PLA2), and lysophosphatidylcholine (LPC) acyltransferase (LPCAT) activities. It has been associated with tumor progression and cancer metastasis, but the mechanisms involved are not clear. We constructed an SNU475 hepatocarcinoma cell line knockout for PRDX6 to study the processes of migration and invasiveness in these mesenchymal cells. They showed lipid peroxidation but inhibition of the NRF2 transcriptional regulator, mitochondrial dysfunction, metabolic reprogramming, an altered cytoskeleton, down-regulation of PCNA, and a diminished growth rate. LPC regulatory action was inhibited, indicating that loss of both the peroxidase and PLA2 activities of PRDX6 are involved. Upstream regulators MYC, ATF4, HNF4A, and HNF4G were activated. Despite AKT activation and GSK3ß inhibition, the prosurvival pathway and the SNAI1-induced EMT program were aborted in the absence of PRDX6, as indicated by diminished migration and invasiveness, down-regulation of bottom-line markers of the EMT program, MMP2, cytoskeletal proteins, and triggering of the "cadherin switch". These changes point to a role for PRDX6 in tumor development and metastasis, so it can be considered a candidate for antitumoral therapies.

Integrated molecular signaling involving mitochondrial dysfunction and alteration of cell metabolism induced by tyrosine kinase inhibitors in cancer.

Cancer cells have unlimited replicative potential, insensitivity to growth-inhibitory signals, evasion of apoptosis, cellular stress, and sustained angiogenesis, invasiveness and metastatic potential. Cancer cells adequately adapt cell metabolism and integrate several intracellular and redox signaling to promote cell survival in an inflammatory and hypoxic microenvironment in order to maintain/expand tumor phenotype. The administration of tyrosine kinase inhibitor (TKI) constitutes the recommended therapeutic strategy in different malignancies at advanced stages. There are important interrelationships between cell stress, redox status, mitochondrial function, metabolism and cellular signaling pathways leading to cell survival/death. The induction of apoptosis and cell cycle arrest widely related to the antitumoral properties of TKIs result from tightly controlled events involving different cellular compartments and signaling pathways. The aim of the present review is to update the most relevant studies dealing with the impact of TKI treatment on cell function. The induction of endoplasmic reticulum (ER) stress and Ca2+ disturbances, leading to alteration of mitochondrial function, redox status and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) signaling pathways that involve cell metabolism reprogramming in cancer cells will be covered. Emphasis will be given to studies that identify key components of the integrated molecular pattern including receptor tyrosine kinase (RTK) downstream signaling, cell death and mitochondria-related events that appear to be involved in the resistance of cancer cells to TKI treatments.

Outcomes of very elderly trauma ICU patients. Results from the Spanish trauma ICU registry.

OBJECTIVE: To analyze outcomes and factors related to mortality among very elderly trauma patients admitted to intensive care units (ICUs) participating in the Spanish trauma ICU registry. DESIGN: A multicenter nationwide registry. Retrospective analysis. November 2012-May 2017. SETTING: Participating ICUs. PATIENTS: Trauma patients aged ≥80 years. INTERVENTIONS: None. MAIN VARIABLES OF INTEREST: The outcomes and influence of limitation of life sustaining therapy (LLST) were analyzed. Comparisons were established using the Wilcoxon test, Chi-squared test or Fisher's exact test as appropriate. Multiple logistic regression analysis was performed to analyze variables related to mortality. A p-value <0.05 was considered statistically significant. RESULTS: The mean patient age was 83.4±3.3 years; 281 males (60.4%). Low-energy falls were the mechanisms of injury in 256 patients (55.1%). The mean ISS was 20.5±11.1, with a mean ICU stay of 7.45±9.9 days. The probability of survival based on the TRISS methodology was 69.8±29.7%. The ICU mortality rate was 15.5%, with an in-hospital mortality rate of 19.2%. The main cause of mortality was intracranial hypertension (42.7%). The ISS, the need for first- and second-tier measures to control intracranial pressure, and being admitted to the ICU for organ donation were independent mortality predictors. LLST was applied in 128 patients (27.9%). Patients who received LLST were older, with more severe trauma, and with more severe brain injury. CONCLUSIONS: Very elderly trauma ICU patients presented mortality rates lower than predicted on the basis of the severity of injury.

Clinical guide for the diagnosis and follow-up of myotonic dystrophy type 1, MD1 or Steinert's disease. / Guía clínica para el diagnóstico y seguimiento de la distrofia miotónica tipo 1, DM1 o enfermedad de Steinert.

BACKGROUND AND OBJECTIVES: Steinert's disease or myotonic dystrophy type 1 (MD1), (OMIM 160900), is the most prevalent myopathy in adults. It is a multisystemic disorder with dysfunction of virtually all organs and tissues and a great phenotypical variability, which implies that it has to be addressed by different specialities with experience in the disease. The knowledge of the disease and its management has changed dramatically in recent years. This guide tries to establish recommendations for the diagnosis, prognosis, follow-up and treatment of the complications of MD1. MATERIAL AND METHODS: Consensus guide developed through a multidisciplinary approach with a systematic literature review. Neurologists, pulmonologists, cardiologists, endocrinologists, neuropaediatricians and geneticists have participated in the guide. RECOMMENDATIONS: The genetic diagnosis should quantify the number of CTG repetitions. MD1 patients need cardiac and respiratory lifetime follow-up. Before any surgery under general anaesthesia, a respiratory evaluation must be done. Dysphagia must be screened periodically. Genetic counselling must be offered to patients and relatives. CONCLUSION: MD1 is a multisystemic disease that requires specialised multidisciplinary follow-up.

Thioredoxin and glutaredoxin regulate metabolism through different multiplex thiol switches.

The aim of the present study was to define the role of Trx and Grx on metabolic thiol redox regulation and identify their protein and metabolite targets. The hepatocarcinoma-derived HepG2 cell line under both normal and oxidative/nitrosative conditions by overexpression of NO synthase (NOS3) was used as experimental model. Grx1 or Trx1 silencing caused conspicuous changes in the redox proteome reflected by significant changes in the reduced/oxidized ratios of specific Cys's including several glycolytic enzymes. Cys91 of peroxiredoxin-6 (PRDX6) and Cys153 of phosphoglycerate mutase-1 (PGAM1), that are known to be involved in progression of tumor growth, are reported here for the first time as specific targets of Grx1. A group of proteins increased their CysRED/CysOX ratio upon Trx1 and/or Grx1 silencing, including caspase-3 Cys163, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Cys247 and triose-phosphate isomerase (TPI) Cys255 likely by enhancement of NOS3 auto-oxidation. The activities of several glycolytic enzymes were also significantly affected. Glycolysis metabolic flux increased upon Trx1 silencing, whereas silencing of Grx1 had the opposite effect. Diversion of metabolic fluxes toward synthesis of fatty acids and phospholipids was observed in siRNA-Grx1 treated cells, while siRNA-Trx1 treated cells showed elevated levels of various sphingomyelins and ceramides and signs of increased protein degradation. Glutathione synthesis was stimulated by both treatments. These data indicate that Trx and Grx have both, common and specific protein Cys redox targets and that down regulation of either redoxin has markedly different metabolic outcomes. They reflect the delicate sensitivity of redox equilibrium to changes in any of the elements involved and the difficulty of forecasting metabolic responses to redox environmental changes.

The regulation of TORC1 pathway by the yeast chaperones Hsp31 is mediated by SFP1 and affects proteasomal activity.

The Saccharomyces cerevisiae heat shock proteins Hsp31-34 are members of DJ-1/ThiJ/Pfpl superfamily that includes human DJ-1 (Park7), a protein involved in heritable Parkinsonism. Although, homologs of these proteins can be found in most organisms their functions are unclear. We have carried out a quantitative proteomics analysis of yeast cells devoid of the whole set of Hsp31 family of proteins, as a model of Parkinson Disease (PD), under conditions of glucose availability and starvation. The protein profile indicates a constitutive activation of the enzyme TORC1 that makes the cells more sensitive to stress conditions. TORC1 activation prevents the cells from diauxic shift and entry into the stationary phase inducing cell death. Sfp1 stays at the helm among the several transcription factors governing the cell adaptation to Hsp31-34 deficiency. We show that Sfp1 remains mainly in the nucleus likely releasing TORC1 from inhibition by cytosolic Sfp1. Impairment of glycolysis leads to increased levels of methylglyoxal and accumulation of glycated proteins. We also show an increase in proteasome subunits in the Hsp31-34 mutant, under the control of Rpn4 transcription factor. This increase is abnormally accompanied by a decrease in proteasomal activity which could lead to accumulation of aberrant proteins and contributing to cell death.

Regulation of Cell Survival, Apoptosis, and Epithelial-to-Mesenchymal Transition by Nitric Oxide-Dependent Post-Translational Modifications.

SIGNIFICANCE: Nitric oxide (NO) is a physiopathological messenger generating different reactive nitrogen species (RNS) according to hypoxic, acidic and redox conditions. Recent Advances: RNS and reactive oxygen species (ROS) promote relevant post-translational modifications, such as nitrosation, nitration, and oxidation, in critical components of cell proliferation and death, epithelial-to-mesenchymal transition, and metastasis. CRITICAL ISSUES: The pro- or antitumoral properties of NO are dependent on local concentration, redox state, cellular status, duration of exposure, and compartmentalization of NO generation. The increased expression of NO synthase has been associated with cancer progression. However, the experimental strategies leading to high intratumoral NO generation have been shown to exert antitumoral properties. The effect of NO and ROS on cell signaling is critically altered by factors modulating tumor progression such as oxygen content, metabolism, and inflammatory response. The review describes the alteration of key components involved in cell survival and death, metabolism, and metastasis induced by RNS- and ROS-related post-translational modifications. FUTURE DIRECTIONS: The identification of the molecular targets affected by nitrosation, nitration, and oxidation, as well as their interactions with other post-translational modifications, will improve the understanding on the complex signaling and cell fate decision in cancer. The therapeutic NO-based strategies have to address the complex crosstalk among NO and ROS with regard to critical components affecting tumor cell survival, metabolism, and metastasis in the progression of cancer, as well as close interaction with ionizing radiation and chemotherapy.

Effect of the route of nutrition and L-alanyl-L-glutamine supplementation in amino acids' concentration in trauma patients.

PURPOSE: Our purpose was to assess the amino acids' (AAs) profile in trauma patients and to assess the effect of the route of nutrition and the exogenous ALA-GLN dipeptide supplementation on plasma AAs' concentration. METHODS: This is a secondary analysis of a previous randomized controlled trial. On day 1 and day 6 after trauma, plasma concentration of 25 AAs was measured using reverse phase high-performance liquid chromatography. Results were analyzed in relation to the route of nutrition and supplementation of ALA-GLN dipeptide. Differences between plasma AAs' concentrations at day 1 and day 6 were evaluated using the Student's t test or Mann-Whitney-Wilcoxon test. One-way ANOVA and the Kruskal-Wallis test were used to compare groups. A two-sided p value less than 0.05 was considered statistically significant. RESULTS: Ninety-eight patients were analyzed. Mean plasma concentrations at day 1 were close to the lower normal level for most AAs. At day 6 we found an increase in the eight essential AAs' concentrations and in 9 out of 17 measured non-essential AAs. At day 6 we found no differences in plasma concentrations for the sum of all AAs (p = .72), glutamine (p = .31) and arginine (p = .23) distributed by the route of nutrition. Administration of ALA-GLN dipeptide increased the plasma concentration of alanine (p = .004), glutamine (p < .001) and citrulline (p = .006). CONCLUSIONS: We found an early depletion of plasma AAs' concentration which partially recovered at day 6, which was unaffected by the route of nutrition. ALA-GLN dipeptide supplementation produced a small increase in plasma levels of glutamine and citrulline.

Epidemiology of RHDV2 (Lagovirus europaeus/GI.2) in free-living wild European rabbits in Portugal.

As the detection of the first outbreak of a novel aetiological agent of rabbit haemorrhagic disease commonly called RHDV2 or RHDVb (Lagovirus europaeus/GI.2, henceforth GI.2) in France in 2010, the virus rapidly spread throughout continental Europe and nearby islands such as Great Britain, Sardinia, Sicily, the Azores and the Canary Islands among others. The outbreaks of this new lagovirus cause important economic losses in rabbitries, and ecological disruptions by affecting the conservation of rabbit-sensitive top predators. We analysed 550 rabbit carcasses collected in the field between May 2013 and March 2016, to investigate the epidemiology of GI.2 in free-living populations and to perform a comparative analysis with the epidemiology of classical rabbit haemorrhagic disease virus forms (RHDV, henceforth GI.1) in Portugal. Rabbits were sexed, aged and liver and blood samples were collected for subsequent RHDV screening and serology. A total of 172 samples were PCR-positive to GI.2, whereas GI.1 strains were not detected in any of the samples. The outbreaks of GI.2 revealed a marked seasonality, with peaks during the breeding season (November-May). We also found that approximately, one-third of free-ranging European rabbits in Portugal have seroconverted to GI.2. We demonstrate that the GI.2 lagovirus is currently widespread in wild populations in Portugal and is affecting a high proportion of adults and juveniles. Therefore, ongoing monitoring and surveillance are required to assess the effects of GI.2 on wild rabbit populations, its evolution, and to guide management actions aimed at mitigating the impacts of rabbit declines in the ecosystem and in rural economies.

Evidence of mixotrophic carbon-capture by n-butanol-producer Clostridium beijerinckii.

Recent efforts to combat increasing greenhouse gas emissions include their capture into advanced biofuels, such as butanol. Traditionally, biobutanol research has been centered solely on its generation from sugars. Our results show partial re-assimilation of CO2 and H2 by n-butanol-producer C. beijerinckii. This was detected as synchronous CO2/H2 oscillations by direct (real-time) monitoring of their fermentation gasses. Additional functional analysis demonstrated increased total carbon recovery above heterotrophic values associated to mixotrophic assimilation of synthesis gas (H2, CO2 and CO). This was further confirmed using 13C-Tracer experiments feeding 13CO2 and measuring the resulting labeled products. Genome- and transcriptome-wide analysis revealed transcription of key C-1 capture and additional energy conservation genes, including partial Wood-Ljungdahl and complete reversed pyruvate ferredoxin oxidoreductase / pyruvate-formate-lyase-dependent (rPFOR/Pfl) pathways. Therefore, this report provides direct genetic and physiological evidences of mixotrophic inorganic carbon-capture by C. beijerinckii.

High purity galacto-oligosaccharides enhance specific Bifidobacterium species and their metabolic activity in the mouse gut microbiome.

Prebiotics are selectively fermented ingredients that result in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon the host health. The aim of this study was to evaluate the influence of a ß(1-4)galacto-oligosaccharides (GOS) formulation consisting of 90% pure GOS (GOS90), on the composition and activity of the mouse gut microbiota. Germ-free mice were colonised with microbiota from four pathogen-free wt 129 mice donors (SPF), and stools were collected during a feeding trial in which GOS90 was delivered orally for 14 days. Pyrosequencing of 16S rDNA amplicons showed that Bifidobacterium and specific Lactobacillus, Bacteroides and Clostridiales were more prevalent in GOS90-fed mice after 14 days, although the prebiotic impact on Bifidobacterium varied among individual mice. Prebiotic feeding also resulted in decreased abundance of Bacteroidales, Helicobacter and Clostridium. High-throughput quantitative PCR showed an increased abundance of Bifidobacterium adolescentis, Bifidobacterium pseudocatenulatum, Bifidobacterium lactis and Bifidobacterium gallicum in the prebiotic-fed mice. Control female mice showed a higher diversity (phylogenetic diversity (PD) = 15.1 ± 3.4 in stools and PD = 13.0 ± 0.6 in intestinal contents) than control males (PD = 7.8 ± 1.6 in stool samples and PD = 9.5 ± 1.0 in intestinal contents). GOS90 did not modify inflammatory biomarkers (interleukin (IL)-6, IL-12, IL-1ß, interferon gamma and tumour necrosis factor alpha). Decreased butyrate, acetate and lactate concentrations in stools of prebiotic fed mice suggested an increase in colonic absorption and reduced excretion. Overall, our results demonstrate that GOS90 is capable of modulating the intestinal microbiome resulting in expansion of the probiome (autochtonous commensal intestinal bacteria considered to have a beneficial influence on health).