A signature of circulating microRNAs predicts the response to treatment with FOLFIRI plus aflibercept in metastatic colorectal cancer patients.

The benefit of adding the antiangiogenic drug aflibercept to FOLFIRI regime in metastatic colorectal cancer (CRC) patients resistant to or progressive on an oxaliplatin-based therapy has been previously demonstrated. However, the absence of validated biomarkers to predict greater outcomes is a major challenge encountered when using antiangiogenic therapies. In this study we investigated profiles of circulating microRNAs (miRNAs) to build predictive models of response to treatment and survival. Plasma was obtained from 98 metastatic CRC patients enrolled in a clinical phase II trial before receiving FOLFIRI plus aflibercept treatment, and the circulating levels of 754 individual miRNAs were quantified using real-time PCR. A distinct signature of circulating miRNAs differentiated responder from non-responder patients. Remarkably, most of these miRNAs were found to target genes that are involved in angiogenic processes. Accordingly, some of these miRNAs had predictive value and entered in predictive models of response to therapy, progression of disease, and survival of patients treated with FOLFIRI plus aflibercept. Among these miRNAs, circulating levels of hsa-miR-33b-5p efficiently discriminated between responder and non-responder patients and predicted the risk of disease progression. Moreover, the combination of circulating VEGF-A and miR-33b-5p levels improved clinical stratification of metastatic CRC patients who were to receive FOLFIRI plus aflibercept treatment. In conclusion, our study supports circulating miRNAs as valuable biomarkers for predicting better outcomes in metastatic CRC patients treated with FOLFIRI plus aflibercept.

Diagnostic potential of NETosis-derived products for disease activity, atherosclerosis and therapeutic effectiveness in Rheumatoid Arthritis patients.

OBJECTIVES: 1) To assess the association of NETosis and NETosis-derived products with the activity of the disease and the development of cardiovascular disease in RA; 2) To evaluate the involvement of NETosis on the effects of biologic therapies such as anti-TNF alpha (Infliximab) and anti-IL6R drugs (Tocilizumab). METHODS: One hundred and six RA patients and 40 healthy donors were evaluated for the occurrence of NETosis. Carotid-intimae media thickness was analyzed as early atherosclerosis marker. Inflammatory and oxidative stress mediators were quantified in plasma and neutrophils. Two additional cohorts of 75 RA patients, treated either with Infliximab (n = 55) or Tocilizumab (n = 20) for six months, were evaluated. RESULTS: NETosis was found increased in RA patients, beside myeloperoxidase and neutrophil elastase protein levels. Cell-free nucleosomes plasma levels were elevated, and strongly correlated with the activity of the disease and the positivity for autoantibodies, alongside inflammatory and oxidative profiles in plasma and neutrophils. Moreover, ROC analyses showed that cell-free nucleosomes levels could identify RA patients showing early atherosclerosis with high specificity. RA patients treated either with IFX or TCZ for six months exhibited decreased generation of NETs. Concomitantly, clinical parameters and serum markers of inflammation were found reduced. Mechanistic in vitro analyses showed that inhibition of NETs extrusion by either DNase, IFX or TCZ, further abridged the endothelial dysfunction and the activation of immune cells, thus influencing the global activity of the vascular system. CONCLUSIONS: NETosis-derived products may have diagnostic potential for disease activity and atherosclerosis, as well as for the assessment of therapeutic effectiveness in RA.

Discovery of potential protein biomarkers of lung adenocarcinoma in bronchoalveolar lavage fluid by SWATH MS data-independent acquisition and targeted data extraction.

UNLABELLED: Lung cancer currently ranks as the neoplasia with the highest global mortality rate. Although some improvements have been introduced in recent years, new advances in diagnosis are required in order to increase survival rates. New mildly invasive endoscopy-based diagnostic techniques include the collection of bronchoalveolar lavage fluid (BALF), which is discarded after using a portion of the fluid for standard pathological procedures. BALF proteomic analysis can contribute to clinical practice with more sensitive biomarkers, and can complement cytohistological studies by aiding in the diagnosis, prognosis, and subtyping of lung cancer, as well as the monitoring of treatment response. The range of quantitative proteomics methodologies used for biomarker discovery is currently being broadened with the introduction of data-independent acquisition (DIA) analysis-related approaches that address the massive quantitation of the components of a proteome. Here we report for the first time a DIA-based quantitative proteomics study using BALF as the source for the discovery of potential lung cancer biomarkers. The results have been encouraging in terms of the number of identified and quantified proteins. A panel of candidate protein biomarkers for adenocarcinoma in BALF is reported; this points to the activation of the complement network as being strongly over-represented and suggests this pathway as a potential target for lung cancer research. In addition, the results reported for haptoglobin, complement C4-A, and glutathione S-transferase pi are consistent with previous studies, which indicates that these proteins deserve further consideration as potential lung cancer biomarkers in BALF. Our study demonstrates that the analysis of BALF proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS), combining a simple sample pre-treatment and SWATH DIA MS, is a useful method for the discovery of potential lung cancer biomarkers. SIGNIFICANCE: Bronchoalveolar lavage fluid (BALF) analysis can contribute to clinical practice with more sensitive biomarkers, thus complementing cytohistological studies in order to aid in the diagnosis, prognosis, and subtyping of lung cancer, as well as the monitoring of treatment response. Here we report a panel of candidate protein biomarkers for adenocarcinoma in BALF. Forty-four proteins showed a fold-change higher than 3.75 among adenocarcinoma patients compared with controls. This report is the first DIA-based quantitative proteomics study to use bronchoalveolar lavage fluid (BALF) as a matrix for discovering potential biomarkers. The results are encouraging in terms of the number of identified and quantified proteins, demonstrating that the analysis of BALF proteins by a SWATH approach is a useful method for the discovery of potential biomarkers of pulmonary diseases.

Nitric oxide and cancer: the emerging role of S-nitrosylation.

Nitric oxide (NO˙) is a short-lived, endogenously produced gas that is highly diffusible across cell membranes and acts as a signaling molecule in the body. The redox state and chemistry of NO˙ facilitate its interaction with various proteins thus regulating various intracellular and intercellular events. One of the key mechanisms by which NO˙ regulates the function of various target proteins is through the coupling of a nitroso moiety from NO-derived metabolites to a reactive cysteine leading to the formation of a S-nitrosothiol (SNO), a process commonly known as S-nitrosylation. S-nitrosylation signaling events within the cell have led to the discovery of many other physiological functions of NO˙ in many other types of cells including cancer cells. Only recently are the diverse roles of S-nitrosylation in cancer beginning to be understood. In the present review we discuss the recent evidence for the diverse roles of NO˙/SNO-related mechanisms in cancer biology and therapy, including the participation of NO˙ in the pathogenesis of cancer, its duality in protecting against or inducing cancer cell death and the contribution of NO˙ to metastatic processes. In addition, NO˙ can be therapeutically used in the reversal of tumor cell resistance to cytotoxic drugs and as a sensitizing agent to chemo- and radiotherapy. Finally, recent studies providing evidence for NO-related mechanisms of epigenetic gene expression regulation will also be discussed. Undoubtedly, new exciting results will contribute to this rapidly expanding area of cancer research.

[Prognostic factors associated with postoperative complications in liver transplant]. / Factores pronósticos de complicaciones postoperatorias en el transplante hepático.

OBJECTIVES: the postoperative evolution of patients submitted to orthotopic liver transplant (OLT) is frequently associated with the appearance of different types of complications such as renal failure, graft rejection, infections, and neurological disorders. These complications are the most significant causes of early morbidity and mortality in patients undergoing OLT. The purpose of the present study was the identification of factors related to the different postoperative complications after OLT. EXPERIMENTAL DESIGN: a prospective study was carried out. PATIENTS: seventy-eight variables were analyzed in 32 consecutive patients undergoing OLT. The factors independently associated with the appearance of postoperative complications were identified using a stepwise logistic regression analysis. RESULTS: the multivariate analysis showed that malondialdehyde and creatinine pretransplant serum levels were associated with the development of renal dysfunction. The pretransplant levels of haemoglobin and the units of platelets administered during surgery were prognostic factors of infections. Acute graft rejection was predicted by ?-glutamyl transpeptidase and total bilirubin serum levels. The pretransplant sodium and glutaredoxin levels in serum were associated with neurological complications. CONCLUSIONS: we propose these markers for the identification of high-risk patients allowing an early surveillance and/or treatment to improve morbidity and survival in patients submitted to OLT.

Rapid induction of NF-kappaB binding during liver cell isolation and culture: inhibition by L-NAME indicates a role for nitric oxide synthase.

This study is the first to demonstrate activation of NF-kappaB binding just 10 minutes into the commonly employed hepatocyte isolation procedure. It is further reported that the anti-oxidant Trolox can prevent the induction of NF-kappaB during the well established hepatocyte isolation procedure but not during their subsequent culture. However both phases of NF-kappaB activation are inhibited by L-NAME intimating a role for NO production, via nitric oxide synthase. These findings demonstrate that at least 2 different signal transduction pathways are operative during hepatocyte isolation and culture. Thus further studies employing Trolox and L-NAME will help delineate how each pathway contributes to the generalised loss of liver function commonly observed in vitro.

The levels of ribonucleotide reductase, thioredoxin, glutaredoxin 1, and GSH are balanced in Escherichia coli K12.

The dithiol forms of thioredoxin and glutaredoxin are hydrogen donors for ribonucleotide reductase. We have determined the intracellular levels of ribonucleotide reductase (RRase), thioredoxin (Trx), glutaredoxin 1 (Grx1), and glutathione (GSH) and the glutathione redox status in new Escherichia coli K12 strains lacking thioredoxin (trxA-), glutaredoxin 1 (grxA-), and/or GSH (gshA-) or overproducing Trx or Grx1 from multicopy plasmids. We propose a regulatory network in which RRase levels are balanced with those of Trx, Grx1, and GSH so that deficiency or overproduction of one component would promote the opposite effect on the others to maintain a balanced supply of deoxyribonucleotides. GSH deficiency strongly increased both Grx1 levels and RRase activity, even more than Trx deficiency. Double gshA-trxA- bacteria were viable, whereas additional deficiency in lipoate synthesis (gshA-trxA-lipA-) caused the inability to grow in minimal medium plates supplemented with acetate plus succinate instead of lipoic acid. Thus, lipoate might be the only substitute of GSH for glutaredoxin reduction in gshA-trxA- cells, although the extremely high Grx1 content (55-fold) of these bacteria suggests that electron transfer from lipoate might be an inefficient reduction mechanism of glutaredoxins. Moreover, the enhanced Grx1 level of gshA-trxA- cells could obviate the need for a large increase in RRase activity, in contrast to grxA-trxA- double mutant cells. Impairment of the sulfate assimilation pathway, leading to very low GSH concentrations, and an oxidized glutathione redox state might explain the inability of grxA-trxA- cells to grow in minimal medium. Restoration of nearly normal levels of both GSH content and redox status cure the growth defect.

Metabolic activation of carcinogenic aromatic amines by fish exposed to environmental pollutants.

Activation of arylamines to mutagenic metabolites by hepatic S9 fractions has been evaluated as a biomaker of fish exposure to pollutants, using gilthead seabream (Sparus aurata), a valuable fish species from the Spanish South Atlantic littoral, as model organism. To obtain maximal sensitivity to the mutagenic action of aromatic amines, a strain of Salmonella typhimurium overproducing O-acetyltransferase was used. Fish were treated with Aroclor 1254, pesticides (malathion and dieldrin), or copper(II), and compared to Aroclor 1254-treated rats. The promutagen activation capabilities of the S9 fractions were further characterized by studying the effect of two monooxygenase inhibitors, alpha-naphthoflavone, a well known inhibitor of aromatic hydrocarbon-inducible forms of cytochrome P450, and methimazole, a substrate for the flavin monooxygenase (FMO) system. This study shows that 2-aminoanthracene (2-AA) and 2-acetylaminofluorene (AAF) activation by gilthead liver is enhanced by treatment of fish with different xenobiotics. The catalyst responsible for this enhanced activation appears to be different for each promutagen and, at least for 2-AA, dependent on the type of xenobiotic. The data presented indicate further that treatment of gilthead with some compounds, such as malathion and dieldrin, enhances the activation of aromatic amines in liver, without inducing ethoxyresorufin-O-deethylase activity. The use of acetyltransferase-overproducing bacteria appears to be a useful tool in the study of arylamine activation by fish liver, where biotransformation capability is lower than in mammals.

Rapid determination of glutathione status in fish liver using high-performance liquid chromatography and electrochemical detection.

A rapid and sensitive method for the detection of reduced (GSH), oxidised (GSSG) and protein-bound (PSSG) glutathione in fish liver, using reversed-phase HPLC with electrochemical detection has been developed. Separation was carried out isocratically at room temperature using 0.020 M sodium phosphate, pH 2.7 as mobile phase. A series dual-channel electrochemical detector was used for the simultaneous determination of GSH and GSSG. PSSG was determined after reduction by 1,4-dithiothreitol. The detection limits found for a 3:1 signal-to-noise ratio were 16.2 and 8.1 pmol for GSH and GSSG, respectively. The results obtained demonstrate that this method could be useful for measurement of the glutathione redox status in fish liver and are consistent with those reported for other fish. The method has been applied to follow the oxidative stress induced in vivo by copper(II) ions in the gilthead seabream fish (Sparus aurata). At longer times after copper(II) injection, the glutathione redox status of the exposed fish returned to a more reduced state, suggesting the existence of adaptive processes.