Prognostic value of gender and primary tumor location in metastatic colon cancer.

Sex might influence prognosis in patients affected by colorectal cancer. We retrospectively studied a cohort of patients affected by metastatic colon cancer (mCC) stratified by sex and primary tumor location. RAS mutational status was also included in the analysis. Overall, 616 patients met the eligibility criteria, 261 women and 355 men. Neither gender, nor RAS mutational status influenced overall survival (OS) in the entire population. As expected, patients with right-sided colon cancer (RCC) had a significant shorter OS compared to those with left-sided colon cancer (LCC) (21.3 vs 33.1 months, p= 0.002). When the analysis was performed stratifying for gender, RCC retained worse prognosis among men (OS 20.5 vs 33.9 months, p= 0.008), but not among women (p= 0.132). Similarly, the presence of RAS mutations had no prognostic effect in women, but was significantly associate with shorter survival in men (OS 29.5 vs 33.7 months, p= 0.046). In addition, when comparing clinical outcome of women or men according to sidedness and RAS mutational status, RCC was associated with dismal prognosis only in men with RAS mutated tumor (OS 17.2 vs 32.3 months, p= 0.008). Our study highlights the importance of gender in the outcome of patients with mCC.

Optimizing the Choice for Adjuvant Chemotherapy in Gastric Cancer.

Advances in the management of gastric cancer have improved patient survival in the last decade. Nonetheless, the number of patients relapsing and dying after a diagnosis of localized gastric cancer is still too high, even in early stages (10% in stage I). Adjuvant systemic chemotherapy has been proven to significantly improve outcomes. In the present article we have critically reviewed the clinical trials that guide the current clinical practice in the adjuvant treatment of patients affected by resectable gastric cancer, focusing on the different approaches worldwide, i.e., adjuvant chemotherapy, adjuvant chemoradiotherapy, and perioperative chemotherapy. We also delineate the clinical-pathological characteristics that are commonly taken into account to identify patients at a higher risk of recurrence and requiring adjuvant chemotherapy, and also describe novel biomarkers and therapeutic agents that might allow personalization of the treatment.

In Vivo Cigarette Smoke Exposure to Examine the Expression of Genes Involved in the Inflammatory Response in the Mouse Uterus.

Cigarette smoke may impair uterine function, but the underlying mechanisms are poorly characterized. In this article, we describe the methodology for whole-body exposure to cigarette smoke together with assessment of the impact of this exposure on the expression of a panel of genes related to stress and toxicity pathways in mouse uteri using an in vivo model. C57BL/6 mice are whole-body-exposed to three cigarettes daily, 7 days/week, for 2 months using a specific rodent ventilator. Uteri are then collected and subjected to qRT-PCR analysis using the Stress & Toxicity PathwayFinder RT2 Profiler PCR Array (Qiagen). Cigarette smoke was found to be associated with an upregulation (≥2-fold) of C-reactive protein (Crp; 2.65-fold, p-value = 0.02), growth arrest and DNA-damage-inducible45γ (Gadd45γ; 2.11-fold, p-value = 0.04), interferon γ (Ifnγ; 2.05-fold, p-value = 0.01), and interleukin1α (Il1α; 7.74-fold, p-value = 0.003) and downregulation of matrix metallopeptidase-9 (Mmp9; -2.42-fold, p-value = 0.01). The protocol used in this study may represent a new experimental model of mouse in vivo mainstream exposure to cigarette smoke. In addition, the resulting overexpression of pro-inflammatory cytokines and genes involved in cell cycle proliferation, together with the downregulation of extracellular matrix metallopeptidases, may represent a toxicological response to cigarette smoke exposure, with potential repercussion for the processes of uterine remodeling and growth that are essential for uterine receptiveness. A recommendation to expand upon this research area is made. © 2021 Wiley Periodicals LLC.

Integrative proteomic and functional analyses provide novel insights into the action of the repurposed drug candidate nitroxoline in AsPC-1 cells.

We recently identified nitroxoline as a repurposed drug candidate in pancreatic cancer (PC) showing a dose-dependent antiproliferative activity in different PC cell lines. This antibiotic is effective in several in vitro and animal cancer models. To date, the mechanisms of nitroxoline anticancer action are largely unknown. Using shotgun proteomics we identified 363 proteins affected by nitroxoline treatment in AsPC-1 pancreatic cancer cells, including 81 consistently deregulated at both 24- and 48-hour treatment. These proteins previously unknown to be affected by nitroxoline were mostly downregulated and interconnected in a single highly-enriched network of protein-protein interactions. Integrative proteomic and functional analyses revealed nitroxoline-induced downregulation of Na/K-ATPase pump and ß-catenin, which associated with drastic impairment in cell growth, migration, invasion, increased ROS production and induction of DNA damage response. Remarkably, nitroxoline induced a previously unknown deregulation of molecules with a critical role in cell bioenergetics, which resulted in mitochondrial depolarization. Our study also suggests that deregulation of cytosolic iron homeostasis and of co-translational targeting to membrane contribute to nitroxoline anticancer action. This study broadens our understanding of the mechanisms of nitroxoline action, showing that the drug modulates multiple proteins crucial in cancer biology and previously unknown to be affected by nitroxoline.

An explorative study identifies miRNA signatures for the diagnosis of non-celiac wheat sensitivity.

Non-celiac wheat sensitivity (NCWS), also referred to as non-celiac gluten sensitivity, is a recently described disorder triggered by wheat/gluten ingestion. NCWS elicits a wide range of symptoms including diarrhoea, intestinal discomfort, and fatigue in analogy with other wheat/gluten-related disorders and celiac disease in particular. From the pathological standpoint, NCWS patients only have a slight increase of intraepithelial lymphocytes, while antibodies to tissue transglutaminase (tTG) and villous atrophy, otherwise diagnostic features of celiac disease, are absent. To date, the diagnosis of NCWS relies on symptoms and exclusion of confounding diseases, since biomarkers are not yet available. Here, the expression levels of selected miRNAs were examined in duodenal biopsies and peripheral blood leukocytes collected from newly diagnosed patients with NCWS and, as controls, from patients with celiac disease and gluten-independent gastrointestinal problems. We identified a few miRNAs whose expression is higher in the intestinal mucosa of patients affected by NCWS in comparison to control patients affect by gluten-independent dyspeptic symptoms (Helicobacter pylori-negative) and celiac disease. The present study provided the first evidence that NCWS patients have a characteristic miRNA expression patterns, such peculiarity could be exploited as a biomarker to the diagnosis of this disease.

Structural Characterization of the Xi Class Glutathione Transferase From the Haloalkaliphilic Archaeon Natrialba magadii.

Xi class glutathione transferases (GSTs) are a recently identified group, within this large superfamily of enzymes, specifically endowed with glutathione-dependent reductase activity on glutathionyl-hydroquinone. Enzymes belonging to this group are widely distributed in bacteria, fungi, and plants but not in higher eukaryotes. Xi class GSTs are also frequently found in archaea and here we focus on the enzyme produced by the extreme haloalkaliphilic archaeon Natrialba magadii (NmGHR). We investigated its function and stability and determined its 3D structure in the apo form by X-ray crystallography. NmGHR displays the same fold of its mesophilic counterparts, is enriched in negatively charged residues, which are evenly distributed along the surface of the protein, and is characterized by a peculiar distribution of hydrophobic residues. A distinctive feature of haloalkaliphilic archaea is their preference for γ-glutamyl-cysteine over glutathione as a reducing thiol. Indeed we found that the N. magadii genome lacks a gene coding for glutathione synthase. Analysis of NmGHR structure suggests that the thiol binding site (G-site) of the enzyme is well suited for hosting γ-glutamyl-cysteine.

Cigarette smoke is associated with altered expression of antioxidant enzymes in granulosa cells from women undergoing in vitro fertilization.

This study was undertaken to evaluate whether cigarette smoke is associated with changes in the expression of antioxidant enzymes in granulosa cells of women undergoing IVF treatments. For this aim, the expression of three antioxidant enzymes (SOD1, SOD2 and catalase) in non-smokers (n = 20) and smokers (n = 20) was analyzed. There was a statistically significant overexpression of SOD2 and catalase mRNA levels in smokers in comparison with non-smokers. Cigarette smoking was associated with a lower fertilization rate, implantation rate and pregnancy rate in comparison with non-smokers. There was no effect on retrieved oocytes number, metaphase II oocytes number, quality of embryos transferred and live birth rate. These findings suggest that cigarette smoke initiates oxidative stress in granulosa cells.

Nucleophosmin mutations alter its nucleolar localization by impairing G-quadruplex binding at ribosomal DNA.

Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex-binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex-binding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.

Fluconazole alters CYP26 gene expression in mouse embryos.

Disruption of embryonal retinoic acid homeostasis has been postulated to represent an etiological factor involved in the onset of fluconazole-induced teratogenesis. In the present study the impact of a teratogenic pulse of fluconazole on the gene expression of cytochrome P450 (CYP) 26 isoforms, which plays a central role in maintaining proper retinoic acid levels by mediating its degradation, was investigated. ICR pregnant mice were orally administered with 0 (vehicle) or 700mg/kg of fluconazole on gestation day 8. Embryos were collected 12, 24 and 48h after treatment. Quantitative real-time reverse-transcription polymerase chain reaction (quantitative real-time RT-PCR) assay was used to quantify the mRNA expression of CYP26a1, CYP26b1 and CYP26c1 in embryos. As result, fluconazole exposure was associated to an up-regulation of CYP26a1, CYP26b1, whereas no significant change was identified for the CYP26c1 isoform. This study demonstrates the capacity of fluconazole to alter CYP26 gene expression in mouse embryos.

Glutathione transferases from Anguilla anguilla liver: identification, cloning and functional characterization.

Glutathione transferases (GSTs) constitute a class of detoxifying enzymes involved in Phase II metabolism. Using GSH-affinity chromatografy followed by HPLC analysis, two GST isoforms were isolated from the Anguilla anguilla liver cytosol. The major GST belongs to the piscine-specific rho class and accounted for about 59% of total GST affinity eluted fraction, while the remaining 41% was represented by a Pi class GST. Both isoforms were cloned, heterologously expressed in Escherichia coli and their enzyme activities were characterized with respect to a broad spectrum of well-known GST substrates. Our data indicate that only a fraction of prototypical GST substrates are conjugated by these enzymes and that Pi class GST has higher specific activity than rho class GST against 1-chloro-2,4-dinitrobenzene (CDNB), ethracrynic acid, 4-nitroquinoline-1-oxide and p-nitrophenyl acetate while trans-2-nonenal is detoxified more efficiently by rho class GST. Analysis of the kinetics parameters of the conjugation against CDNB indicated that the utilization ratio K(cat)/K(m) is slightly higher for rho class GST with respect to pi class GSTs. Finally, to determine the potential for environmental inhibition of the GST isoforms, we examined the effect of the widely used herbicide atrazine as an inhibitor of catalytic activity. The inhibition studies revealed that atrazine was an effective inhibitor of GST-CDNB catalytic activities of both isoforms at micromolar concentrations, suggesting the sensitivity of these isoforms to pesticide inhibition at environmentally relevant concentrations.

The forgotten variables of DNA array hybridization.

The reproducibility and reliability of DNA array measurements have been repeatedly questioned during the years. A reassessment of fundamental variables of nucleic acid hybridization might help to solve some of these problems. The hybridization equilibrium, t(1/2), is 41 days for a target human genome. Similar hybridization t(1/2) are expected for whole-transcriptome chips hybridized with tissue cDNA. This implies that most studies on mammalian cells have not been performed under equilibrium conditions. Non-equilibrium binding introduces a stochastic factor into hybridization dynamics; in other words, hybridization will prevail at different spots in different experiments, everything else being equal. A careful re-evaluation of results obtained under non-equilibrium conditions might prove fruitful and help to explain instances where findings conflict.

Sigma-class glutathione transferase from Xenopus laevis: molecular cloning, expression, and site-directed mutagenesis.

The structural gene for glutathione transferase (XlGSTS1-1) in the amphibia Xenopus laevis has been cloned from an embryo library and its nucleotide sequence has been determined. Open reading frame analysis indicated that xlgsts1 gene encodes the smallest protein of sigma class GST so far identified as being composed of only 194 amino acid residues. The recombinant XlGSTS1-1 shows a narrow range of substrate specificity as well as a significantly lower 1-chloro-2,4-dinitrobenzene conjugation capacity than that of squid sigma class GST. To compare the structural and functional differences between the squid and amphibian enzymes, several site-specific mutations were introduced in XlGSTS1-1, i.e., Ser100Asn, Phe102Tyr, Trp143Leu, Phe146Leu, and Trp148Cys. The results obtained indicate that Trp143 and Trp148 are more important determinants for the structural stability of XlGSTS1-1 rather than for its substrate specificity.

A novel amphibian Pi-class glutathione transferase isoenzyme from Xenopus laevis: importance of phenylalanine 111 in the H-site.

Screening of a liver tumour cDNA library from Xenopus laevis resulted in the isolation of a full-length cDNA clone encoding a novel Pi-class amphibian glutathione transferase (GST) isoenzyme (designated as XlGSTP1-1). The gene encodes a protein of 212 amino acids with a calculated molecular mass of 24428 Da. The product of the gene has been overexpressed in Escherichia coli and characterized. XlGSTP1-1 has one of the highest specific activities towards 1-chloro-2,4-dinitrobenzene (1310 micromol/min per mg of protein) obtained with any GST. A notable feature of XlGSTP1-1 is the presence in the H-site of Phe(111) and Pro(208) in place of tyrosine and glycine residues respectively, present in other mammalian Pi-class GSTs. Site-directed mutagenesis indicate that Phe(111) is involved in substrate specificity of XlGSTP1-1. We provide evidence showing that XlGSTP1-1 is present only in the embryo and its expression might be associated with cellular proliferation.