Biological and clinical relevance of quantitative global methylation of repetitive DNA sequences in chronic lymphocytic leukemia

Global DNA hypomethylation affecting repeat sequences has been reported in different cancer types. Herein, we investigated the methylation levels of repetitive DNA elements in chronic lymphocytic leukemia (CLL), their correlation with the major cytogenetic and molecular features, and clinical relevance in predicting therapy-free survival (TFS). A quantitative bisulfite-PCR Pyrosequencing method was used to evaluate methylation of Alu, long interspersed nuclear elements-1 (LINE-1) and satellite-α (SAT-α) sequences in 77 untreated early-stage (Binet A) CLL patients. Peripheral B-cells from 7 healthy donors were used as controls. Methylation levels (median %5mC) were lower in B-CLLs compared with controls (21.4 vs. 25.9; 66.8 vs. 85.7; 84.0, vs. 88.2 for Alu, LINE-1 and SAT-α, respectively) (p < 0.001). Among CLL patients, a significant association was observed with 17p13.1 deletion (16.8 vs. 22.4; 51.2 vs. 68.5; 52.6 vs. 85.0, for Alu, LINE-1 and SAT-α) but not with other major genetic lesions, IgVH mutation status, CD38 or ZAP-70 expression. Follow-up analyses showed that lower SAT-α methylation levels appeared to be an independent prognostic marker significantly associated with shorter TFS. Our study extended previous limited evidences in methylation of repetitive sequences in CLL suggesting an important biological and clinical relevance in the disease.     

Efficacy of antihyperglycemic therapies and the influence of baseline hemoglobin A(1C): a meta-analysis of the liraglutide development program

ObjectiveTo compare Iiraglutide versus common antihyperglycemic treatments in reducing hemoglobin A_1c (A1C) values across multiple levels of baseline glycemic control and in reaching glycemic targets.MethodsPooled patient data from 7 phase 3, multinational, randomized controlled trials in patients with type 2 diabetes were stratified by baseline A1C values into 5 categories: ≤ 7.5%, > 7.5% to 8.0%, > 8.0% to 8.5%, > 8.5% to 9.0%, and > 9.0%. The changes in A1C from baseline to week 26 of treatment and patient proportions reaching A1C targets of < 7.0% and ≤ 6.5% were compared between liraglutide (1.8 mg daily) and sitagliptin, glimepiride, rosiglitazone, exenatide, and insulin glargine across all baseline A1C categories.ResultsIrrespective of treatment, reductions in A1C levels were generally greater in groups with higher baseline A1C values. After 26 weeks of treatment, liraglutide produced the greatest reductions in A1C values across all baseline categories, ranging from 0.7% to 1.8% (baseline A1C categories ≤ 7.5% to > 9.0%, respectively), followed by insulin glargine (0.3% to 1.5%) and then by glimepiride (0.4% to 1.3%). Generally, larger percentages of patients achieved the A1C target of ≤ 6.5% with liraglutide therapy across all baseline categories (from 62% of patients with A1C values ≤ 7.5% to 10% of patients with A1C values > 9.0%) in comparison with other treatments (ranging from 49% to 0% of patients, respectively). Similarly, greater proportions of patients also reached the A1C target of < 7.0% with liraglutide therapy across all baseline categories (from 83% of patients with A1C values ≤ 7.5% to 25% of patients with A1C values > 9.0%) versus comparators (from 74% to 5% of patients, respectively).ConclusionAcross a wide spectrum of baseline A1C categories, liraglutide is an efficacious treatment option for patients with type 2 diabetes. (Endocr Pract. 2011;17: 906-913)     

Proteomic analysis of cellular response to novel proapoptotic agents related to atypical retinoids in human IGROV-1 ovarian carcinoma cells

Novel agents characterized by the scaffold of the atypical retinoid ST1926, but containing different chemical functions (carboxylic or hydroxamic acid), exhibit potent proapoptotic activity. In the present paper, we show that the treatment of the IGROV-1 ovarian cancer cell line with compounds sharing structural features with ST1926 (ST1898, ST3595, ST3056) determines a strong inhibition of proliferation mainly due to apoptotic cell death. In an effort to understand the mechanism of action of these compounds, we performed a proteomics analysis of IGROV-1 total lysates and nuclear extracts. Using this approach, we found that deregulation of calcium homeostasis, oxidative stress, cytoskeleton reorganization, and deregulation of proteasome function may represent important pathways involved in response of IGROV-1 cells to the studied compounds. The most prominent effect was down-regulation of factors involved in protein degradation, an event more marked in cells treated with ST3595. In addition, we identified proteins specifically modulated by each treatment, including prohibitin and cochaperone P23 (ST1898), pre-mRNA splicing factor SF2p32 and clathrin light chain (ST3595), as well as Far upstream element (FUSE) binding protein 1 and DNA-binding protein B (ST3056). By identifying proteins modulated by novel proapoptotic agents, this study provides insights into critical aspects of their mechanism of action.     

Development of a classification and ranking method for the identification of possible biomarkers in two-dimensional gel-electrophoresis based on principal component analysis and variable selection procedures

The identification of biomarkers is one of the leading research areas in proteomics. When biomarkers have to be searched for in spot volume datasets produced by 2D gel-electrophoresis, problems may arise related to the large number of spots present in each map and the small number of samples available in each class (control/pathological). In such cases multivariate methods are usually exploited together with variable selection procedures, to provide a set of possible biomarkers: they are however usually aimed to the selection of the smallest set of variables (spots) providing the best performances in prediction. This approach seems not to be suitable for the identification of potential biomarkers since in this case all the possible candidate biomarkers have to be identified to provide a general picture of the "pathological state": in this case exhaustivity has to be preferred to provide a complete understanding of the mechanisms underlying the pathology. We propose here a ranking and classification method, "Ranking-PCA", based on Principal Component Analysis and variable selection in forward search: the method selects one variable at a time as the one providing the best separation of the two classes investigated in the space given by the relevant PCs. The method was applied to an artificial dataset and a real case-study: Ranking-PCA exhaustively identified the potential biomarkers and provided reliable and robust results.     

Fusarium oxysporum degradation and detoxification of a new textile-glycoconjugate azo dye (GAD)

Degradation and detoxification of textile dyes are of interest due to the huge environmental impact of such chemicals. An isolate of Fusarium oxysporum was used to degrade and to detoxify a new chemical class of textile dyes called Glycoconjugate Azo Dye (GAD). After 6 d of growth in a liquid batch culture, the fungus degraded the dye and the culture medium at the end of incubation period showed a ?100% detoxification compared to the initial dye solution. Increasing the initial fungal inoculum, the dye was totally decolourized after 24 h of incubation. The degradation ability was found to be common among various isolates of F. oxysporum suggesting this as a specific trait of this species. Degrading rate was enhanced in concomitancy to the glucose depletion and the beginning of the stationary phase of growth, suggesting that the shift from the primary to the secondary metabolism may be the trigger of the degradation pathway. The Daphnia magna acute toxicity test demonstrated a strong detoxification of GAD-4 by F. oxysporum, resulting in non-toxic metabolite production. Fusarium oxysporum could, therefore, be taken into consideration to develop new remediation strategies of textile effluents.     

HF-EPR, Raman, UV/VIS light spectroscopic, and DFT studies of the ribonucleotide reductase R2 tyrosyl radical from Epstein-Barr virus

Epstein-Barr virus (EBV) belongs to the gamma subfamily of herpes viruses, among the most common pathogenic viruses in humans worldwide. The viral ribonucleotide reductase small subunit (RNR R2) is involved in the biosynthesis of nucleotides, the DNA precursors necessary for viral replication, and is an important drug target for EBV. RNR R2 generates a stable tyrosyl radical required for enzymatic turnover. Here, the electronic and magnetic properties of the tyrosyl radical in EBV R2 have been determined by X-band and high-field/high-frequency electron paramagnetic resonance (EPR) spectroscopy recorded at cryogenic temperatures. The radical exhibits an unusually low g₁-tensor component at 2.0080, indicative of a positive charge in the vicinity of the radical. Consistent with these EPR results a relatively high C-O stretching frequency associated with the phenoxyl radical (at 1508 cm⁻¹) is observed with resonance Raman spectroscopy. In contrast to mouse R2, EBV R2 does not show a deuterium shift in the resonance Raman spectra. Thus, the presence of a water molecule as a hydrogen bond donor moiety could not be identified unequivocally. Theoretical simulations showed that a water molecule placed at a distance of 2.6 Å from the tyrosyl-oxygen does not result in a detectable deuterium shift in the calculated Raman spectra. UV/VIS light spectroscopic studies with metal chelators and tyrosyl radical scavengers are consistent with a more accessible dimetal binding/radical site and a lower affinity for Fe²⁺ in EBV R2 than in Escherichia coli R2. Comparison with previous studies of RNR R2s from mouse, bacteria, and herpes viruses, demonstrates that finely tuned electronic properties of the radical exist within the same RNR R2 Ia class.     

In-depth proteomic analysis of non-alcoholic beverages with peptide ligand libraries. I: Almond milk and orgeat syrup

Combinatorial peptide ligand libraries, both commercial and home-made, have been adopted to investigate the proteome of non-alcoholic beverages, in order to assess their genuineness and detect also trace proteins, in search of potential allergens. Two such beverages have been studied: almond milk and orgeat syrup. In the first product we have been able to identify 132 unique protein species, the deepest investigation so far of the almond proteome. In the second beverage, a handful of proteins (just 14) have been detected, belonging to a bitter almond extract. In both cases, the genuineness of such products has been verified, as well as the fact that almond milk, judging on the total protein and fat content, must have been produced with 100g ground almonds per litre of beverage, as required by authorities. On the contrary, cheap orgeat syrups produced by local supermarkets and sold as their own brands, where found not to contain any residual proteins, suggesting that they contained only synthetic aromas and no natural plant extracts. This could be the starting point for investigating the myriad of beverages that in the last decades have invaded the shelves of supermarkets the world over, whose genuineness and natural origin have never been properly assessed.