A new approach for evaluating in vivo anti-leukemic activity using the SCE assay. An application on three newly synthesised anti-tumour steroidal esters

Three newly synthesised steroidal esteric derivatives of nitrogen mustard (compounds 1-3) were comparatively studied on a molar basis regarding their ability to induce sister chromatid exchanges (SCEs) in normal human lymphocytes in vitro and therapeutic effects on leukemia P388 bearing mice. Compounds 1 and 3 are modified steroidal esters of p-methyl-m-N,N-bis(2-chloroethyl)amino benzoic acid, and compound 2 is a modified steroidal ester of chlorambucil. All compounds induced statistically significant increases in SCEs and decreases in proliferation rate indices (PRIs) of cultured human lymphocytes and significantly increased the life span of P388 bearing mice. In this study, the doses applied for therapeutic purposes upon leukemia P388 bearing mice in vivo were derived from cytogenetic observations in normal human lymphocytes in vitro. A substantially better therapeutic effect was obtained compared to the effect achieved after the use of quite higher doses related with LD(10) values. We have demonstrated that the order of anti-tumour effectiveness of the treatment schedules of the three newly synthesised compounds tested (at doses derived from cytogenetic observations) coincides with the order of the cytogenetic effects they induce. The SCE assay appears to have an application in the clinical prediction of tumour sensitivity to potential chemotherapeutics.     

Mannosylglycerate stabilizes staphylococcal nuclease with restriction of slow β-sheet motions

Mannosylglycerate is a compatible solute typical of thermophilic marine microorganisms that has a remarkable ability to protect proteins from thermal denaturation. This ionic solute appears to be a universal stabilizing agent, but the extent of protection depends on the specific protein examined. To understand how mannosylglycerate confers protection, we have been studying its influence on the internal motions of a hyperstable staphylococcal nuclease (SNase). Previously, we found a correlation between the magnitude of protein stabilization and the restriction of fast backbone motions. We now report the effect of mannosylglycerate on the fast motions of side-chains and on the slower unfolding motions of the protein. Side-chain motions were assessed by (13)CH(3) relaxation measurements and model-free analysis while slower unfolding motions were probed by H/D exchange measurements at increasing concentrations of urea. Side-chain motions were little affected by the presence of different concentrations of mannosylglycerate or even by the presence of urea (0.25M), and show no correlation with changes in the thermodynamic stability of SNase. Native hydrogen exchange experiments showed that, contrary to reports on other stabilizing solutes, mannosylglycerate restricts local motions in addition to the global motions of the protein. The protein unfolding/folding pathway remained undisturbed in the presence of mannosylglycerate but the solute showed a specific effect on the local motions of β-sheet residues. This work reinforces the link between solute-induced stabilization and restriction of protein motions at different timescales, and shows that the solute preferentially affects specific structural elements of SNase.     

Integration Analysis of Three Omics Data Using Penalized Regression Methods: An Application to Bladder Cancer

Omics data integration is becoming necessary to investigate the genomic mechanisms involved in complex diseases. During the integration process, many challenges arise such as data heterogeneity, the smaller number of individuals in comparison to the number of parameters, multicollinearity, and interpretation and validation of results due to their complexity and lack of knowledge about biological processes. To overcome some of these issues, innovative statistical approaches are being developed. In this work, we propose a permutation-based method to concomitantly assess significance and correct by multiple testing with the MaxT algorithm. This was applied with penalized regression methods (LASSO and ENET) when exploring relationships between common genetic variants, DNA methylation and gene expression measured in bladder tumor samples. The overall analysis flow consisted of three steps: (1) SNPs/CpGs were selected per each gene probe within 1Mb window upstream and downstream the gene; (2) LASSO and ENET were applied to assess the association between each expression probe and the selected SNPs/CpGs in three multivariable models (SNP, CPG, and Global models, the latter integrating SNPs and CPGs); and (3) the significance of each model was assessed using the permutation-based MaxT method. We identified 48 genes whose expression levels were significantly associated with both SNPs and CPGs. Importantly, 36 (75%) of them were replicated in an independent data set (TCGA) and the performance of the proposed method was checked with a simulation study. We further support our results with a biological interpretation based on an enrichment analysis. The approach we propose allows reducing computational time and is flexible and easy to implement when analyzing several types of omics data. Our results highlight the importance of integrating omics data by applying appropriate statistical strategies to discover new insights into the complex genetic mechanisms involved in disease conditions.     

Variants of the human PPARG locus and the susceptibility to chronic periodontitis

Apart from its regulatory function in lipid and glucose metabolism, peroxisome proliferator-activated receptor (PPAR)γ has impact on the regulation of inflammation and bone metabolism. The aim of the study was to investigate the association of five polymorphisms (rs10865710, rs2067819, rs3892175, rs1801282, rs3856806) within the PPARG gene with chronic periodontitis. The study population comprised 402 periodontitis patients and 793 healthy individuals. Genotyping of the PPARG gene polymorphisms was performed by PCR and melting curve analysis. Comparison of frequency distribution of genotypes between individuals with periodontal disease and healthy controls for the polymorphism rs3856806 showed a P-value of 0.04 but failed to reach significance after correction for multiple testing (P??0.90). A 3-site analysis (rs2067819-rs1801282-rs3856860) revealed five haplotypes with a frequency of ≥1% among cases and controls. Following adjustment for age, gender and smoking, none of the haplotypes was significantly different between periodontitis and healthy controls after Bonferroni correction. This study could not show a significant association between PPARG gene variants and chronic periodontitis.     

Respiratory and immune response to maximal physical exertion following exposure to secondhand smoke in healthy adults

We assessed the cardiorespiratory and immune response to physical exertion following secondhand smoke (SHS) exposure through a randomized crossover experiment. Data were obtained from 16 (8 women) non-smoking adults during and following a maximal oxygen uptake cycling protocol administered at baseline and at 0-, 1-, and 3- hours following 1-hour of SHS set at bar/restaurant carbon monoxide levels. We found that SHS was associated with a 12% decrease in maximum power output, an 8.2% reduction in maximal oxygen consumption, a 6% increase in perceived exertion, and a 6.7% decrease in time to exhaustion (P<0.05). Moreover, at 0-hours almost all respiratory and immune variables measured were adversely affected (P<0.05). For instance, FEV(1) values at 0-hours dropped by 17.4%, while TNF-α increased by 90.1% (P<0.05). At 3-hours mean values of cotinine, perceived exertion and recovery systolic blood pressure in both sexes, IL4, TNF-α and IFN-γ in men, as well as FEV(1)/FVC, percent predicted FEV(1), respiratory rate, and tidal volume in women remained different compared to baseline (P<0.05). It is concluded that a 1-hour of SHS at bar/restaurant levels adversely affects the cardiorespiratory and immune response to maximal physical exertion in healthy nonsmokers for at least three hours following SHS.