Combined Genotoxic Effects of a Polycyclic Aromatic Hydrocarbon (B(a)P) and an Heterocyclic Amine (PhIP) in Relation to Colorectal Carcinogenesis

Colorectal neoplasia is the third most common cancer worldwide. Environmental factors such as diet are known to be involved in the etiology of this cancer. Several epidemiological studies have suggested that specific neo-formed mutagenic compounds related to meat consumption are an underlying factor involved in the association between diet and colorectal cancer. Heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs) are known mutagens and possible human carcinogens formed at the same time in meat during cooking processes. We studied the genotoxicity of the model PAH benzo(a)pyrene (B(a)P) and HCA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), alone or in mixture, using the mouse intestinal cell line Apc^Min/+, mimicking the early step of colorectal carcinogenesis, and control Apc^+/+ cells. The genotoxicity of B(a)P and PhIP was investigated using both cell lines, through the quantification of B(a)P and PhIP derived DNA adducts, as well as the use of a genotoxic assay based on histone H2AX phosphorylation quantification. Our results demonstrate that heterozygous Apc mutated cells are more effective to metabolize B(a)P. We also established in different experiments that PhIP and B(a)P were more genotoxic on Apc^Min/+ cells compared to Apc^+/+. Moreover when tested in mixture, we observed a combined genotoxicity of B(a)P and PhIP on the two cell lines, with an increase of PhIP derived DNA adducts in the presence of B(a)P. Because of their genotoxic effects observed on heterozygous Apc mutated cells and their possible combined genotoxic effects, both B(a)P and PhIP, taken together, could be implicated in the observed association between meat consumption and colorectal cancer.     

The Calpain/Calpastatin System Has Opposing Roles in Growth and Metastatic Dissemination of Melanoma

Conventional calpains are ubiquitous cysteine proteases whose activity is promoted by calcium signaling and specifically limited by calpastatin. Calpain expression has been shown to be increased in human malignant cells, but the contribution of the calpain/calpastatin system in tumorigenesis remains unclear. It may play an important role in tumor cells themselves (cell growth, migration, and a contrario cell death) and/or in tumor niche (tissue infiltration by immune cells, neo-angiogenesis). In this study, we have used a mouse model of melanoma as a tool to gain further understanding of the role of calpains in tumor progression. To determine the respective importance of each target, we overexpressed calpastatin in tumor and/or host in isolation. Our data demonstrate that calpain inhibition in both tumor and host blunts tumor growth, while paradoxically increasing metastatic dissemination to regional lymph nodes. Specifically, calpain inhibition in melanoma cells limits tumor growth in vitro and in vivo but increases dissemination by amplifying cell resistance to apoptosis and accelerating migration process. Meanwhile, calpain inhibition restricted to host cells blunts tumor infiltration by immune cells and angiogenesis required for antitumor immunity, allowing tumor cells to escape tumor niche and disseminate. The development of highly specific calpain inhibitors with potential medical applications in cancer should take into account the opposing roles of the calpain/calpastatin system in initial tumor growth and subsequent metastatic dissemination.     

Subpopulations of Staphylococcus aureus Clonal Complex 121 Are Associated with Distinct Clinical Entities

We investigated the population structure of Staphylococcus aureus clonal complex CC121 by mutation discovery at 115 genetic housekeeping loci from each of 154 isolates, sampled on five continents between 1953 and 2009. In addition, we pyro-sequenced the genomes from ten representative isolates. The genome-wide SNPs that were ascertained revealed the evolutionary history of CC121, indicating at least six major clades (A to F) within the clonal complex and dating its most recent common ancestor to the pre-antibiotic era. The toxin gene complement of CC121 isolates was correlated with their SNP-based phylogeny. Moreover, we found a highly significant association of clinical phenotypes with phylogenetic affiliations, which is unusual for S. aureus. All isolates evidently sampled from superficial infections (including staphylococcal scalded skin syndrome, bullous impetigo, exfoliative dermatitis, conjunctivitis) clustered in clade F, which included the European epidemic fusidic-acid resistant impetigo clone (EEFIC). In comparison, isolates from deep-seated infections (abscess, furuncle, pyomyositis, necrotizing pneumonia) were disseminated in several clades, but not in clade F. Our results demonstrate that phylogenetic lineages with distinct clinical properties exist within an S. aureus clonal complex, and that SNPs serve as powerful discriminatory markers, able to identify these lineages. All CC121 genomes harboured a 41-kilobase prophage that was dissimilar to S. aureus phages sequenced previously. Community-associated MRSA and MSSA from Cambodia were extremely closely related, suggesting this MRSA arose in the region.     

COX-2-Derived Prostanoids and Oxidative Stress Additionally Reduce Endothelium-Mediated Relaxation in Old Type 2 Diabetic Rats

Endothelial dysfunction in resistance arteries alters end organ perfusion in type 2 diabetes. Superoxides and cyclooxygenase-2 (COX-2) derivatives have been shown separately to alter endothelium-mediated relaxation in aging and diabetes but their role in the alteration of vascular tone in old diabetic subjects is not clear, especially in resistance arteries. Consequently, we investigated the role of superoxide and COX-2-derivatives on endothelium-dependent relaxation in 3 and 12 month-old Zucker diabetic fatty (ZDF) and lean (LZ) rats. Mesenteric resistance arteries were isolated and vascular tone was investigated using wire-myography. Endothelium (acetylcholine)-dependent relaxation was lower in ZDF than in LZ rats (60 versus 84% maximal relaxation in young rats and 41 versus 69% in old rats). Blocking NO production with L-NAME was less efficient in old than in young rats. L-NAME had no effect in old ZDF rats although eNOS expression level in old ZDF rats was similar to that in old LZ rats. Superoxide level and NADPH-oxidase subunits (p67phox and gp91phox) expression level were greater in ZDF than in LZ rats and were further increased by aging in ZDF rats. In young ZDF rats reducing superoxide level with tempol restored acetylcholine-dependent relaxation to the level of LZ rats. In old ZDF rats tempol improved acetylcholine-dependent relaxation without increasing it to the level of LZ rats. COX-2 (immunolabelling and Western-blot) was present in arteries of ZDF rats and absent in LZ rats. In old ZDF rats arterial COX-2 level was higher than in young ZDF rats. COX-2 blockade with NS398 restored in part acetylcholine-dependent relaxation in arteries of old ZDF rats and the combination of tempol and NS398 fully restored relaxation in control (LZ rats) level. Accordingly, superoxide production and COX-2 derivatives together reduced endothelium-dependent relaxation in old ZDF rats whereas superoxides alone attenuated relaxation in young ZDF or old LZ rats.     

827Spatio-Temporal Quantification of FRET in Living Cells by Fast Time-Domain FLIM: A Comparative Study of Non-Fitting Methods

Förster Resonance Energy Transfer (FRET) measured with Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique to investigate spatio-temporal regulation of protein-protein interactions in living cells. When using standard fitting methods to analyze time domain FLIM, the correct estimation of the FRET parameters requires a high number of photons and therefore long acquisition times which are incompatible with the observation of dynamic protein-protein interactions. Recently, non-fitting strategies have been developed for the analysis of FLIM images: the polar plot or “phasor” and the minimal fraction of interacting donor mf_D. We propose here a novel non-fitting strategy based on the calculation of moments. We then compare the performance of these three methods when shortening the acquisition time: either by reducing the number of counted photons N or the number of temporal channels N_ch, which is particularly adapted for the original fast-FLIM prototype presented in this work that employs the time gated approach. Based on theoretical calculations, Monte Carlo simulations and experimental data, we determine the domain of validity of each method. We thus demonstrate that the polar approach remains accurate for a large range of conditions (low N, N_ch or small fractions of interacting donor f_D). The validity domain of the moments method is more restricted (not applicable when f_D<0.25 or when N_ch = 4) but it is more precise than the polar approach. We also demonstrate that the mf_D is robust in all conditions and it is the most precise strategy; although it does not strictly provide the fraction of interacting donor. We show using the fast-FLIM prototype (with an acquisition rate up to 1 Hz) that these non-fitting strategies are very powerful for on-line analysis on a standard computer and thus for quantifying automatically the spatio-temporal activation of Rac-GTPase in living cells by FRET.     

Identification of Ovarian Cancer Metastatic miRNAs

Serous epithelial ovarian cancer (EOC) patients often succumb to aggressive metastatic disease, yet little is known about the behavior and genetics of ovarian cancer metastasis. Here, we aim to understand how omental metastases differ from primary tumors and how these differences may influence chemotherapy. We analyzed the miRNA expression profiles of primary EOC tumors and their respective omental metastases from 9 patients using miRNA Taqman qPCR arrays. We find 17 miRNAs with differential expression in omental lesions compared to primary tumors. miR-21, miR-150, and miR-146a have low expression in most primary tumors with significantly increased expression in omental lesions, with concomitant decreased expression of predicted mRNA targets based on mRNA expression. We find that miR-150 and miR-146a mediate spheroid size. Both miR-146a and miR-150 increase the number of residual surviving cells by 2–4 fold when challenged with lethal cisplatin concentrations. These observations suggest that at least two of the miRNAs, miR-146a and miR-150, up-regulated in omental lesions, stimulate survival and increase drug tolerance. Our observations suggest that cancer cells in omental tumors express key miRNAs differently than primary tumors, and that at least some of these microRNAs may be critical regulators of the emergence of drug resistant disease.     

On the Use of a Test to Exhaustion Specific to Tennis (TEST) with Ball Hitting by Elite Players

PurposeWe aimed to a) introduce a new Test to Exhaustion Specific to Tennis (TEST) and compare performance (test duration) and physiological responses to those obtained during the 20-m multistage shuttle test (MSST), and b) determine to which extent those variables correlate with performance level (tennis competitive ranking) for both test procedures.MethodsTwenty-seven junior players (8 males, 19 females) members of the national teams of the French Tennis Federation completed MSST and TEST, including elements of the game (ball hitting, intermittent activity, lateral displacement), in a randomized order. Cardiorespiratory responses were compared at submaximal (respiratory compensation point) and maximal loads between the two tests.ResultsAt the respiratory compensation point oxygen uptake (50.1 ± 4.7 vs. 47.5 ± 4.3 mL.min^-1.kg^-1, p = 0.02), but not minute ventilation and heart rate, was higher for TEST compared to MSST. However, load increment and physiological responses at exhaustion did not differ between the two tests. Players’ ranking correlated negatively with oxygen uptake measured at submaximal and maximal loads for both TEST (r = -0.41; p = 0.01 and -0.55; p = 0.004) and MSST (r = -0.38; P = 0.05 and -0.51; p = 0.1).ConclusionUsing TEST provides a tennis-specific assessment of aerobic fitness and may be used to prescribe aerobic exercise in a context more appropriate to the game than MSST. Results also indicate that VO₂ values both at submaximal and maximal load reached during TEST and MSST are moderate predictors of players competitive ranking.