Potentiation of polarized intestinal Caco-2 cell responsiveness to probiotics complexed with secretory IgA.

The precise mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple consequences that remain poorly understood at the molecular level. Deciphering such events can provide valuable information as to the mode of action of commensal and probiotic microorganisms in the gastrointestinal environment. Potential roles of such microorganisms along the privileged target represented by the mucosal immune system include maturation prior, during and after weaning, and the reduction of inflammatory reactions in pathogenic conditions. Using human intestinal epithelial Caco-2 cell grown as polarized monolayers, we found that association of a Lactobacillus or a Bifidobacterium with nonspecific secretory IgA (SIgA) enhanced probiotic adhesion by a factor of 3.4-fold or more. Bacteria alone or in complex with SIgA reinforced transepithelial electrical resistance, a phenomenon coupled with increased phosphorylation of tight junction proteins zonula occludens-1 and occludin. In contrast, association with SIgA resulted in both enhanced level of nuclear translocation of NF-κB and production of epithelial polymeric Ig receptor as compared with bacteria alone. Moreover, thymic stromal lymphopoietin production was increased upon exposure to bacteria and further enhanced with SIgA-based complexes, whereas the level of pro-inflammatory epithelial cell mediators remained unaffected. Interestingly, SIgA-mediated potentiation of the Caco-2 cell responsiveness to the two probiotics tested involved Fab-independent interaction with the bacteria. These findings add to the multiple functions of SIgA and underscore a novel role of the antibody in interaction with intestinal bacteria.

Identification and characterization of novel classes of macrophage migration inhibitory factor (MIF) inhibitors with distinct mechanisms of action.

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC(50) values in the range of 0.2-15.5 microm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro(1); 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.

Addressing non-communicable diseases in the Seychelles: towards a comprehensive plan of action.

This article reviews the different steps taken during the past 20 years for the prevention and control of non-communicable diseases (NCDs) in the Seychelles. National surveys revealed high levels of several cardiovascular risk factors and prompted an organized response, starting with the creation of an NCD unit in the Ministry of Health. Information campaigns and nationwide activities raised awareness and rallied increasingly broad and high-level support. Significant policy was developed including comprehensive tobacco legislation and a School Nutrition Policy that bans soft drinks in schools. NCD guidelines were developed and specialized 'NCD nurses' were trained to complement doctors in district health centers. Decreasing smoking prevalence is evidence of success, but the raising so-called diabesity epidemic calls for an integrated multi-sector policy to mould an environment conducive to healthy behaviors. Essential components of these efforts include: effective surveillance mechanisms supplemented by focused research; generating broad interest and consensus; mobilizing leadership and commitment at all levels; involving local and international expertise; building on existing efforts; and seeking integrated, multi-disciplinary and multi-sector approaches.

Risk Assessement of Herbicide Mixtures

Lake Geneva is one of the largest European lakes with a surface area of 580 km2. Its catchment area covers 7400 km2, of which approximately 20% is arable land. Monitoring campaigns have been carried out in 2004 and 2005 to determine the contamination of the lake by pesticides. The results highlight the widespread presence of herbicides in water, the measured concentrations for most substances remaining constant in 2004 and 2005. However, for some individual herbicides the concentrations increased drastically (e.g., the herbicide foramsulfuron). We assessed the environmental risk of the herbicides detected in the lake using water quality criteria recently determined for the Swiss environmental protection agency. Furthermore, we assessed the risk of herbicide mixtures, grouped based upon their mode of action. Generally, the risk estimated for all single substances is low, except for some sulfonylurea compounds. For these substances, the measured concentrations are higher than the predicted no-effect concentration. Impact on the flora of the lake can therefore not be excluded. When mixtures of pesticides with similar mode of action are taken into account, the risk remains lower than the mixture water quality criteria for all groups, but can reach as high as one third of this quality criteria. A further step would therefore be to assess the risk of the total pesticide mixture, including similar and dissimilar modes of action.

Indications, perspectives et limites de la stimulation électrique épidurale en cas d'artériopathie périphérique ou coronarienne [Indications, limits and future of epidural spinal cord electrical stimulation for coronary and peripheral artery disease]

The electrical stimulation of the dorsal columns of the spinal cord exerts a dual analgesic and vasodilatory effect on ischemic tissues. It is increasingly considered a valuable method to treat severe and otherwise intractable coronary and peripheral artery disease. The quality of the results depends from both a strict selection of the patients by vascular specialists and the frequency and quality of the follow-up controls. However the indications, limits, mode of action and results of spinal cord stimulation are still poorly understood. This article, based on a personal experience of 164 implantations for peripheral and coronary artery disease, aims to draw attention to this technique and to provide information on recent and future developments.

Integrating nuclear receptor mobility in models of gene regulation.

The mode of action of nuclear receptors in living cells is an actively investigated field but much remains hypothetical due to the lack, until recently, of methods allowing the assessment of molecular mechanisms in vivo. However, these last years, the development of fluorescence microscopy methods has allowed initiating the dissection of the molecular mechanisms underlying gene regulation by nuclear receptors directly in living cells or organisms. Following our analyses on peroxisome proliferator activated receptors (PPARs) in living cells, we discuss here the different models arising from the use of these tools, that attempt to link mobility, DNA binding or chromatin interaction, and transcriptional activity.

Comprehensive approach for the detection of antifungal compounds using a susceptible strain of Candida albicans and confirmation of in vivo activity with the Galleria mellonella model.

An efficient screening strategy for the identification of potentially interesting low-abundance antifungal natural products in crude extracts that combines both a sensitive bioautography assay and high performance liquid chromatography (HPLC) microfractionation was developed. This method relies on high performance thin layer chromatography (HPTLC) bioautography with a hypersusceptible engineered strain of Candida albicans (DSY2621) for bioactivity detection, followed by the evaluation of wild type strains in standard microdilution antifungal assays. Active extracts were microfractionated by HPLC in 96-well plates, and the fractions were subsequently submitted to the bioassay. This procedure enabled precise localisation of the antifungal compounds directly in the HPLC chromatograms of the crude extracts. HPLC-PDA-mass spectrometry (MS) data obtained in parallel to the HPLC antifungal profiles provided a first chemical screening about the bioactive constituents. Transposition of the HPLC analytical conditions to medium-pressure liquid chromatography (MPLC) allowed the efficient isolation of the active constituents in mg amounts for structure confirmation and more extensive characterisation of their biological activities. The antifungal properties of the isolated natural products were evaluated by their minimum inhibitory concentration (MIC) in a dilution assay against both wild type and engineered strains of C. albicans. The biological activity of the most promising agents was further evaluated in vitro by electron microscopy and in vivo in a Galleria mellonella model of C. albicans infection. The overall procedure represents a rational and comprehensive means of evaluating antifungal activity from various perspectives for the selection of initial hits that can be explored in more in-depth mode-of-action studies. This strategy is illustrated by the identification and bioactivity evaluation of a series of antifungal compounds from the methanolic extract of a Rubiaceae plant, Morinda tomentosa, which was used as a model in these studies.

Physiological ligands of PPARs in inflammation and lipid homeostasis

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that are involved in many physiological processes, such as inflammation and energy homeostasis. In general, PPARs must be activated by ligands to stimulate the expression of their target genes. These agonists can be synthetic molecules, such as drugs used to treat hypertriglyceridemia and insulin resistance, or natural physiological ligands, such as fatty acids and eicosanoids. Although recent work has uncovered a surprisingly broad variety of natural molecules capable of activating PPARs, relatively little is known about their mode of action in an in vivo physiological context. The action of physiological ligands in situations of food deprivation and abundance, especially with respect to their intervention in the inflammatory response, and in both lipid homeostasis and inflammation resolution will be reviewed.

Cilengitide modulates attachment and viability of human glioma cells, but not sensitivity to irradiation or temozolomide in vitro.

Cilengitide is a cyclic peptide antagonist of integrins alphavbeta3 and alphavbeta5 that is currently being evaluated as a novel therapeutic agent for recurrent and newly diagnosed glioblastoma. Its mode of action is thought to be mainly antiangiogenic but may include direct effects on tumor cells, notably on attachment, migration, invasion, and viability. In this study we found that, at clinically relevant concentrations, cilengitide (1-100 microM) induces detachment in some but not all glioma cell lines, while the effect on cell viability is modest. Detachment induced by cilengitide could not be predicted by the level of expression of the cilengitide target molecules, alphavbeta3 and alphavbeta5, at the cell surface. Glioma cell death induced by cilengitide was associated with the generation of caspase activity, but caspase activity was not required for cell death since ectopic expression of cytokine response modifier (crm)-A or coexposure to the broad-spectrum caspase inhibitor zVAD-fmk was not protective. Moreover, forced expression of the antiapoptotic protein marker Bcl-X(L) or altering the p53 status did not modulate cilengitide-induced cell death. No consistent effects of cilengitide on glioma cell migration or invasiveness were observed in vitro. Preliminary clinical results indicate a preferential benefit from cilengitide added to temozolomide-based radiochemotherapy in patients with O(6)-methylguanine DNA methyltransferase (MGMT) gene promoter methylation. Accordingly, we also examined whether the MGMT status determines glioma cell responses to cilengitide alone or in combination with temozolomide. Neither ectopic expression of MGMT in MGMT-negative cells nor silencing the MGMT gene in MGMT-positive cells altered glioma cell responses to cilengitide alone or to cilengitide in combination with temozolomide. These data suggest that the beneficial clinical effects derived from cilengitide in vivo may arise from altered perfusion, which promotes temozolomide delivery to glioma cells.

Entre résistance et contestation. La genèse du squat comme mode d'action

Cette contribution vise plusieurs objectifs théoriques : questionner les implicites du concept de répertoire d'action pour proposer une typification des modes d'action, indiquer des pistes pour une analyse conjointe des répertoires instrumentaux et discursifs mis en oeuvre par les organisations, reconsidérer les ruptures historiques mises au jour par Charles Tilly, et enfin réfléchir aux conditions organisationnelles et contextuelles favorisant l'usage de formes d'actions directes. Elle se fonde empiriquement sur l'étude de la genèse d'un mode d'action spécifique, le squat, entre 1880 et 1914, au moyen d'un travail d'archives et d'un travail d'étymologie. On montre notamment que le passage de la « résistance » à la « contestation », dans laquelle prédomine le « revendiqué », suppose une forme particulière de rapport au politique qui, au début de ce siècle, constituait en soi un enjeu de luttes important entre différents groupes.

Inferring the mode of origin of polyploid species from next-generation sequence data.

Many eukaryote organisms are polyploid. However, despite their importance, evolutionary inference of polyploid origins and modes of inheritance has been limited by a need for analyses of allele segregation at multiple loci using crosses. The increasing availability of sequence data for nonmodel species now allows the application of established approaches for the analysis of genomic data in polyploids. Here, we ask whether approximate Bayesian computation (ABC), applied to realistic traditional and next-generation sequence data, allows correct inference of the evolutionary and demographic history of polyploids. Using simulations, we evaluate the robustness of evolutionary inference by ABC for tetraploid species as a function of the number of individuals and loci sampled, and the presence or absence of an outgroup. We find that ABC adequately retrieves the recent evolutionary history of polyploid species on the basis of both old and new sequencing technologies. The application of ABC to sequence data from diploid and polyploid species of the plant genus Capsella confirms its utility. Our analysis strongly supports an allopolyploid origin of C. bursa-pastoris about 80 000 years ago. This conclusion runs contrary to previous findings based on the same data set but using an alternative approach and is in agreement with recent findings based on whole-genome sequencing. Our results indicate that ABC is a promising and powerful method for revealing the evolution of polyploid species, without the need to attribute alleles to a homeologous chromosome pair. The approach can readily be extended to more complex scenarios involving higher ploidy levels.

Epigenetic regulatory elements associate with specific histone modifications to prevent silencing of telomeric genes.

In eukaryotic cells, transgene expression levels may be limited by an unfavourable chromatin structure at the integration site. Epigenetic regulators are DNA sequences which may protect transgenes from such position effect. We evaluated different epigenetic regulators for their ability to protect transgene expression at telomeres, which are commonly associated to low or inconsistent expression because of their repressive chromatin environment. Although to variable extents, matrix attachment regions (MARs), ubiquitous chromatin opening element (UCOE) and the chicken cHS4 insulator acted as barrier elements, protecting a telomeric-distal transgene from silencing. MARs also increased the probability of silent gene reactivation in time-course experiments. Additionally, all MARs improved the level of expression in non-silenced cells, unlike other elements. MARs were associated to histone marks usually linked to actively expressed genes, especially acetylation of histone H3 and H4, suggesting that they may prevent the spread of silencing chromatin by imposing acetylation marks on nearby nucleosomes. Alternatively, an UCOE was found to act by preventing deposition of repressive chromatin marks. We conclude that epigenetic DNA elements used to enhance and stabilize transgene expression all have specific epigenetic signature that might be at the basis of their mode of action.

Molecular characterization of a human matrix attachment region epigenetic regulator.

Matrix attachment regions (MAR) generally act as epigenetic regulatory sequences that increase gene expression, and they were proposed to partition chromosomes into loop-forming domains. However, their molecular mode of action remains poorly understood. Here, we assessed the possible contribution of the AT-rich core and adjacent transcription factor binding motifs to the transcription augmenting and anti-silencing effects of human MAR 1-68. Either flanking sequences together with the AT-rich core were required to obtain the full MAR effects. Shortened MAR derivatives retaining full MAR activity were constructed from combinations of the AT-rich sequence and multimerized transcription factor binding motifs, implying that both transcription factors and the AT-rich microsatellite sequence are required to mediate the MAR effect. Genomic analysis indicated that MAR AT-rich cores may be depleted of histones and enriched in RNA polymerase II, providing a molecular interpretation of their chromatin domain insulator and transcriptional augmentation activities.