Reciprocal Regulation of Shh Trafficking and H2O2 Levels via a Noncanonical BOC-Rac1 Pathway.

Among molecules that bridge environment, cell metabolism, and cell signaling, hydrogen peroxide (H2O2) recently appeared as an emerging but central player. Its level depends on cell metabolism and environment and was recently shown to play key roles during embryogenesis, contrasting with its long-established role in disease progression. We decided to explore whether the secreted morphogen Sonic hedgehog (Shh), known to be essential in a variety of biological processes ranging from embryonic development to adult tissue homeostasis and cancers, was part of these interactions. Here, we report that H2O2 levels control key steps of Shh delivery in cell culture: increased levels reduce primary secretion, stimulate endocytosis and accelerate delivery to recipient cells; in addition, physiological in vivo modulation of H2O2 levels changes Shh distribution and tissue patterning. Moreover, a feedback loop exists in which Shh trafficking controls H2O2 synthesis via a non-canonical BOC-Rac1 pathway, leading to cytoneme growth. Our findings reveal that Shh directly impacts its own distribution, thus providing a molecular explanation for the robustness of morphogenesis to both environmental insults and individual variability.

The role of organization of care in GPs' prevention practice.

BACKGROUND: General practitioners (GPs) do not systematically include preventive recommendations in their practice, and some characteristics of health care organization are associated with more systematic prevention. But the characteristics of health care organization may act in a nonuniform manner depending on the type of preventive care. Thus, one characteristic can be positively associated with one type of preventive care and negatively associated with another. Our aim was to investigate the association between health care organization in general practice and different areas of preventive care (immunization and addiction prevention), in search of nonuniform associations. METHODS: We used a representative survey of 1,813 French GPs conducted in 2009. Four preventive care practices were studied: immunization through flu and HPV vaccination, and prevention of addictive behaviors concerning tobacco and alcohol use.Characteristics of GPs' health care organization and the social context of their practice were collected (spatial accessibility to GPs and socioeconomic level of the area of practice). We constructed mixed models to study associations and interactions between the organization variables and preventive care. RESULTS: Four out of five characteristics of GPs' organization have uneven impacts on different types of preventive care (p-interaction < 10-4). For example, number of daily consultations is associated with better immunization prevention but with poorer prevention counseling in addictive behaviors. In contrast, working with digital medical files is uniformly associated with both types of preventive care (OR = 1.29 [1.15-1.45]; P < 10-4). CONCLUSION: An approach centered on specific types of preventive care should help deepen our understanding of prevention and possibly help to identify a new typology for preventive care.

Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells.

Real-time imaging of bacterial virulence factor dynamics is hampered by the limited number of fluorescent tools suitable for tagging secreted effectors. Here, we demonstrated that the fluorogenic reporter FAST could be used to tag secreted proteins, and we implemented it to monitor infection dynamics in epithelial cells exposed to the human pathogen Listeria monocytogenes (Lm). By tracking individual FAST-labelled vacuoles after Lm internalisation into cells, we unveiled the heterogeneity of residence time inside entry vacuoles. Although half of the bacterial population escaped within 13 minutes after entry, 12% of bacteria remained entrapped over an hour inside long term vacuoles, and sometimes much longer, regardless of the secretion of the pore-forming toxin listeriolysin O (LLO). We imaged LLO-FAST in these long-term vacuoles, and showed that LLO enabled Lm to proliferate inside these compartments, reminiscent of what had been previously observed for Spacious Listeria-containing phagosomes (SLAPs). Unexpectedly, inside epithelial SLAP-like vacuoles (eSLAPs), Lm proliferated as fast as in the host cytosol. eSLAPs thus constitute an alternative replication niche in epithelial cells that might promote the colonization of host tissues.

HCV and HBV prevalence based on home blood self-sampling and screening history in the general population in 2016: contribution to the new French screening strategy.

BACKGROUND: The advent of effective direct-acting antivirals (DAAs), has prompted an assessment of the French Hepatitis C virus (HCV) screening strategy, which historically targeted high-risk groups. One of the options put forward is the implementation of combined (i.e., simultaneous) HCV, Hepatitis B virus (HBV) and HIV screening for all adults at least once during their lifetime ("universal combined screening"). However, recent national survey-based data are lacking to guide decision-making regarding which new strategy to implement. Accordingly, we aimed to provide updated data for both chronic hepatitis C (CHC) and B (CHB) prevalence and for HCV and HBV screening history, using data from the BaroTest and 2016 Health Barometer (2016-HB) studies, respectively. METHODS: 2016-HB was a national cross-sectional phone based health survey conducted in 2016 among 20,032 randomly selected individuals from the general population in mainland France. BaroTest was a virological sub-study nested in 2016-HB. Data collected for BaroTest were based on home blood self-sampling on dried blood spots (DBS). RESULTS: From 6945 analyzed DBS, chronic hepatitis C (CHC) and B (CHB) prevalence was estimated at 0.30% (95% Confidence Interval (CI): 0.13-0.70) and 0.30% (95% CI: 0.13-0.70), respectively. The proportion of individuals aware of their status was estimated at 80.6% (95% CI: 44.2-95.6) for CHC and 17.5% (95% CI: 4.9-46.4) for CHB. Universal combined screening would involve testing between 32.6 and 85.3% of 15-75 year olds according to whether we consider only individuals not previously tested for any of the three viruses, or also those already tested for one or two of the viruses. CONCLUSIONS: Our data are essential to guide decision-making regarding which new HCV screening recommendation to implement in France. They also highlight that efforts are still needed to achieve the WHO's targets for eliminating these diseases. Home blood self-sampling may prove to be a useful tool for screening and epidemiological studies.

General practitioners who never perform Pap smear: the medical offer and the socio-economic context around their office could limit their involvement in cervical cancer screening.

BACKGROUND: In France, with the growing scarcity of gynecologists and a globally low and socially differentiated coverage of cervical cancer screening (CCS), general practitioners (GPs) are valuable resources to improve screening services for women. Still all GPs do not perform Pap smears. In order to promote this screening among GPs, the characteristics of physicians who never perform CCS should be more precisely specified. Besides already-known individual characteristics, the contextual aspects of the physicians' office, such as gynecologist density in the area, could shape GPs gynecological activities. METHODS: To analyze county (département) characteristics of GPs' office associated with no performance of CCS, we used a representative sample of 1063 French GPs conducted in 2009 and we constructed mixed models with two levels, GP and county. RESULTS: Almost 35% (n = 369) of the GPs declared never performing CCS. GPs working in counties with a poor GP-density per inhabitants were more likely to perform CCS (odds ratio (OR) = 0.52 for each increase of density by 1 GP per 10,000 inhabitants, 95% confidence interval (CI) = 0.37-0.74). On the contrary, GPs working in counties with an easier access to a gynecologist were more likely not to perform CCS (OR = 1.06 for each increase of density by 1 gynecologist per 100,000 women, 95%CI = 1.03-1.10 and OR = 2.02 if the first gynecologist is reachable in less than 15 min, 95%CI = 1.20-3.41) as well as GPs working in areas with a poverty rate above the national average (OR = 1.66, 95%CI = 1.09-2.54). These contextual characteristics explain most of the differences between counties concerning rates of not performing CCS. CONCLUSIONS: Specific programs should be developed for GPs working in contexts unfavorable to their involvement in CCS.

Single-Molecule Localization Microscopy with the Fluorescence-Activating and Absorption-Shifting Tag (FAST) System.

We develop and employ the Fluorescence-Activating and absorption-Shifting Tag (FAST) system for super-resolution (SR) imaging and single-molecule tracking based on single-molecule localizations. The fast off rate of fluorogen binding, combined with its spatially well-separated labeling of the densely expressed FAST fusion proteins, allowed single-molecule measurements to be performed in both living and fixed cells. The well-separated fluorescence localization density was achieved by either reversibly controlling the fluorogen concentration or by irreversibly photobleaching the FAST-fluorogen complex. The experimentally determined resolution of 28 nm allowed us to resolve Ensconsin-labeled microtubules and to track single molecules in mitochondria. Our results demonstrate that FAST is well-suited for single-molecule localization microscopy (SMLM). The small size and the availability of spectrally distinct fluorogens present unique advantages of the FAST system as a potential orthogonal labeling strategy that could be applied in conjunction with existing super-resolution dyes and photoactivatable proteins in versatile imaging applications.

Live cell super resolution imaging by radial fluctuations using fluorogen binding tags.

Fluorescence-Activating and absorption-Shifting Tag (FAST) is a novel genetically encoded optical highlighter probe. Since the fluorescence of FAST originates from the stochastic and reversible diffusive association of a fluorogenic ligand, we investigate the application of FAST using Super-Resolution Radial Fluctuations (SRRF) to achieve routine imaging below the diffraction limit in a widefield epifluorescence microscope. We show that intensity fluctuation analysis like SRRF allows the imaging of FAST-tagged proteins with sub - 100 nm resolution in live cells. FAST co-labeled with conventional fluorophores enables real time multicolour 2D and 3D super-resolution imaging, indicating that FAST can be used for the observation of sub-diffraction limited structures in both living and fixed samples.

Vaccine hesitancy in the French population in 2016, and its association with vaccine uptake and perceived vaccine risk-benefit balance.

BackgroundVaccine hesitancy (VH) is prominent in France. Objectives: This study aimed to estimate the prevalence and socio-demographic correlates of VH in sub-groups of the French population and to investigate the association of VH with both vaccine uptake and perceived risk-benefit balance (RBB) for four vaccines. Methods: During the 2016 Health Barometer - a national cross-sectional telephone survey in a representative sample of the French population - parents of 1-15 year-old children, parents of 11-15 year-old girls and elderly people aged 65-75 years were asked about VH (using three questions adapted from the World Health Organization definition), vaccine uptake and perceived RBB for measles and hepatitis B (children's parents), human papillomavirus (girls' parents) and seasonal influenza (elderly people) vaccines. Results: A total of 3,938 parents including 959 girls' parents - and 2,418 elderly people were interviewed. VH prevalence estimates were 46% (95% confidence interval (CI): 44-48) among parents, 48% (95%CI: 45-51) among girls' parents and 35% (95% CI: 33-36) among elderly people, with higher estimates associated with high education level, children's age (10-15 years), and, for the elderly, poor perception of health status. VH was associated with uncertainty about and/or an unfavourable perception of vaccines' RBB for the four vaccines and with lower self-reported vaccine uptake, except for human papillomavirus vaccine in girls. Results were confirmed by multivariable analysis. Conclusion: Further research is needed to study the association between VH and vaccine uptake for other vaccines, and to design and validate measurement tools to monitor VH over time.

Contextual determinants of participation in cervical cancer screening in France, 2010.

BACKGROUND: Some contextual factors associated with participation in cervical cancer screening are reported in the literature, but few studies have examined their combined effect. Our objective was to assess the role of contextual characteristics, separately and in combination, in participation in cervical cancer screening in France. METHODS: Marginal Poisson regression models - taking into account the correlation between women in a given commune - were conducted using data from the Baromètre Santé 2010 survey. The characteristics of the commune of residence of the women studied were the potential spatial accessibility to general practitioners (GP) and gynecologists, the agglomeration category, and the socioeconomic level. RESULTS: The analyses were performed in 3380 women, 88.2% of whom were up to date with their cervical cancer screening. Once the individual characteristics were taken into account, the screening participation rate was similar in all the communes, with the exception of those with poor access to a gynecologist and good access to a GP, where the rate was 6% lower (95%CI: 0.5-11%) than in the communes with good access to both GP and gynecologist. The same association with accessibility was observed in small agglomerations. Compared to women living in the more advantaged communes, the screening participation rate was 8% (2-12%) lower in those living in the more disadvantaged ones, except when accessibility to both types of physician was high. DISCUSSION: We observed an association between potential spatial accessibility to care in women's residential communities and their cervical cancer screening practices, in particular in small agglomerations, rural communes, and more disadvantaged communes.

Disengagement of general practitioners in cervical cancer screening.

In the absence of organized cervical cancer screening (CCS) programs, gynecologists remain principal actors in obtaining a Pap smear, followed by general practitioners (GPs). In France, with the growing scarcity of gynecologists and social inequalities in access to opportunistic screening, GPs are valuable resources for women's gynecologic follow-up. We aimed to investigate the characteristics of GPs who do not perform CCS, analyzing the effect of GPs' sex and their evolution over time. On the basis of data from three cross-sectional surveys conducted among representative samples of French GPs in 1998, 2002, and 2009 (n=5199), we constructed univariate and multivariate logistic mixed models (level 2: county, level 1: GP) with random intercept stratified on GPs' sex to investigate the characteristics of the GPs associated with no practice of CCS ever. Almost one-third of all GPs did not perform CCS ever and it increased with time. Male GPs were always more likely not to perform it (odds ratio=0.50, 95% confidence interval=0.42-0.59). The percentage of GPs not performing CCS increased more markedly among male than among female GPs, and increased more among the youngest age group. Increasingly fewer GPs engage in CCS when the growing scarcity of medical gynecologists calls for more participation. Female GPs remain significantly more active in CCS than male GPs. The participation in CCS is determined differently according to the practitioner's sex.

'Auto-click' functionalization for diversified copper(I) and gold(I) NHCs.

Azide-tagged Cu(I)-NHC reacts in an 'auto-click' process to furnish complexes functionalized by 1,2,3-triazoles bearing diverse substituents. The resulting Cu(I) complexes are amenable to further transmetallation to Au(I). The whole strategy proceeds with mild conditions and constitutes an efficient entry to functionalised metal-NHCs with biorelevant moieties.

Hepatitis B knowledge, perceptions and practices in the French general population: the room for improvement.

BACKGROUND: Little is known about the knowledge, perceptions and prevention practices of the French general population with respect to Hepatitis B virus (HBV) infection. This article describes this population's knowledge of HBV, their perceptions of the disease, and associated screening and vaccination practices. It compares these indicators with those observed in the same population for HIV, an infection with a chronic course and transmission modes resembling those of HBV. METHODS: A module on hepatitis B was added into the HIV KABP (Knowledge, Attitudes, Beliefs and Practices) survey which was carried out telephonically in 2010 among a random sample of 9,014 individuals aged between 18-69 and living in metropolitan France. RESULTS: Compared with HIV, the general population was less aware that needle exchange during intravenous drug use and sexual relationships are HBV transmission modes (HBV: 89.9% and 69.7%; HIV: 99.1% and 99.4%). The fear of both illnesses was similar at 20.3%. The individual perceived risk of infection was higher for HBV than for HIV with, respectively, 60.8% and 40.3% of respondents believing they had an equal or greater risk of being infected than the average person. However, the percentage of those reporting HBV screening during their lifetime (27.4%) was half that for HIV screening (61.4%). In multivariate analysis, HBV screening was reported more often by individuals born in areas with high HBV endemicity (OR = 2.1 [95% CI: 1.5-2.9]) than by those born in low HBV endemicity areas, and more often by those who reported they had taken drugs intravenously during their lifetime (OR = 2.2 [95% CI: 1.2-4.2]) than those who did not report such behavior. Almost one in two respondents (47%) reported HBV vaccination. The intermediate or high endemicity groups did not report vaccination more often than those born in low endemicity areas nor did those reporting intravenously drug use compared with those who did not. CONCLUSIONS: This study highlights very contrasting levels of knowledge, perceptions and practices regarding HBV and HIV in the French general population. Our results demonstrate the need to improve the general and high-risk populations' knowledge of HBV, in particular concerning sexual transmission, in order to improve screening and vaccination practices.

Anticancer activity of silver-N-heterocyclic carbene complexes: caspase-independent induction of apoptosis via mitochondrial apoptosis-inducing factor (AIF).

Fourteen silver(I) complexes bearing N-heterocyclic carbene (NHC) ligands were prepared and evaluated for anticancer activity. Some of these were found to exhibit potent antiproliferative activity toward several types of human cancer cell lines, including drug-resistant cell lines, with IC(50) values in the nanomolar range. An initial investigation into the mechanism of cell death induced by this family of silver(I) complexes was carried out. Cell death was shown to result from the activation of apoptosis without involvement of primary necrosis. In HL60 cells, silver-NHCs induce depolarization of the mitochondrial membrane potential (ΔΨ(m)) and likely allow the release of mitochondrial proteins to elicit early apoptosis. This effect is not related to the overproduction of reactive oxygen species (ROS). In addition, apoptosis is not associated with the activation of caspase-3, but is triggered by the translocation of apoptosis-inducing factor (AIF) and caspase-12 from mitochondria and the endoplasmic reticulum, respectively, into the nucleus to promote DNA fragmentation and ultimately cell death. No modification in cell-cycle distribution was observed, indicating that silver-NHCs are not genotoxic. Finally, the use of a fluorescent complex showed that silver-NHCs target mitochondria. Altogether, these results demonstrate that silver-NHCs induce cancer cell death independent of the caspase cascade via the mitochondrial AIF pathway.

Use of copper(I) catalyzed azide alkyne cycloaddition (CuAAC) for the preparation of conjugated pyrrolo[2,3-a]carbazole Pim kinase inhibitors.

We have previously demonstrated that pyrrolo[2,3-a]carbazole-3-carbaldehydes are potent Pim kinase inhibitors with in vitro antiproliferative activities. In the present study, we report the synthesis of new pyrrolocarbazoles substituted at the N-10 position. When their ability to inhibit Pim kinase activities were evaluated in in vitro assays, we observed that this nitrogen atom can be substituted without loss of Pim-1 and Pim-3 inhibitory potencies. Moreover, when we added a fluorescent dansyl group (compound 13), we were able to show that 13 penetrates the plasma membrane and enters the cytoplasm.

Advances in metal-carbene complexes as potent anti-cancer agents.

This critical review is an updated survey of metal-carbenes as potential anticancer chemotherapeutics. We report on the recent advances in the discovery of N-heterocyclic carbenes, acyclic diamino carbenes and abnormal NHCs associated with metals from groups 10 and 11 that displayed antiproliferative activity and emphasize, when possible, their molecular target(s) and their mechanism of action.

A platinum Chugaev carbene complex as a potent anticancer agent.

A platinum Chugaev complex was synthesised and fully characterized by multinuclear NMR spectroscopy and X-ray crystallography. This cis bis acyclic diamino carbene complex acts as a cytotoxic compound and behaves as a cisplatin equivalent by interacting with supercoiled DNA and thiols. Stability of the ligand is also discussed.

Genetically encoded photocontrol of protein localization in mammalian cells.

Precise photochemical control of protein function can be achieved through the site-specific introduction of caging groups. Chemical and enzymatic methods, including in vitro translation and chemical ligation, have been used to photocage proteins in vitro. These methods have been extended to allow the introduction of caged proteins into cells by permeabilization or microinjection, but cellular delivery remains challenging. Since lysine residues are key determinants for nuclear localization sequences, the target of key post-translational modifications (including ubiquitination, methylation, and acetylation), and key residues in many important enzyme active sites, we were interested in photocaging lysine to control protein localization, post-translational modification, and enzymatic activity. Photochemical control of these important functions mediated by lysine residues in proteins has not previously been demonstrated in living cells. Here we synthesized 1 and evolved a pyrrolysyl-tRNA synthetase/tRNA pair to genetically encode the incorporation of this amino acid in response to an amber codon in mammalian cells. To exemplify the utility of this amino acid, we caged the nuclear localization sequences (NLSs) of nucleoplasmin and the tumor suppressor p53 in human cells, thus mislocalizing the proteins in the cytosol. We triggered protein nuclear import with a pulse of light, allowing us to directly quantify the kinetics of nuclear import.

[Representations and attitudes toward cancer in the French general population]. / Représentations et attitudes du public vis-à-vis du cancer.

Cancer has become a major public health issue. It is thus crucial to measure the general population's behaviours, opinions and perceptions about cancer and its associated risk factors. This article describes some of the main findings of a 2005 French survey (n = 4,046). Cancer is considered by a large majority to be the most serious disease, far before HIV/AIDS and cardiovascular diseases. The carcinogenic risk that is associated to main risk factors, such as sun exposure, tobacco-smoking and alcohol use appears to be well-known. However, many people justify dangerous behaviours with strongly-anchored beliefs, which maintain dangerous behaviours for health on the long-term. What's more, the perception of risk proliferation can also generate risk denial. Because self-exempting beliefs are still widespread within the general opinion, it is essential to continue public health information campaigns dedicated to cancer prevention, so as to induce better prevention practices within the general population and to reduce stigmatisation and isolation experienced by cancer patients. If risk denial is not systematically a consequence of a lack of information, it is generally associated to a cognitive construction that gives coherence to behaviours.