Éditeur(s) : Elsevier
Résumé : Because Vibrio aestuarianus is known to cause serious infections in Pacific oyster Crassostrea gigas, a real-time PCR assay was developed targeting the dnaJ gene of this bacterium. Only V. aestuarianus strains isolated from C. gigas mortality events in different geographic areas and the reference strain tested positive, whereas no amplification products was obtained with type strains belonging to 23 other species of Vibrio. Sensitivity and reproducibility of the method were assessed using either seawater or oyster homogenate samples spiked with one V aestuarianus strain. All these samples were stored at -20 degrees C in order to mimic retrospective or grouped natural sample analysis without quantification bias due to prolonged freezing. Analysis of standard curves revealed excellent correlation values between light microscopy cell enumerations and PCR Threshold Cycle (Ct) values, and acceptable PCR reaction efficiencies for all type of samples. Quantification curves of both sample types were equivalent, with a detection level as low as 1.6 V. aestuarianus cells in the PCR reaction tube, corresponding to 1.6.10(2) cells ml(-1) and 1.6.10(2) cells mg(-1) in seawater and entire oyster samples, respectively, taking into account the dilution factor used for appropriate template DNA preparation. Comparison of PCR assay reproducibility according to the complexity of samples revealed that seawater samples gave more reproducible quantification measures than samples from oyster homogenate, with precision of measured Ct values inferior to 0.4 and 0.6 respectively at 99% confidence. Use of the real-time PCR assay allowed us to monitor V. aestuarianus load in oysters naturally infected with this pathogen. Furthermore, we were able to detect V aestuarianus in samples of seawater in which oysters had been reared and in algal cultures used for feeding oysters. Because of the rapidity and reliability of the real-time PCR assay method used in this study, just a few hours are needed compared with the two days required using the classic culture method, this technique will be particularly valuable in mollusc pathology laboratories, for monitoring the source and course of infections by V. aestuarianus in pathogenesis and epidemiologic studies, as well as for designing appropriate prophylactic control measures. (C) 2009 Elsevier B.V. All rights reserved.
Journal of Microbiological Methods (0167-7012) (Elsevier), 2009-05 , Vol. 77 , N. 2 , P. 191-197

Droits : 2009 Elsevier B.V. All rights reserved.
http://archimer.ifremer.fr/doc/2009/publication-6447.pdf
DOI:10.1016/j.mimet.2009.01.021
http://archimer.ifremer.fr/doc/00000/6447/

Éditeur(s) : HAL CCSD
Résumé : International audience
This article describes a previously unreported mutation at position 210 (C210T) of the mitochondrial large subunit ribosomal RNA (mtLSUrRNA) gene of Pneumocystis jirovecii, which led to a false-negative result of a real-time polymerase chain reaction (PCR) assay. Since the aforementioned real-time PCR assay is widely used in France, a French multicenter study was conducted to estimate the mutation frequency and its potential impact on the routine diagnosis of Pneumocystis pneumonia (PCP). Through analysis of data obtained from eight centers, the mutation frequency was estimated at 0.28%. This low frequency should not call into question the routine use of this PCR assay. Nonetheless, the occurrence of the false-negative PCR result provides arguments for maintaining microscopic techniques combined to PCR assays to achieve PCP diagnosis.
Medical Mycology

hal-01477200
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01477200
DOI : 10.1093/mmy/myw051
PUBMED : 27489302

Éditeur(s) : HAL CCSD American Society for Microbiology
Résumé : International audience
Molecular diagnosis of congenital toxoplasmosis or disseminated toxoplasmosis is based mainly on PCR. The repeated DNA element rep529 has become the main DNA target used in most PCR methods, whether laboratory developed or commercial. In this multicenter study, we evaluated the Toxoplasma ELITe MGB (Elitech) commercial kit by comparison with three reference quantitative PCR assays (RAs) used routinely in three proficient laboratories of the French National Reference Center for Toxoplasmosis network, using Toxoplasma calibrated suspensions diluted to obtain a range of concentrations from 0.1 to 10,000 parasites/ml. These suspensions were extracted with either the DNA extraction kit (EXTRAblood; Elitech) recommended by the manufacturer or the QIAamp DNA minikit (Qiagen). The Toxoplasma ELITe MGB assay was also evaluated on a panel of 128 clinical samples, including 56 amniotic fluid samples, 55 placenta samples, and various other samples, of which 95 originated from patients with proven toxoplasmosis. The ELITe MGB assay amplified low-concentration replicates (<10 parasites/ml) of calibrated suspensions less frequently than the RAs of 2/3 laboratories. Additionally, the combination of EXTRAblood and Toxoplasma ELITe MGB yielded poorer sensitivity than the combination of QIAamp DNA minikit and ELITe MGB for low parasite concentrations (P < 0.001 for 1 parasite/ml). On clinical samples, the sensitivity and the specificity of the commercial assay were 89% and 100%, respectively. The sensitivity ranged from 79% (placenta samples) to 100% (amniotic fluid samples). Overall, this study shows that the Toxoplasma ELITe MGB assay is suitable for the diagnosis of toxoplasmosis from non-cell-rich or non-hemoglobin-rich samples and that the EXTRAblood kit is not optimal.
ISSN: 0095-1137

hal-01526432
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01526432
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01526432/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01526432/file/Evaluation%20of%20the%20Toxoplasma.pdf
DOI : 10.1128/JCM.02379-16

Éditeur(s) : Elsevier
Résumé : Shrimp farming is a small but growing industry in New Caledonia. Since 1993, "Syndrome 93" has been affecting New Caledonian shrimp farming industry every cold season, causing severe epizootic mortalities in grow-out ponds and significant losses. Highly pathogenic strains of Vibrio penaeicida are considered the etiological agent of the disease in Litopenaeus stylirostris. On one hand, studies demonstrated that healthy shrimp may carry V penaeicida for weeks with a high overall prevalence, regardless of any seasonal pattern or temperature conditions. On the other hand, larvae are free of V penaeicida and are also resistant to experimental infection. V penaeicida is frequently detected in incoming water pumped from the bays, which was shown, by a molecular typing study, to be the infectious source. This particular epidemiological pattern highlights the major role of the factors that trigger and aggravate the disease in grow-out ponds, where shrimp populations carry the pathogen all year round. In order to gain a better understanding of "Syndrome 93" epidemiology, quantification of V penaeicida both in shrimp and the shrimp farm ecosystem is necessary. This article describes the steps in the successful development of a real-time PCR quantification assay of V penaeicida in shrimp haemolymph, seawater (from ponds or bays) and sediment pore water, including the choice of an accurate extraction technique. The entire detection method; including sample processing, DNA extraction and real-time PCR amplification, can be completed within 4 h. (c) 2006 Elsevier B.V All rights reserved.
Journal of Microbiological Methods (0167-7012) (Elsevier), 2006-10 , Vol. 67 , N. 1 , P. 27-35

Droits : 2006 Elsevier
http://archimer.ifremer.fr/doc/2006/publication-1903.pdf
DOI:10.1016/j.mimet.2006.02.013
http://archimer.ifremer.fr/doc/00000/1903/

Éditeur(s) : HAL CCSD Lippincott, Williams & Wilkins
Résumé : International audience
Purpose of review Toxoplasmosis in immunocompromised patients is associated with a high mortality rate. Molecular techniques are important tools to diagnose acute disease in immunocompromised patients, but there are various methods with variable efficiency. Some of them have been validated for the diagnosis of congenital toxoplasmosis, but the impact of their use has not been evaluated in immunocompromised patients. Recent findings Toxoplasmosis is of increasing importance in non-HIV immunocompromised patients. In addition, the picture of disease shows greater severity in South America, both in immunocompetent study participants and in congenitally infected infants. These epidemiological differences could influence the sensitivity of diagnostic methods. This review analyzes recent data on molecular diagnosis and compares them with older ones, in light of progress gained in molecular techniques and of recent epidemiological findings. Most recent studies were conducted in South America and used PCR targeting the B1 gene. PCR on blood could allow diagnosing a significant proportion of patients with ocular toxoplasmosis in Brazil. Summary Quantitative PCR methods with specific probes should be used to improve sensitivity and warrant specificity. Performance of quantitative PCR targeting the repeated 529 bp sequence for the diagnosis of toxoplasmosis in immunocompromised patients needs evaluation in field studies in South America and in western countries.
ISSN: 0951-7375

hal-01390970
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01390970
DOI : 10.1097/QCO.0000000000000275
PUBMED : 27191201

Éditeur(s) : Elsevier
Résumé : In a previous study, we demonstrated the existence of an emerging cluster of Vibrio nigripulchritudo that proved to be associated with shrimp mortality events in New Caledonia. Using sequence polymorphisms evidenced in this previous MultiLocus Sequence Typing study, we developed two new quantitative PCR assays permitting the detection and quantification of V. nigripulchritudo at the genospecies level using SYBR Green I chemistry and at the emerging cluster level using Fluorescence Resonance Energy Transfer technology with hybridization probes. The use of this molecular diagnostic tool evidenced the colonization of the shrimp pond ecosystem by the pathogenic cluster at least at the onset of the disease. This new tool will allow better investigation of the dynamics of this bacterial pathogen in the shrimp farm ecosystem. (C) 2007 Elsevier B.V. All rights reserved.
Journal of Microbiological Methods (0167-7012) (Elsevier), 2007-07 , Vol. 70 , N. 1 , P. 30-38

Droits : 2007 Elsevier B.V. All rights reserved
http://archimer.ifremer.fr/doc/2007/publication-2729.pdf
DOI:10.1016/j.mimet.2007.03.007
http://archimer.ifremer.fr/doc/00000/2729/

Éditeur(s) : Academic Press Ltd- Elsevier Science Ltd
Résumé : The understanding of reciprocal interactions between Crassostrea gigas and Vibrio sp., whether these be virulent or avirulent, is vital for the development of methods to improve the health status of cultured oysters. We describe an original non-invasive experimental infection technique using cohabitation, designed to explore these interactions. Using real-time PCR techniques we examined the dynamics of virulent and avirulent Vibrio sp. in oyster hemolymph and tank seawater, and made a parallel study of the expression of four genes involved in oyster immune defense: Cg-BPI, Cg-EcSOD, Cg-IκB, Cg-TIMP. No mortality occurred in control animals, but oysters put in cohabitation for 2–48 h with animals previously infected by two Vibrio pathogens suffered mortalities from 2 to 16 days post-cohabitation. Our results show that virulent Vibrio infect healthy individuals after only 2 h of cohabitation, with values ranging from 4.5×102 to 2×104 cells ml−1 hemolymph. Simultaneously, an approximate ten-fold increase of the total Vibrio population was observed in control animals, with a 6.6–78.5-fold up-expression of targeted genes. In contrast, oysters exposed to harmful bacteria had mean expression levels strongly down-regulated by a factor of 9.2–29 (depending on the gene) compared with control animals. Although oysters were still found to be infected by virulent Vibrio after 6–48 h of cohabitation, no significant differences were noted when comparing levels of each transcript in control and infected oysters at the same sampling times during this period: the important differences were noted before 6 h cohabitation. Taken together, our data support (1) the hypothesis that virulent Vibrio disturbs the immune response of this invertebrate host both rapidly and significantly, although this occurs specifically during an early and transient period during the first 6 h of cohabitation challenge, and that (2) expression of targeted genes is not correlated with vibriosis resistance.
Fish & Shellfish Immunology (1050-4648) (Academic Press Ltd- Elsevier Science Ltd), 2011-02 , Vol. 30 , N. 2 , P. 691-699

Droits : 2011 Elsevier Ltd All rights reserved.
http://archimer.ifremer.fr/doc/00025/13604/10681.pdf
DOI:10.1016/j.fsi.2010.12.017
http://archimer.ifremer.fr/doc/00025/13604/

Éditeur(s) : Inter-Research
Résumé : Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) were purified from diseased freshwater prawns M rosenbergii and used to infect healthy post-larvae (PL) by an immersion method. Three groups of prawns were challenged with various combined doses of MrNV and XSV. Signs of white-tail disease (WTD) were observed in Groups 1 and 2, which had been challenged with combinations containing relatively high proportions of MrNV and low proportions of XSV. By contrast there was little sign of WTD in Group 3, which had been challenged with a higher proportion of XSV than MrNV. A 2-step Taqman real-time RT-PCR was developed and applied to quantify viral copy numbers in each challenged PL. Results showed that genomic copies of both viruses were much higher in Groups 1 and 2 than they were in Group 3, indicating that MrNV plays a key role in WTD of M rosenbergii. The linear correlation between MrNV and XSV genome copies in infected prawns demonstrated that XSV is a satellite virus, dependent on MrNV, but its role in pathogenicity of WTD remains unclear.
Diseases of aquatic organisms (0177-5103) (Inter-Research), 2006-07 , Vol. 71 , N. 1 , P. 11-17

Droits : Inter-Research 2006
http://archimer.ifremer.fr/doc/2006/publication-3568.pdf
DOI:10.3354/dao071011
http://archimer.ifremer.fr/doc/00000/3568/

Éditeur(s) : Elsevier Science Inc
Résumé : The sex-determining system of Crassostrea gigas is still poorly known, especially regarding the potential influence of temperature. In order to address this question, mRNA expressions of actors of the molecular cascade (Cg-DMI, Cg-SoxE, Cg-beta-catenin, Cg-Foxl2/Cg-FoxI2os) and of Oyvlg, a germ cell marker, were investigated by real-time PCR in spat grown at different temperatures (18, 22, 25 and 28 degrees C). In parallel, gonadic differentiation, gametogenesis and sex ratios were assessed by histology at each of these temperatures. Whatever the temperature, Cg-DMI, Cg-SoxE, Cg-beta-catenin and Oyvlg expressions peaked at the same developmental stage, always after Cg-Foxl2/Cg-Foxl2os (around 40-44 dpf for spat grown at 18 degrees C). Temperatures increased the kinetics of first gonadic differentiation and gametogenesis. At 25 degrees C a significant switch occurred in sex ratio towards males and in the balance of expression between male and female genes, in favor of males. A slight gametogenesis disturbance was also observed. These results strengthen the hypotheses about the sex-determining time window and molecular cascade governing the development of C gigas, with notably the involvement of Cg-Foxl2/Cg-Foxl2os in the very early steps. They also suggest an influence of temperature on the oyster's sex determination which, associated to genetic control, would induce a mixed sex determination system (GSD +TSD). (C) 2013 Elsevier Inc. All rights reserved.
Comparative Biochemistry And Physiology A-molecular & Integrative Physiology (1095-6433) (Elsevier Science Inc), 2013-05 , Vol. 165 , N. 1 , P. 61-69

Droits : 2013 Elsevier Inc. All rights reserved
http://archimer.ifremer.fr/doc/00136/24767/22874.pdf
DOI:10.1016/j.cbpa.2013.02.007
http://archimer.ifremer.fr/doc/00136/24767/

Éditeur(s) : HAL CCSD BioMed Central
Résumé : International audience
UNLABELLED: ABSTRACT: BACKGROUND: Genomics, transcriptomics and proteomics of glioblastoma multiforme (GBM) have recently emerged as possible tools to discover therapeutic targets and biomarkers for new therapies including immunotherapy. It is well known that macroscopically complete surgical excision, radiotherapy and chemotherapy have therapeutic limitations to improve survival in these patients. In this study, we used a differential proteomic-based technique (2D-Difference Gel Electrophoresis) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify proteins that may serve as brain tumor antigens in new therapeutic assays. Five samples of patients presenting a GBM and five samples of microscopically normal brain tissues derived from brain epileptic surgery specimen were labeled and run in 2D-PAGE (Two-Dimensional Polyacrylamide Gel Electrophoresis) with an internal pool sample on each gel. Five gels were matched and compared with DIA (Difference In-gel Analysis) software. Differential spots were picked, in-gel digested and peptide mass fingerprints were obtained. RESULTS: From 51 protein-spots significantly up-regulated in GBM samples, mass spectrometry (MS) identified twenty-two proteins. The differential expression of a selected protein set was first validated by western-blotting, then tested on large cohorts of GBM specimens and non-tumor tissues, using immunohistochemistry and real-time RT-PCR. CONCLUSIONS: Our results confirmed the importance of previously described proteins in glioma pathology and their potential usefulness as biological markers but also revealed some new interesting targets for future therapies.
ISSN: 1477-5956

inserm-00585020
http://www.hal.inserm.fr/inserm-00585020
http://www.hal.inserm.fr/inserm-00585020/document
http://www.hal.inserm.fr/inserm-00585020/file/1477-5956-9-16.pdf
DOI : 10.1186/1477-5956-9-16

Éditeur(s) : HAL CCSD Taylor & Francis
Résumé : International audience
The functional organization of the circadian system and the location of the main circadian oscillators vary through phylogeny. Present study investigates by in situ hybridization the anatomical location of the clock gene gPer1b in forebrain and midbrain, pituitary, and in two peripheral locations, the anterior intestine and liver, in a teleost fish, the goldfish (Carassius auratus). Moreover, the daily expression profiles of this gene were also studied by quantitative Real Time-PCR. Goldfish were maintained under a 12L–12D photoperiod and fed daily at 2 h after lights were switched on. A wide distribution of gPer1b mRNA in goldfish brain and pituitary was found in telencephalon, some hypothalamic nuclei (including the homologous to mammalian SCN), habenular nucleus, optic tectum, cerebellum and torus longitudinalis. Moreover, gPer1b expression was observed, for the first time in teleosts, in the pituitary, liver and anterior intestine. Day/night differences in gper1b mRNA abundance were found by in situ hybridization, with higher signal at nighttime that correlates with the results obtained by RT-PCR, where a rhythmic gPer1b expression was found in all tissues with acrophases at the end of the night. Amplitudes of gper1b rhythms vary among tissues, being higher in liver and intestine than in the brain, maybe because different cues entrain clocks in these locations. These results support the existence of functional clocks in many central and peripheral locations in goldfish coordinated, ticking at the same time
ISSN: 0742-0528

hal-01197353
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01197353
DOI : 10.3109/07420528.2015.1049615
PUBMED : 26171989

Éditeur(s) : Université de Pau et des Pays de l'Adour
Résumé : In a context of decline of European eel (Anguilla anguilla L.) and search for biological markers of habitat use and quality, a multidisciplinary approach was engaged on the Adour estuary and its associated wetlands. Two axes were developed. The first axe aimed at highlighting the successive presence of the species in different ecosystems based on the otolith elemental composition. The second aimed at the evaluation of the chemical pressure underwent by eels based on the contaminant bioconcentration in soft tissues and the early molecular response to the chemical stress. With a view to the analyses of otolith elemental composition taking into account the 3D structure, the low elemental concentration in the matrix and keeping the finest spatial resolution, four micro sampling strategies and multi elemental analyses using femtosecond laser ablation ICP-MS were tested (crater, 1D scan, 2D scan, flash ablation). The strategy showing the best compromise according to the objectives (continuous recording, multi elemental approach) was applied on otolith of yellow eels from the Adour. The simultaneous use of Sr and Ba profiles, after validation, represents a new tool, easy, fast and reliable for the reconstruction of freshwater and brackish ecosystems use by eel avoiding misinterpretation due to vaterite inclusions. Three major behaviors were highlighted on the Adour: a residency in brackish water, a residency in freshwater and nomad behavior. The last one is illustrated by a shift to brackish water after three to four years spent in freshwater. Simultaneously, the study of inorganic and organic contamination of eel soft tissues showed that sampled wetlands are moderately contaminated. These results were confirmed by the first estimation, in wild conditions, of the early response to the chemical stress using the hepatic expression of CYP1A1 gene measured by real time RT-PCR. Conversely, in the downstream part of the Adour estuary, Hg loads in eels, especially methyl mercury, and PCBs analyses indicate values that need further investigations considering the European norms of consumption. The comparison of the different markers used (heavy metal and organochlorinated compounds contamination of soft tissues, condition index) support a lower quality of the estuary despite higher eel growth rate on this site. This study supports the use of numerous markers in integrated studies in order to understand and specify the relationships between eels and their continental growth zones.
Dans un contexte de déclin de l'anguille européenne (Anguilla anguilla L.) et de recherche de marqueurs biologiques retraçant la fréquentation des divers habitats et renseignant sur la qualité de ceux-ci, une approche pluridisciplinaire a été mise en place sur l'estuaire de l'Adour et ses zones humides. Deux axes ont été privilégiés. Le premier cherchait à visualiser la présence successive de l'espèce dans différents écosystèmes en se basant sur la composition élémentaire des otolithes. Le deuxième avait pour but d'évaluer la pression chimique inorganique et organique subie par l'anguille dans les zones d'études en s'appuyant sur la bioconcentration des contaminants dans les tissus mous et la réponse moléculaire précoce au stress chimique. En vue des analyses de la composition élémentaire des otolithes et pour répondre au mieux à la structure en trois dimensions, à la faible concentration des éléments dans la matrice tout en conservant une résolution spatiale la plus fine possible, quatre stratégies de micro-échantillonnage et d'analyses multiélémentaires par ablation laser femtoseconde ICP-MS ont été testées (cratère, scan 1D, scan 2D, ablation flash). La méthodologie présentant le meilleur compromis par rapport aux objectifs fixés (enregistrement continu, approche multiélémentaire) a ensuite été appliquée sur les otolithes d'anguilles jaunes du Bas-Adour. L'utilisation simultanée des profils continus de Sr et de Ba, après validation, constitue un outil facile d'utilisation, rapide et fiable pour reconstruire les historiques de fréquentation des différents écosystèmes estuariens et dulçaquicoles par l'anguille tout en s'affranchissant des problèmes potentiels liés aux inclusions de vatérite, un polymorphe du carbonate de calcium. Trois grands types de comportement ont pu être mis en évidence sur l'Adour : résidence en eau saumâtre, résidence en eau douce et nomadisme. Ce dernier est plus particulièrement illustré par un mouvement marqué vers l'eau saumâtre pour des individus ayant passé de trois à quatre ans dans l'eau douce. Parallèlement, l'étude de la contamination organique et inorganique des tissus mous de l'anguille a permis de déduire à la faible contamination des zones humides échantillonnées. La première estimation en condition naturelle de la réponse précoce au stress chimique par évaluation de l'expression hépatique du gène CYP1A1 par RT-PCR en temps réel confirme ces résultats. En revanche, dans la partie estuarienne aval de l'Adour, les niveaux en Hg, présent en majorité sous sa forme méthylée dans les anguilles, et l'analyse des PCBs indiquent des valeurs qui suscitent un examen plus approfondi au regard des normes européennes en vigueur. La confrontation des différents descripteurs (contamination des tissus mous par les métaux lourds et organochlorés, indice de condition) utilisés conforte la qualité moindre de la zone estuarienne malgré des performances de croissance de l'anguille favorisées sur ces sites. Ainsi, cette étude supporte l'intérêt de la mise en oeuvre de descripteurs multiples utilisés (niveaux de métaux lourds et organochlorés dans les tissus mous, réponse moléculaire au stress chimique, microchimie de l'otolithe, indice de condition, taux de croissance) dans des approches intégrées pour comprendre et préciser les relations existantes entre l'anguille et ses zones de croissance continentales.

Droits : info:eu-repo/semantics/openAccess
http://archimer.ifremer.fr/doc/2009/these-7381.pdf
http://archimer.ifremer.fr/doc/00000/7381/

Éditeur(s) : HAL CCSD
Résumé : International audience
Kisspeptins are well known as mediators of the coordinated communication between the brain-pituitary axis and the gonads in many vertebrates. To test the hypothesis that gonadal steroids regulate kiss1 and kiss2 mRNA expression in European sea bass (a teleost fish), we examined the brains of gonad-intact (control) and castrated animals, as well as castrated males (GDX) and ovariectomized females (OVX) that received testosterone (T) and estradiol (E2) replacement, respectively, during recrudescence. In GDX males, low expression of kiss1 mRNA is observed by in situ hybridization in the caudal hypothalamus (CH) and the mediobasal hypothalamus (MBH), although hypothalamic changes in kiss1 mRNA levels were not statistically different among the groups, as revealed by real-Time PCR. However, T strongly decreased kiss2 expression levels in the hypothalamus, which was documented in the MBH and the nucleus of the lateral recess (NRLd) in GDX T-Treated sea bass males. Conversely, it appears that E2 evokes lowkiss1mRNAin the CH, while there were cells expressing kiss2 in the MBH and NRLd in these OVX females. These results demonstrate that kisspeptin neurons are presumably sensitive to the feedback actions of sex steroids in the sea bass, suggesting that the MBH represents a major site for sex steroid actions on kisspeptins in this species. Also, recent data provide evidence that both positive and negative actions occur in key factors involved in sea bass reproductive function, including changes in the expression of gnrh-1/gonadotropin, cyp19b, er and ar genes and sex steroid and gonadotropin plasma levels in this teleost fish. © 2016. Published by The Company of Biologists Ltd.
Journal of Experimental Biology

hal-01406072
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01406072
DOI : 10.1242/jeb.137364
PUBMED : 27591305

Éditeur(s) : HAL CCSD
Résumé : International audience
In French Guiana, located on the northeastern coast of South America, bats of different species are very numerous. The infection of bats and their ticks with zoonotic bacteria, especially Rickettsia species, is so far unknown. In order to improve knowledge of these zoonotic pathogens in this French overseas department, the presence and diversity of tick-borne bacteria was investigated with molecular tools in bat ticks. In the beginning of 2013, 32 bats were caught in Saint-Jean-du-Maroni, an area close to the coast of French Guiana, and the ticks of these animals were collected. A total of 354 larvae of Argasidae soft ticks (Ornithodoros hasei) from 12 bats (Noctilio albiventris) were collected and 107 of them were analysed. DNA was extracted from the samples and quantitative real-time PCR was carried out to detect Rickettsia spp., Bartonella spp., Borrelia spp. and Coxiella burnetii. All tested samples were negative for Bartonella spp., Borrelia spp. and Coxiella burnetii. Rickettsia DNA was detected in 31 (28.9%) ticks. An almost entire (1118 base pairs long) sequence of the gltA gene was obtained after the amplification of some positive samples on conventional PCR and sequencing. A Bayesian tree was constructed using concatenated rrs, gltA, ompA, ompB, and gene D sequences. The study of characteristic sequences shows that this Rickettsia species is very close (98.3-99.8%) genetically to R. peacockii. Nevertheless, the comparative analysis of sequences obtained from gltA, ompA, ompB, rrs and gene D fragments demonstrated that this Rickettsia is different from the other members of the spotted fever group. The sequences of this new species were deposited in GenBank as Candidatus Rickettsia wissemanii. This is the first report showing the presence of nucleic acid of Rickettsia in Ornithodoros hasei ticks from South American bats. (C) 2016 Elsevier GmbH. All rights reserved.
TICKS AND TICK-BORNE DISEASES

hal-01459758
https://hal.archives-ouvertes.fr/hal-01459758
DOI : 10.1016/j.ttbdis.2016.09.004

Éditeur(s) : HAL CCSD Wiley-Blackwell
Résumé : International audience
AIMS: Virus detection has often been difficult due to a low concentration in water. In this study, we developed a new procedure based on concentration of virus particles on an innovative support: poly-L-lysine dendrigrafts (DGL), coupled with directed nucleic acid extraction and real-time PCR quantification. METHODS AND RESULTS: This method was evaluated using the bacteriophage MS2 as a model virus. This virus exhibited the size and structural properties of human pathogenic enteric viruses and has often been used to assess new supports of concentration. Moreover, this bacteriophage is also a faecal contamination indicator. In this study, many water filtration conditions were tested (volume of water, concentration, etc.), and more than 80% of bacteriophage were recovered after filtration on polymer, in most conditions. We demonstrated that the method was linear (slope = 0*99 ± 0*04 and Y intercept when x = -0*02 ± 0*28), valid (as manipulators, tested concentrations, volumes of sample and batch of polymer did not have any influence on concentration) and sensitive (allowing to concentrate up to 16,600-fold 1 l of sample and to detect and quantify down to 750 GC l(-1) and 7500 GC l(-1), respectively). CONCLUSIONS: To conclude, this support exhibits high interest to retain viruses and to allow to detect low concentration of virus in water. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives valuable advance in the methods of concentration and diagnosis of virus in water.
Journal of Applied Microbiology

hal-00828987
https://hal.archives-ouvertes.fr/hal-00828987
DOI : 10.1111/jam.12209
PUBMED : 23551794

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
Ghrelin is a gut-brain peptide hormone, which binds to the growth hormone secretagogue receptor (GHS-R) to regulate a wide variety of biological processes in fish. Despite these prominent physiological roles, no studies have reported the anatomical distribution of preproghrelin transcripts using in situ hybridization in a non-mammalian vertebrate, and its mapping within the different encephalic areas remains unknown. Similarly, no information is available on the possible 24-h variations in the expression of preproghrelin and its receptor in any vertebrate species. The first aim of this study was to investigate the anatomical distribution of ghrelin and GHS-R1a ghrelin receptor subtype in brain and gastrointestinal tract of goldfish (Carassius auratus) using immunohistochemistry and in situ hybridization. Our second aim was to characterize possible daily variations of preproghrelin and ghs-r1 mRNA expression in central and peripheral tissues using real-time reverse transcription-quantitative PCR. Results show ghrelin expression and immunoreactivity in the gastrointestinal tract, with the most abundant signal observed in the mucosal epithelium. These are in agreement with previous findings on mucosal cells as the primary synthesizing site of ghrelin in goldfish. Ghrelin receptor was observed mainly in the hypothalamus with low expression in telencephalon, pineal and cerebellum, and in the same gastrointestinal areas as ghrelin. Daily rhythms in mRNA expression were found for preproghrelin and ghs-r1 in hypothalamus and pituitary with the acrophase occurring at nighttime. Preproghrelin, but not ghs-r1a, displayed a similar daily expression rhythm in the gastrointestinal tract with an amplitude 3-fold higher than the rest of tissues. Together, these results described for the first time in fish the mapping of preproghrelin and ghrelin receptor ghs-r1a in brain and gastrointestinal tract of goldfish, and provide the first evidence for a daily regulation of both genes expression in such locations, suggesting a possible connection between the ghrelinergic and circadian systems in teleosts
ISSN: 1932-6203

hal-01225511
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01225511
DOI : 10.1371/journal.pone.0141043
PUBMED : 26506093

Éditeur(s) : HAL CCSD Wiley-Blackwell
Résumé : International audience
Virus detection has often been difficult due to a low concentration in water. In this study, we developed a new procedure based on concentration of virus particles on an innovative support: poly-L-lysine dendrigrafts (DGL), coupled with directed nucleic acid extraction and real-time PCR quantification. Methods and Results: This method was evaluated using the bacteriophage MS2 as a model virus. This virus exhibited the size and structural properties of human pathogenic enteric viruses and has often been used to assess new supports of concentration. Moreover, this bacteriophage is also a faecal contamination indicator. In this study, many water filtration conditions were tested (volume of water, concentration, etc.), and more than 80% of bacteriophage were recovered after filtration on polymer, in most conditions. We demonstrated that the method was linear (slope = 0 99 0 04 and Y intercept when x = 0 02 0 28), valid (as manipulators, tested concentrations, volumes of sample and batch of polymer did not have any influence on concentration) and sensitive (allowing to concentrate up to 16 600-fold 1 l of sample and to detect and quantify down to 750 GC l 1 and 7500 GC l 1, respectively). Conclusions: To conclude, this support exhibits high interest to retain viruses and to allow to detect low concentration of virus in water. Significance and Impact of the Study: This study gives valuable advance in the methods of concentration and diagnosis of virus in water.
Journal of Applied Microbiology

hal-00932328
https://hal.archives-ouvertes.fr/hal-00932328
DOI : 10.1111/jam.12209

Éditeur(s) : HAL CCSD BioMed Central
Résumé : International audience
BACKGROUND: Hepatocellular carcinoma (HCC) is a particularly severe disease characterized by a high rate of recurrence and death even after surgical resection. Molecular characterization of HCC helps refine prognosis and may facilitate the development of improved therapy. Phosphatidylinositol 4-kinases have recently been identified as cellular factors associated with cancer. Also, phosphatidylinositol 4-kinase type IIIα (PI4KA) is necessary for the propagation of the hepatitis C virus, a major etiological factor for HCC. METHODS: Reverse transcription, quantitative real-time PCR was used to assay PI4KA mRNA. The expression levels were investigated both in a collection of molecularly and clinically characterized hepatic tissues from 344 patients with diverse liver diseases and in human hepatocyte cell lines whose proliferative and differentiation status was controlled by specific culture conditions. Analytical microarray data for 60 HCC and six normal liver tissue samples were exploited to study correlations between PI4KA mRNA levels and cell proliferation markers in vivo. Postoperative disease-specific survival and time to recurrence in a set of 214 patients with HCC were studied by univariate and multivariate analyses. RESULTS: PI4KA mRNA was more abundant in HCC than normal healthy tissues. This upregulation correlated significantly with both poor differentiation and the active proliferation rate in HCC. These associations were confirmed with in vitro models. Moreover, patients with HCC who had been treated by surgical resection and had higher PI4KA mRNA concentrations in their tumor tissue exhibited a higher risk of tumor recurrence (median time: 20 months versus 49 months, P = 0.0012) and shorter disease-specific survival (first quartile time: 16 months versus 48 months, P = 0.0004). Finally, the abundance of PI4KA mRNA proved to be an independent prognostic marker of survival for cases of HCC (hazard ratio = 2.36, P = 0.0064). CONCLUSIONS: PI4KA mRNA could be used as a new molecular marker to improve established prognostic models for HCC. These findings also indicate possible new lines of research for the development of innovative therapeutic approaches targeting PI4KA.
ISSN: 1471-2407

inserm-00934294
http://www.hal.inserm.fr/inserm-00934294
http://www.hal.inserm.fr/inserm-00934294/document
http://www.hal.inserm.fr/inserm-00934294/file/1471-2407-14-7.pdf
DOI : 10.1186/1471-2407-14-7

Éditeur(s) : HAL CCSD BioMed Central
Résumé : International audience
BACKGROUND: The orphan receptors COUP-TF (chicken ovalbumin upstream promoter transcription factor) I and II are members of the nuclear receptor superfamily that play distinct and critical roles in vertebrate organogenesis. The involvement of COUP-TFs in cancer development has recently been suggested by several studies but remains poorly understood. METHODS: MCF-7 breast cancer cells overexpressing COUP-TFI and human breast tumors were used to investigate the role of COUP-TFI in the regulation of CXCL12/CXCR4 signaling axis in relation to cell growth and migration. We used Immunofluorescence, western-blot, RT-PCR, Formaldehyde-assisted Isolation of Regulatory Elements (FAIRE) assays, as well as cell proliferation and migration assays. RESULTS: Previously, we showed that COUP-TFI expression is enhanced in breast cancer compared to normal tissue. Here, we report that the CXCL12/CXCR4 signaling pathway, a crucial pathway in cell growth and migration, is an endogenous target of COUP-TFI in breast cancer cells. The overexpression of COUP-TFI in MCF-7 cells inhibits the expression of the chemokine CXCL12 and markedly enhances the expression of its receptor, CXCR4. Our results demonstrate that the modification of CXCL12/CXCR4 expression by COUP-TFI is mediated by the activation of epithelial growth factor (EGF) and the EGF receptor. Furthermore, we provide evidence that these effects of COUP-TFI increase the growth and motility of MCF-7 cells in response to CXCL12. Cell migration toward a CXCL12 gradient was inhibited by AMD3100, a specific antagonist of CXCR4, or in the presence of excess CXCL12 in the cell culture medium. The expression profiles of CXCR4, CXCR7, CXCL12, and COUP-TFI mRNA in 82 breast tumors and control non-tumor samples were measured using real-time PCR. CXCR4 expression was found to be significantly increased in the tumors and correlated with the tumor grade, whereas the expression of CXCL12 was significantly decreased in the tumors compared with the healthy samples. Significantly higher COUP-TFI mRNA expression was also detected in grade 1 tumors. CONCLUSIONS: Together, our mechanistic in vitro assays and in vivo results suggest that a reduction in chemokine CXCL12 expression, with an enhancement of CXCR4 expression, provoked by COUP-TFI, could be associated with an increase in the invasive potential of breast cancer cells.
ISSN: 1471-2407

hal-01024753
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01024753
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01024753/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01024753/file/1471-2407-14-407.pdf
DOI : 10.1186/1471-2407-14-407

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
CXCL-test is a method that uses the estrogen-dependent secretion of the natural endogenous chemokine CXCL12 to evaluate the estrogenic activity of molecules. CXCL12 chemokine is involved in the estrogen dependent proliferation of breast cancer cells. Its measure is an indicator of cell proliferation and is used as an alternative test to classical proliferation test. Here we aimed to optimize this test, first to increase the number of tested molecules in a single assay and then to decrease the number of intermediate steps. The optimized CXCL-test was finally used for the evaluation of the estrogenic potency of emerging chemical pollutants: the UV filter benzophenones (BPs). The effect of BPs on CXCL12 secretion was also validated by real time quantitative RT-PCR. The optimized CXCL-test allowed a fast and direct assessment of estrogenic potency of molecules. The estrogenic activities of benzophenones were characterized and divided in two groups. The first one contains weak estrogenic compounds (BP, BP1, BP2, BP3, 234BP and 2344′BP). The second one contains medium estrogenic compounds (4BP, 44′BP, BP8, THB).
ISSN: 0045-6535

hal-01477205
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01477205
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01477205/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01477205/file/Rapid%20assessment%20of%20estrogenic%20compounds_accepted.pdf
DOI : 10.1016/j.chemosphere.2017.01.037