UCAGenomiX related publications

Du to our strong expertise in "omics" experiments and in microRNAs topics we decided to separate into 3 categories the related publications into which the Functional genomics Platform of Nice-Sophia-Antipolis is involved :
  1. Expression studies (DNA microarrays and high-throughput sequencing experiments)
  2. MicroRNA studies
  3. Miscellaneous

Magnone Virginie

 magnone@ipmc.cnrs.fr
 04 93 95 77 90
 660 route des lucioles 06560 Valbonne - Sophia-Antipolis

19 publications found

1. The "one airway, one disease" concept in light of Th2 inflammation.
Eur Respir J. 2018 Sep 6. pii: 1800437. doi: 10.1183/13993003.00437-2018.
Giovannini-Chami L, Paquet A, Sanfiorenzo C, Pons N, Cazareth J, Magnone V, Lebrigand K, Chevalier B, Vallauri A, Julia V, Marquette CH, Marcet B, Leroy S, Barbry P
Université Côte d'Azur, Hôpitaux pédiatriques de Nice CHU-Lenval, Pediatric Pulmonology and Allergology Department, Nice, France. Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Sophia Antipolis, France. Université Côte d'Azur, CHU de Nice, FHU Oncoage, Pulmonology Department, Nice, France.

In line with the pathophysiological continuum described between nose and bronchus in allergic respiratory diseases, we assessed whether nasal epithelium could mirror the Th2 status of bronchial epithelium.Nasal and bronchial cells were collected by brushings from patients with allergic rhinitis and asthma (AR, n=12), isolated allergic rhinitis (R, n=14) and healthy controls (C, n=13). Cellular composition was assessed by flow cytometry. Gene expression was analysed by RNA sequencing. Th2, Th17 and interferon signatures were derived from the literature.Infiltration by polymorphonuclear neutrophils in nose excluded 30% of the initial cohort. All bronchial samples from AR group were Th2-high. Nasal samples gene expression profile from the AR group correctly predicted the paired bronchial sample Th2 status in 71% of cases. Nevertheless, nasal cells did not appear as a reliable surrogate of the Th2 response, in particular due to a more robust influence of the interferon response in 14/26 nasal samples. Th2 scores correlated with mast cells counts (p<0.001) and numbers of sensitizations (p=0.006 and 0.002), while Th17 scores correlated with PMN counts (p<0.014).The large variability in nasal cell composition and type of inflammation restricts its use as a surrogate for assessing bronchial Th2 inflammation in AR patients.
Pubmed link : 30190271

2. Characterization of microRNAs from Arabidopsis galls highlights a role for miR159 in the plant response to the root-knot nematode Meloidogyne incognita
New Phytol. 2017 Sep 14. doi: 10.1111/nph.14717.
Medina C, da Rocha M, Magliano M, Ratpopoulo A, Revel B, Marteu N, Magnone V, Lebrigand K, Cabrera J, Barcala M, Silva AC, Millar A, Escobar C, Abad P, Favery B, Jaubert-Possamai S
INRA, Université Côte d'Azur, CNRS, ISA, 400 route des Chappes, BP167, 06903, Sophia Antipolis, France. UCA Genomix, Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UMR6097, Sophia Antipolis, France. Facultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-La Mancha, Avda. Carlos III S/N, Edificio Sabatini, E-45071, Toledo, Spain. Plant Science Division, Research School of Biology, Australian National University, Canberra, 2601, ACT, Australia.

Root knot nematodes (RKN) are root parasites that induce the genetic reprogramming of vascular cells into giant feeding cells and the development of root galls. MicroRNAs (miRNAs) regulate gene expression during development and plant responses to various stresses. Disruption of post-transcriptional gene silencing in Arabidopsis ago1 or ago2 mutants decrease the infection rate of RKN suggesting a role for this mechanism in the plant-nematode interaction. By sequencing small RNAs from uninfected Arabidopsis roots and from galls 7 and 14 d post infection with Meloidogyne incognita, we identified 24 miRNAs differentially expressed in gall as putative regulators of gall development. Moreover, strong activity within galls was detected for five miRNA promoters. Analyses of nematode development in an Arabidopsis miR159abc mutant had a lower susceptibility to RKN, suggesting a role for the miR159 family in the plant response to M. incognita. Localization of mature miR159 within the giant and surrounding cells suggested a role in giant cell and gall. Finally, overexpression of miR159 in galls at 14 d post inoculation was associated with the repression of the miR159 target MYB33 which expression is restricted to the early stages of infection. Overall, these results implicate the miR159 in plant responses to RKN.
Pubmed link : 28906559

3. A new long noncoding RNA (LncRNA) is induced in cutaneous squamous cell carcinoma and downregulates several anticancer and cell-differentiation genes in mouse.
J Biol Chem. 2017 Jun 8. pii: jbc.M117.776260. doi: 10.1074/jbc.M117.776260. [Epub ahead of print]
Ponzio G, Rezzonico R, Bourget I, Allan R, Nottet N, Popa A, Magnone V, Rios G, Mari B, Barbry P
Université Côte d’Azur, CNRS, IPMC, France. Université Côte d'Azur, CNRS, INSERM, IRCAN, France.

Keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. Although some of the early events involved in this pathology have been identified, the subsequent steps leading to tumor development are poorly defined. We demonstrate here that the development of mouse tumors induced by the concomitant application of a carcinogen and a tumor promoter (7,12 dimethylbenz[a]anthracene [DMBA] and 12-O-tetradecanoylphorbol-13-acetate [TPA], respectively) is associated with the upregulation of a previously uncharacterized long noncoding RNA (lncRNA), termed AK144841. We found that AK144841 expression was absent from normal skin and was specifically stimulated in tumors and highly tumorigenic cells. We also found that AK144841 exists in two variants, one consisting of a large 2-kb transcript composed of four exons and one of a 1.8-kb transcript lacking the second exon. Gain- and loss-of-function studies indicated that AK144841 mainly inhibited gene expression, specifically downregulating the expression of genes of the late-cornified-envelope-1 (Lce1) family involved in epidermal terminal differentiation and of anticancer genes such as Cgref1, Brsk1, Basp1, Dusp5, Btg2, Anpep, Dhrs9, Stfa2, Tpm1, SerpinB2, Cpa4, Crct1, Cryab, Il24, Csf2, and Rgs16. Interestingly, the lack of the second exon significantly decreased AK144841's inhibitory effect on gene expression. We also noted that high AK144841 expression correlated with a low expression of the aforementioned genes and with the tumorigenic potential of cell lines. These findings suggest that AK144841 could contribute to the dedifferentiation program of tumor-forming keratinocytes and to molecular cascades leading to tumor development.
Pubmed link : 28596382

4. Eμ and 3'RR IgH enhancers show hierarchic unilateral dependence in mature B-cells
Sci Rep. 2017 Mar 27;7(1):442. doi: 10.1038/s41598-017-00575-0.
Saintamand A, Vincent-Fabert C, Marquet M, Ghazzaui N, Magnone V, Pinaud E, Cogné M, Denizot Y
CNRS UMR 7276, CRIBL, Université de Limoges, Limoges, France. alexis.saintamand@live.fr. INSERM U1236, Université Rennes 1, Rennes, France. alexis.saintamand@live.fr. CNRS UMR 7276, CRIBL, Université de Limoges, Limoges, France. CNRS et Université de Nice Sophia Antipolis, Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097, Sophia, Antipolis, France. CNRS UMR 7276, CRIBL, Université de Limoges, Limoges, France. yves.denizot@unilim.fr.

Enhancer and super-enhancers are master regulators of cell fate. While they act at long-distances on adjacent genes, it is unclear whether they also act on one another. The immunoglobulin heavy chain (IgH) locus is unique in carrying two super-enhancers at both ends of the constant gene cluster: the 5'Eμ super-enhancer promotes VDJ recombination during the earliest steps of B-cell ontogeny while the 3' regulatory region (3'RR) is essential for late differentiation. Since they carry functional synergies in mature B-cells and physically interact during IgH locus DNA looping, we investigated if they were independent engines of locus remodelling or if their function was more intimately intermingled, their optimal activation then requiring physical contact with each other. Analysis of chromatin marks, enhancer RNA transcription and accessibility in Eμ- and 3'RR-deficient mice show, in mature activated B-cells, an unilateral dependence of this pair of enhancers: while the 3'RR acts in autonomy, Eμ in contrast likely falls under control of the 3'RR.
Pubmed link : 28348365

5. Characterizing isomiR variants within the microRNA-34/449 family
FEBS Lett. 2017 Mar;591(5):693-705. doi: 10.1002/1873-3468.12595. Epub 2017 Feb 28
Mercey O, Popa A, Cavard A, Paquet A, Chevalier B, Pons N, Magnone V, Zangari J, Brest P, Zaragosi LE, Ponzio G, Lebrigand K, Barbry P, Marcet B
CNRS, IPMC, Université Côte d'Azur, Sophia-Antipolis, Valbonne, France. CNRS, INSERM, IRCAN, FHU-OncoAge, Université Côte d'Azur, Sophia-Antipolis, Valbonne, France.

miR-34/449 microRNAs are conserved regulators of multiciliated cell differentiation. Here, we evidence and characterize expression of two isomiR variant sequences from the miR-34/449 family in human airway epithelial cells. These isomiRs differ from their canonical counterparts miR-34b and miR-449c by one supplemental uridine at their 5'-end, leading to a one-base shift in their seed region. Overexpression of canonical miR-34/449 or 5'-isomiR-34/449 induces distinct gene expression profiles and biological effects. However, some target transcripts and functional activities are shared by both canonical microRNAs and isomiRs. Indeed, both repress important targets that result in cell cycle blockage and Notch pathway inhibition. Our findings suggest that 5'-isomiR-34/449 may represent additional mechanisms by which miR-34/449 family finely controls several pathways to drive multiciliogenesis.
Pubmed link : 28192603

6. miR-200 family controls late steps of postnatal forebrain neurogenesis via Zeb2 inhibition.
Sci Rep. 2016 Oct 21;6:35729. doi: 10.1038/srep35729.
Beclin C, Follert P, Stappers E, Barral S, Nathalie C, de Chevigny A, Magnone V, Lebrigand K, Bissels U, Huylebroeck D, Bosio A, Barbry P, Seuntjens E, Cremer H
IBDM, Aix-Marseille Université, CNRS, UMR7288, 13288 Marseille, France. Laboratory of Molecular Biology, Dept Development and Regeneration, KULeuven, 3000 Leuven, Belgium. Miltenyi Biotec GmbH, Bergisch Gladbach, Germany. CNRS and University Nice Sophia Antipolis, IPMC, Sophia Antipolis, France. Dept Cell Biology, Erasmus MC, 3015 CN Rotterdam, The Netherlands. GIGA-Neurosciences, Université de Liège, 4000 Liège, Belgium.

During neurogenesis, generation, migration and integration of the correct numbers of each neuron sub-type depends on complex molecular interactions in space and time. MicroRNAs represent a key control level allowing the flexibility and stability needed for this process. Insight into the role of this regulatory pathway in the brain is still limited. We performed a sequential experimental approach using postnatal olfactory bulb neurogenesis in mice, starting from global expression analyses to the investigation of functional interactions between defined microRNAs and their targets. Deep sequencing of small RNAs extracted from defined compartments of the postnatal neurogenic system demonstrated that the miR-200 family is specifically induced during late neuronal differentiation stages. Using in vivo strategies we interfered with the entire miR-200 family in loss- and gain-of-function settings, showing a role of miR-200 in neuronal maturation. This function is mediated by targeting the transcription factor Zeb2. Interestingly, so far functional interaction between miR-200 and Zeb2 has been exclusively reported in cancer or cultured stem cells. Our data demonstrate that this regulatory interaction is also active during normal neurogenesis.
Pubmed link : 27767083

7. Deciphering the importance of the palindromic architecture of the immunoglobulin heavy-chain 3' regulatory region.
Nat Commun. 2016 Feb 17;7:10730. doi: 10.1038/ncomms10730.
Saintamand A, Vincent-Fabert C, Garot A, Rouaud P, Oruc Z, Magnone V, Cogné M, Denizot Y
Université de Limoges, CRIBL, UMR CNRS 7276, Limoges 87025, France. CNRS et Université de Nice Sophia Antipolis, Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097, Sophia Antipolis 06560, France. Institut Universitaire de France, Paris 75231, France.

The IgH 3' regulatory region (3'RR) controls class switch recombination (CSR) and somatic hypermutation (SHM) in B cells. The mouse 3'RR contains four enhancer elements with hs1,2 flanked by inverted repeated sequences and the centre of a 25-kb palindrome bounded by two hs3 enhancer inverted copies (hs3a and hs3b). hs4 lies downstream of the palindrome. In mammals, evolution maintained this unique palindromic arrangement, suggesting that it is functionally significant. Here we report that deconstructing the palindromic IgH 3'RR strongly affects its function even when enhancers are preserved. CSR and IgH transcription appear to be poorly dependent on the 3'RR architecture and it is more or less preserved, provided 3'RR enhancers are present. By contrast, a 'palindromic effect' significantly lowers VH germline transcription, AID recruitment and SHM. In conclusion, this work indicates that the IgH 3'RR does not simply pile up enhancer units but also optimally exposes them into a functional architecture of crucial importance.
Pubmed link : 26883548

8. Mantle cell lymphoma-like lymphomas in c-myc-3'RR/p53+/- mice and c-myc-3'RR/Cdk4R24C mice: differential oncogenic mechanisms but similar cellular origin.
Oncotarget. 2012 May;3(5):586-93.
Rouaud P, Fiancette R, Vincent-Fabert C, Magnone V, Cogné M, Dubus P, Denizot Y
UMR CNRS 7276, Faculté de Médecine, Limoges, France.

Mantle cell lymphoma (MCL) is a malignant lymphoproliferative B-cell disorder that does not occur spontaneously in mice but experimental mice model have been developed. Recently two different mice models prone to develop MCL-like lymphomas were generated: c-myc-3'RR/Cdk4(R24C) mice and c-myc-3'RR/p53+/- mice. Comparison of their gene expression profiles does not highlight specific differences other than those in relation with their specific mutational status (i.e., Cdk4(R24C) mutation or p53 mutations). We propose that similarly to typical human MCL and its blastoid or cyclin-D1 variants that correspond to the same genetic entity, MCL-like lymphomas of c-myc-3'RR/ p53+/- mice and c-myc-3'RR/Cdk4(R24C) mice represent a spectrum of the same entity.
Pubmed link : 22592113

9. A defect of the INK4-Cdk4 checkpoint and Myc collaborate in blastoid mantle cell lymphoma-like lymphoma formation in mice.
Am J Pathol. 2012 Apr;180(4):1688-701. doi: 10.1016/j.ajpath.2012.01.004. Epub 2012 Feb 9.
Vincent-Fabert C, Fiancette R, Rouaud P, Baudet C, Truffinet V, Magnone V, Guillaudeau A, Cogné M, Dubus P, Denizot Y
Faculty of Medicine, UMR CNRS 6101, Limoges, France.

Mantle cell lymphoma (MCL) is a B-cell malignancy characterized by a monoclonal proliferation of lymphocytes with the co-expression of CD5 and CD43, but not of CD23. Typical MCL is associated with overexpression of cyclin D1, and blastoid MCL variants are associated with Myc (alias c-myc) translocations. In this study, we developed a murine model of MCL-like lymphoma by crossing Cdk4(R24C) mice with Myc-3'RR transgenic mice. The Cdk4(R24C) mouse is a knockin strain that expresses a Cdk4 protein that is resistant to inhibition by p16(INK4a) as well as other INK4 family members. Ablation of INK4 control on Cdk4 does not affect lymphomagenesis, B-cell maturation, and functions in Cdk4(R24C) mice. Additionally, B cells were normal in numbers, cell cycle activity, mitogen responsiveness, and Ig synthesis in response to activation. By contrast, breeding Cdk4(R24C) mice with Myc-3'RR transgenic mice prone to develop aggressive Burkitt lymphoma-like lymphoma (CD19(+)IgM(+)IgD(+) cells) leads to the development of clonal blastoid MCL-like lymphoma (CD19(+)IgM(+)CD5(+)CD43(+)CD23(-) cells) in Myc/Cdk4(R24C) mice. Western blot analysis revealed high amounts of Cdk4/cyclin D1 complexes as the main hallmark of these lymphomas. These results indicate that although silent in nonmalignant B cells, a defect in the INK4-Cdk4 checkpoint can participate in lymphomagenesis in conjunction with additional alterations of cell cycle control, a situation that might be reminiscent of the development of human blastoid MCL.
Pubmed link : 22326754

10. A p53 defect sensitizes various stages of B cell development to lymphomagenesis in mice carrying an IgH 3' regulatory region-driven c-myc transgene.
J Immunol. 2011 Dec 1;187(11):5772-82. doi: 10.4049/jimmunol.1102059. Epub 2011 Oct 28.
Fiancette R, Rouaud P, Vincent-Fabert C, Laffleur B, Magnone V, Cogné M, Denizot Y
Faculté de Médecine, Unité Mixte de Recherche 6101, Centre National de la Recherche Scientifique, 87025 Limoges, France.

Although c-myc is classically described as the driving oncogene in Burkitt's lymphoma (BL), deregulation and mutations of c-myc have been reported in multiple solid tumors and in other mature B cell malignancies such as mantle cell lymphoma (MCL), myeloma, and plasma cell lymphoma (PCL). After translocation into the IgH locus, c-myc is constitutively expressed under the control of active IgH enhancers. Those located in the IgH 3' regulatory region (3'RR) are master control elements of class switch recombination and of the transcriptional burst associated with plasma cell differentiation. c-myc-3'RR mice are prone to lymphomas with rather homogeneous, most often BL-like, phenotypes with incomplete penetrance (75% tumor incidence) and long latencies (10-12 mo). To reproduce c-myc-induced mature B cell lymphomagenesis in the context of an additional defect often observed in human lymphomas, we intercrossed c-myc-3'RR with p53(+/-) mice. Double transgenic c-myc-3'RR/p53(+/-) mice developed lymphoma with short latency (2-4 mo) and full penetrance (100% tumor incidence). The spectrum of B lymphomas occurring in c-myc-3'RR/p53(+/-) mice was widened, including nonactivated (CD43(-)) BL, activated (CD43(+)) BL, MCL-like lymphoma, and PCL, thus showing that 3'RR-mediated deregulation of c-myc can promote various types of B lymphoproliferation in cells that first acquired a p53 defect. c-myc/p53(+/-) mice closely reproduce many features of BL, MCL, and PCL and provide a novel and efficient model to dissect the molecular events leading to c-myc-induced lymphomagenesis and an important tool to test potential therapeutic agents on malignant B cells featuring various maturation stages.
Pubmed link : 22039300

11. Adaptations to endosymbiosis in a cnidarian-dinoflagellate association: differential gene expression and specific gene duplications.
PLoS Genet. 2011 Jul;7(7):e1002187. doi: 10.1371/journal.pgen.1002187. Epub 2011 Jul 21.
Ganot P, Moya A, Magnone V, Allemand D, Furla P, Sabourault C
Université de Nice-Sophia-Antipolis, Nice, France.

Trophic endosymbiosis between anthozoans and photosynthetic dinoflagellates forms the key foundation of reef ecosystems. Dysfunction and collapse of symbiosis lead to bleaching (symbiont expulsion), which is responsible for the severe worldwide decline of coral reefs. Molecular signals are central to the stability of this partnership and are therefore closely related to coral health. To decipher inter-partner signaling, we developed genomic resources (cDNA library and microarrays) from the symbiotic sea anemone Anemonia viridis. Here we describe differential expression between symbiotic (also called zooxanthellate anemones) or aposymbiotic (also called bleached) A. viridis specimens, using microarray hybridizations and qPCR experiments. We mapped, for the first time, transcript abundance separately in the epidermal cell layer and the gastrodermal cells that host photosynthetic symbionts. Transcriptomic profiles showed large inter-individual variability, indicating that aposymbiosis could be induced by different pathways. We defined a restricted subset of 39 common genes that are characteristic of the symbiotic or aposymbiotic states. We demonstrated that transcription of many genes belonging to this set is specifically enhanced in the symbiotic cells (gastroderm). A model is proposed where the aposymbiotic and therefore heterotrophic state triggers vesicular trafficking, whereas the symbiotic and therefore autotrophic state favors metabolic exchanges between host and symbiont. Several genetic pathways were investigated in more detail: i) a key vitamin K-dependant process involved in the dinoflagellate-cnidarian recognition; ii) two cnidarian tissue-specific carbonic anhydrases involved in the carbon transfer from the environment to the intracellular symbionts; iii) host collagen synthesis, mostly supported by the symbiotic tissue. Further, we identified specific gene duplications and showed that the cnidarian-specific isoform was also up-regulated both in the symbiotic state and in the gastroderm. Our results thus offer new insight into the inter-partner signaling required for the physiological mechanisms of the symbiosis that is crucial for coral health.
Pubmed link : 21811417

12. miR-210 is overexpressed in late stages of lung cancer and mediates mitochondrial alterations associated with modulation of HIF-1 activity.
Cell Death Differ. 2010 Oct 1.
Puissegur MP, Mazure NM, Bertero T, Pradelli L, Grosso S, Robbe-Sermesant K, Maurin T, Lebrigand K, Cardinaud B, Hofman V, Fourre S, Magnone V, Ricci JE, Pouyssegur J, Gounon P, Hofman P, Barbry P, Mari B
[1] Institut de Pharmacologie Moleculaire et Cellulaire, CNRS UMR6097, Sophia Antipolis, France [2] University of Nice Sophia-Antipolis, Nice, France.

Following the identification of a set of hypoxia-regulated microRNAs (miRNAs), recent studies have highlighted the importance of miR-210 and of its transcriptional regulation by the transcription factor hypoxia-inducible factor-1 (HIF-1). We report here that miR-210 is overexpressed at late stages of non-small cell lung cancer. Expression of miR-210 in lung adenocarcinoma A549 cells caused an alteration of cell viability associated with induction of caspase-3/7 activity. miR-210 induced a loss of mitochondrial membrane potential and the apparition of an aberrant mitochondrial phenotype. The expression profiling of cells overexpressing miR-210 revealed a specific signature characterized by enrichment for transcripts related to 'cell death' and 'mitochondrial dysfunction', including several subunits of the electron transport chain (ETC) complexes I and II. The transcript coding for one of these ETC components, SDHD, subunit D of succinate dehydrogenase complex (SDH), was validated as a bona fide miR-210 target. Moreover, SDHD knockdown mimicked miR-210-mediated mitochondrial alterations. Finally, miR-210-dependent targeting of SDHD was able to activate HIF-1, in line with previous studies linking loss-of-function SDH mutations to HIF-1 activation. miR-210 can thus regulate mitochondrial function by targeting key ETC component genes with important consequences on cell metabolism, survival and modulation of HIF-1 activity. These observations help explain contradictory data regarding miR-210 expression and its putative function in solid tumors.
Pubmed link : 20885442

13. Innovative approach for transcriptomic analysis of obligate intracellular pathogen: selective capture of transcribed sequences of Ehrlichia ruminantium.
BMC Mol Biol. 2009 Dec 24;10:111.
Emboule L, Daigle F, Meyer DF, Mari B, Pinarello V, Sheikboudou C, Magnone V, Frutos R, Viari A, Barbry P, Martinez D, Lefrançois T, Vachiery N
UMR 15 CIRAD-INRA, Controle des maladies animales exotiques et emergentes, Site de Duclos, Prise d'Eau 97170, Petit Bourg, Guadeloupe. loic.emboule@cirad.fr

BACKGROUND: Whole genome transcriptomic analysis is a powerful approach to elucidate the molecular mechanisms controlling the pathogenesis of obligate intracellular bacteria. However, the major hurdle resides in the low quantity of prokaryotic mRNAs extracted from host cells. Our model Ehrlichia ruminantium (ER), the causative agent of heartwater, is transmitted by tick Amblyomma variegatum. This bacterium affects wild and domestic ruminants and is present in Sub-Saharan Africa and the Caribbean islands. Because of its strictly intracellular location, which constitutes a limitation for its extensive study, the molecular mechanisms involved in its pathogenicity are still poorly understood. RESULTS: We successfully adapted the SCOTS method (Selective Capture of Transcribed Sequences) on the model Rickettsiales ER to capture mRNAs. Southern Blots and RT-PCR revealed an enrichment of ER's cDNAs and a diminution of ribosomal contaminants after three rounds of capture. qRT-PCR and whole-genome ER microarrays hybridizations demonstrated that SCOTS method introduced only a limited bias on gene expression. Indeed, we confirmed the differential gene expression between poorly and highly expressed genes before and after SCOTS captures. The comparative gene expression obtained from ER microarrays data, on samples before and after SCOTS at 96 hpi was significantly correlated (R2 = 0.7). Moreover, SCOTS method is crucial for microarrays analysis of ER, especially for early time points post-infection. There was low detection of transcripts for untreated samples whereas 24% and 70.7% were revealed for SCOTS samples at 24 and 96 hpi respectively. CONCLUSIONS: We conclude that this SCOTS method has a key importance for the transcriptomic analysis of ER and can be potentially used for other Rickettsiales. This study constitutes the first step for further gene expression analyses that will lead to a better understanding of both ER pathogenicity and the adaptation of obligate intracellular bacteria to their environment.
Pubmed link : 20034374

14. Genetic differences among Staphylococcus aureus isolates from dairy ruminant species: a single-dye DNA microarray approach
Vet Microbiol 133, 105-114
Vautor E, Magnone V, Rios G, Le Brigand K, Bergonier D, Lina G, Meugnier H, Barbry P, Thiery R, Pepin M
Agence Française de Securite Sanitaire des Aliments (AFSSA), Unite Pathologie des Petits Ruminants, Sophia-Antipolis, France. e.vautor@sophia.afssa.fr

Staphylococcus aureus is recognized worldwide as a major pathogen causing clinical or subclinical intramammary infections in lactating sheep, goats and cows. The present study was carried out to compare 65 S. aureus isolates mainly obtained from nasal carriage and subclinical mastitis in dairy sheep and 43 isolates obtained from subclinical mastitis from 22 goats and 21 cows. A DNA microarray, containing probes against 190 true or putative virulence factors, was used to detect the presence of the virulence genes. Their presence/absence was independently assessed by PCR for the genes of interest. Sheep isolates obtained from the nostrils or the udders did not show any significant tissue specific virulence factor. The dominant pulse-field electrophoresis profile (OV/OV'), associated with spa clonal complex spa-CC 1773, matched mainly with the agr group III and was only found in ovine and caprine isolates. This clone was more specifically characterized by the prevalence of the following virulence genes: lpl4, ssl6, bsaA1, bsaB, bsaP, SAV0812. Moreover, seven virulence-associated genes (lpl1, sel, sec, tst, lukF-PV-like component, lukM, SAV0876) were associated with isolates from small ruminants, while the egc cluster, fhuD1, abiF and SAV2496 with bovine isolates. This genomic study suggests the existence of lineage- and host-specific genes leading to the development of host-specific pathogenic traits of S. aureus isolates.
Pubmed link : 18640795

15. Easy and fast detection and genotyping of high-risk human papillomavirus by dedicated DNA microarrays.
J Virol Methods. 2006 Nov;137(2):236-44. Epub 2006 Aug 1.
Albrecht V, Chevallier A, Magnone V, Barbry P, Vandenbos F, Bongain A, Lefebvre JC, Giordanengo V
Laboratoire de Virologie, Hopital de l'Archet, BP 3079, 151 route de Saint-Antoine de Ginestiere, 06200 Nice Cedex 3, France.

Persistent cervical high-risk human papillomavirus (HPV) infection is correlated with an increased risk of developing a high-grade cervical intraepithelial lesion. A two-step method was developed for detection and genotyping of high-risk HPV. DNA was firstly amplified by asymmetrical PCR in the presence of Cy3-labelled primers and dUTP. Labelled DNA was then genotyped using DNA microarray hybridization. The current study evaluated the technical efficacy of laboratory-designed HPV DNA microarrays for high-risk HPV genotyping on 57 malignant and non-malignant cervical smears. The approach was evaluated for a broad range of cytological samples: high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL) and atypical squamous cells of high-grade (ASC-H). High-risk HPV was also detected in six atypical squamous cells of undetermined significance (ASC-US) samples; among them only one cervical specimen was found uninfected, associated with no histological lesion. The HPV oligonucleotide DNA microarray genotyping detected 36 infections with a single high-risk HPV type and 5 multiple infections with several high-risk types. Taken together, these results demonstrate the sensitivity and specificity of the HPV DNA microarray approach. This approach could improve clinical management of patients with cervical cytological abnormalities.
Pubmed link : 16879879

16. An open-access long oligonucleotide microarray resource for analysis of the human and mouse transcriptomes.
Nucleic Acids Res. 2006 Jul 19;34(12):e87
Le Brigand K, Russell R, Moreilhon C, Rouillard JM, Jost B, Amiot F, Magnone V, Bole-Feysot C, Rostagno P, Virolle V, Defamie V, Dessen P, Williams G, Lyons P, Rios G, Mari B, Gulari E, Kastner P, Gidrol X, Freeman TC, Barbry P
CNRS, Institut de Pharmacologie Moleculaire et Cellulaire, UMR6097, 660, route des Lucioles, F-06560 Sophia Antipolis, France.

Two collections of oligonucleotides have been designed for preparing pangenomic human and mouse microarrays. A total of 148,993 and 121,703 oligonucleotides were designed against human and mouse transcripts. Quality scores were created in order to select 25,342 human and 24,109 mouse oligonucleotides. They correspond to: (i) a BLAST-specificity score; (ii) the number of expressed sequence tags matching each probe; (iii) the distance to the 3' end of the target mRNA. Scores were also used to compare in silico the two microarrays with commercial microarrays. The sets described here, called RNG/MRC collections, appear at least as specific and sensitive as those from the commercial platforms. The RNG/MRC collections have now been used by an Anglo-French consortium to distribute more than 3500 microarrays to the academic community. Ad hoc identification of tissue-specific transcripts and a approximately 80% correlation with hybridizations performed on Affymetrix GeneChiptrade mark suggest that the RNG/MRC microarrays perform well. This work provides a comprehensive open resource for investigators working on human and mouse transcriptomes, as well as a generic method to generate new microarray collections in other organisms. All information related to these probes, as well as additional information about commercial microarrays have been stored in a freely-accessible database called MEDIANTE.
Pubmed link : 17384016

17. Live Staphylococcus aureus and bacterial soluble factors induce different transcriptional responses in human airway cells.
Physiol Genomics. 2005 Feb 10;20(3):244-55. Epub 2004 Dec 14.Click here to read
Moreilhon C, Gras D, Hologne C, Bajolet O, Cottrez F, Magnone V, Merten M, Groux H, Puchelle E, Barbry P
Institut de Pharmacologie Moleculaire et Cellulaire UMR 6097 Centre National de la Recherche Scientifique, Universite de Nice-Sophia Antipolis, Valbonne, France.

To characterize the response of respiratory epithelium to infection by Staphylococcus aureus (S. aureus), human airway cells were incubated for 1 to 24 h with a supernatant of a S. aureus culture (bacterial supernatant), then profiled with a pangenomic DNA microarray. Because an upregulation of many genes was noticed around 3 h, three independent approaches were then used to characterize the host response to a 3-h contact either with bacterial supernatant or with live bacteria: 1) a DNA microarray containing 4,200 sequence-verified probes, 2) a semiquantitative RT-PCR with a set of 537 pairs of validated primers, or 3) ELISA assay of IL-8, IL-6, TNFalpha, and PGE(2). Among others, Fos, Jun, and EGR-1 were upregulated by the bacterial supernatant and by live bacteria. Increased expression of bhlhb2 and Mig-6, promoter regions which harbor HIF responding elements, was explained by an increased expression of the HIF-1alpha protein. Activation of the inducible form of cyclooxygenase, COX-2, and of the interleukins IL-1, IL-6, and IL-8, as well as of the NF-kappaB pathway, was observed preferentially in cells in contact with bacterial supernatant. Early infection was characterized by an upregulation of anti-apoptotic genes and a downregulation of pro-apoptotic genes. This correlated with a necrotic, rather than apoptotic cell death. Overall, this first global description of an airway epithelial infection by S. aureus demonstrates a larger global response to bacterial supernatant (in term of altered genes and variation factors) than to exponentially growing live bacteria.
Pubmed link : 15598879

18. Early gene expression in wounded human keratinocytes revealed by DNA microarray analysis.
Comp Funct Genomics. 2003;4(1):47-55.
Dayem MA, Moreilhon C, Turchi L, Magnone V, Christen R, Ponzio G, Barbry P
Laboratoire de Physiologie Genomique des Eucaryotes CNRS/UNSA UMR 6097 IPMC F-06560 Sophia Antipolis France.

WOUND HEALING INVOLVES SEVERAL STEPS: spreading of the cells, migration and proliferation. We have profiled gene expression during the early events of wound healing in normal human keratinocytes with a home-made DNA microarray containing about 1000 relevant human probes. An original wounding machine was used, that allows the wounding of up to 40% of the surface of a confluent monolayer of cultured cells grown on a Petri dish (compared with 5% with a classical 'scratch' method). The two aims of the present study were: (a) to validate a limited number of genes by comparing the expression levels obtained with this technique with those found in the literature; (b) to combine the use of the wounding machine with DNA microarray analysis for large-scale detection of the molecular events triggered during the early stages of the wound-healing process. The time-courses of RNA expression observed at 0.5, 1.5, 3, 6 and 15 h after wounding for genes such as c-Fos, c-Jun, Egr1, the plasminogen activator PLAU (uPA) and the signal transducer and transcription activator STAT3, were consistent with previously published data. This suggests that our methodologies are able to perform quantitative measurement of gene expression. Transcripts encoding two zinc finger proteins, ZFP36 and ZNF161, and the tumour necrosis factor alpha-induced protein TNFAIP3, were also overexpressed after wounding. The role of the p38 mitogen-activated protein kinase (p38MAPK) in wound healing was shown after the inhibition of p38 by SB203580, but our results also suggest the existence of surrogate activating pathways.
Pubmed link : 18629100

19. TWIK-2, an inactivating 2P domain K+ channel
J Biol Chem. 2000 Sep 15;275(37):28722-30.
Patel AJ, Maingret F, Magnone V, Fosset M, Lazdunski M, Honore E
Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UPR 411, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France.

We cloned human and rat TWIK-2 and expressed this novel 2P domain K(+) channel in transiently transfected COS cells. TWIK-2 is highly expressed in the gastrointestinal tract, the vasculature, and the immune system. Rat TWIK-2 currents are about 15 times larger than human TWIK-2 currents, but both exhibit outward rectification in a physiological K(+) gradient and mild inward rectification in symmetrical K(+) conditions. TWIK-2 currents are inactivating at depolarized potentials, and the kinetic of inactivation is highly temperature-sensitive. TWIK-2 shows an extremely low conductance, which prevents the visualization of discrete single channel events. The inactivation and rectification are intrinsic properties of TWIK-2 channels. In a physiological K(+) gradient, TWIK-2 is half inhibited by 0.1 mm Ba(2+), quinine, and quinidine. Finally, cysteine 53 in the M1P1 external loop is required for functional expression of TWIK-2 but is not critical for subunit self-assembly. TWIK-2 is the first reported 2P domain K(+) channel that inactivates. The base-line, transient, and delayed activities of TWIK-2 suggest that this novel 2P domain K(+) channel may play an important functional role in cell electrogenesis.
Pubmed link : 10887187