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Genetic mapping and genome-wide studies provide evidence for the association of several genetic polymorphisms with malaria, a complex pathological disease with multiple severity degrees. We have previously described Berr1and Berr2 as candidate genes identified in the WLA/Pas inbreed mouse strain predisposing to resistance to cerebral malaria (CM) induced by P. berghei ANKA. We report in this study the phenotypic and functional characteristics of a congenic strain we have derived for Berr2WLA allele on the C57BL/6JR (B6) background. B6.WLA-Berr2 was found highly resistant to CM compared to C57BL/6JR susceptible mice. The mechanisms associated with CM resistance were analyzed by combining genotype, transcriptomic and immune response studies. We found that B6.WLA-Berr2 mice showed a reduced parasite sequestration and blood-brain barrier disruption with low CXCR3+ T cell infiltration in the brain along with altered glial cell response upon P. berghei ANKA infection compared to B6. In addition, we have identified the CD300f, belonging to a family of Ig-like encoding genes, as a potential candidate associated with CM resistance. Microglia cells isolated from the brain of infected B6.WLA-Berr2 mice significantly expressed higher level of CD300f compared to CMS mice and were associated with inhibition of inflammatory response.

International audience; The P2X purinergic receptor 7 (P2RX7) is a poorly selective ATP‐gated ion channel. Although P2RX7 binds ATP with relatively low affinity, prolonged activation can lead to nonselective membrane pore formation. Indeed, brief exposure to ATP triggers a rapid Ca2+ influx, whereas prolonged exposure to high ATP concentrations results in the passage of larger organic molecules. P2RX7 is involved in the physiopathology of a number of diseases and has notably emerged as a potential therapeutic target in inflammation, neuropathic pain, Alzheimer's disease, and cancer—prompting growing interest in the synthesis of novel P2RX7 modulators and the development of reliable, stringent screening methods. In the present study, we developed methods based on conventional flow cytometry, imaging flow cytometry and spectral flow cytometry and used them to measure P2RX7's activity upon activation by 3'‐O‐(4‐benzoyl)benzoyl ATP. We also demonstrated the use of the highly sensitive DNA‐intercalating dye TO‐PRO‐3 to determine P2RX7's large pore activity. The simultaneous quantification of calcium influx (Fluo‐3 AM), large pore opening (TO‐PRO‐3), and viability (propidium iodide) is a very efficient method for low‐ to medium‐throughput screening of P2RX7 modulators. Agonist and antagonist potencies can be accurately evaluated. Spectral cytometry notably enabled us to assay several biological activities while correcting for the intrinsic fluorescence of the screened compounds—otherwise a well‐known limitation of fluorescence‐based screening. Hence, spectral cytometry appears to be a useful, novel tool for drug candidate screening.

International audience; The escalating burden of antibiotic drug resistance necessitates research into novel classes of antibiotics and their mechanism of action. Pyrrolomycins are a family of potent natural product antibiotics with nanomolar activity against Gram-positive bacteria, yet with an elusive mechanism of action. In this work, we dissect the apparent Gram-positive specific activity of pyrrolomycins and show that Gram-negative bacteria are equally sensitive to pyrrolomycins when drug efflux transporters are removed and that albumin in medium plays a large role in pyrrolomycin activity. The selection of resistant mutants allowed for the characterization and validation of a number of mechanisms of resistance to pyrrolomycins in both Staphylococcus aureus and an Escherichia coli ΔtolC mutant, all of which appear to affect compound penetration rather than being target associated. Imaging of the impact of pyrrolomycin on the E. coli ΔtolC mutant using scanning electron microscopy showed blebbing of the bacterial cell wall often at the site of bacterial division. Using potentiometric probes and an electrophysiological technique with an artificial bilayer lipid membrane, it was demonstrated that pyrrolomycins C and D are very potent membrane-depolarizing agents, an order of magnitude more active than conventional carbonyl cyanide m-chlorophenylhydrazone (CCCP), specifically disturbing the proton gradient and uncoupling oxidative phosphorylation via protonophoric action. This work clearly unveils the until-now-elusive mechanism of action of pyrrolomycins and explains their antibiotic activity as well as mechanisms of innate and acquired drug resistance in bacteria.

Background: Malaria is a complex pathological disease. Genetic mapping and genome-wide studies provide evidence for the association of several genetic polymorphisms with malaria severity degrees. However, the genetic factors that predispose to CM remain poorly understood. We previously studied the genetic determinism of experimental CM (ECM) using the mouse malaria parasite P. berghei ANKA (PbA). We described two loci, Berr1WLA and Berr1WLA, associated with resistance to ECM (CMR) in WLA/Pas mice. In this study, we analyzed the consequences induced by PbA infection in these new congenic mouse strains. Methods: We derived two congenic strains on the C57BL/6JR background for Berr1WLA and Berr1WLA alleles, which were partially and highly CMR, respectively compared to C57BL/6JR CMS mice. The mechanisms associated with the CMR phenotype were studied by combining genotype, transcriptomic and immune response analyses. Findings: Upon P. berghei ANKA infection, mice bearing the Berr1WLA allele showed reduced parasite load, blood brain barrier disruption and diminished CXCR3+ T cell infiltration in the brain along with altered astrocyte, microglia, and monocyte response and decreased pro-inflammatory factors production. We have identified the CD300f as a potential candidate gene associated with CM resistance. We found that brain monocytes and microglial cells from B6.W-Berr2 compared to B6 mice highly expressed the CD300f receptors upon PbA infection. Interpretation: This report underlines the value of using congenic mouse strains to identify novel genetic factors in the etiology of CM and provides a unique model for studying the immunopathological processes associated to severe malaria. Fundaing Statement: This work was supported by ARCir “Dynamique” Region Nord-Pas de Calais and the LabEx PARAFRAP: ANR-11-LABX-0024. TK is a recipient of PRESTIGE (Sanction No.2014-1- 0043: Marie Curie Actions / FP7 / PCOFUND-GA-2013-609102) and Region Haut-de-France (CNRS - Sanction No.147894) Post-doctoral Fellowship and Fondation des Treilles (Young Rresearcher Prize 2016). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: All animal work was conducted in strict accordance with the Directive 2010/63/EU of the European Parliament and Council ‘On the protection of animals used for scientific purposes’. All protocols were approved by the Ethical Committee of Institute Pasteur of Lille and the French Ministry of Agriculture (permit number A 75-15-28) and performed in compliance with the NIH Animal Welfare Assurance #A5476-01 issued on 02/07/2007. All efforts were taken to minimize animal suffering.

// Guillaume Brocqueville 1 , Renee S. Chmelar 2 , Helene Bauderlique-Le Roy 1 , Emeric Deruy 3 , Lu Tian 1 , Robert L. Vessella 4 , Norman M. Greenberg 2, 5 , Larry R. Rohrschneider 2, * , Roland P. Bourette 1 1 University of Lille, CNRS, Institut Pasteur de Lille, UMR 8161-M3T-Mechanisms of Tumorigenesis and Targeted Therapies, SIRIC ONCOLille, F-59000 Lille, France 2 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA 3 BioImaging Center Lille, Institut Pasteur de Lille, University of Lille, F-59000 Lille, France 4 Department of Urology, University of Washington, Seattle, WA 98195, USA 5 Present address: NMG Scientific Consulting, North Potomac, MD 20878, USA * Deceased Correspondence to: Roland P. Bourette, e-mail: roland.bourette@ibl.cnrs.fr Keywords: stem cells, prostate, s-SHIP, epithelial cells, transgenic mouse Received: November 04, 2015 Accepted: March 28, 2016 Published: April 12, 2016 ABSTRACT Isolation of prostate stem cells (PSCs) is crucial for understanding their biology during normal development and tumorigenesis. In this aim, we used a transgenic mouse model expressing GFP from the stem cell-specific s-SHIP promoter to mark putative stem cells during postnatal prostate development. Here we show that cells identified by GFP expression are present transiently during early prostate development and localize to the basal cell layer of the epithelium. These prostate GFP + cells are a subpopulation of the Lin – CD24 + Sca-1 + CD49f + cells and are capable of self-renewal together with enhanced growth potential in sphere-forming assay in vitro , a phenotype consistent with that of a PSC population. Transplantation assays of prostate GFP + cells demonstrate reconstitution of prostate ducts containing both basal and luminal cells in renal grafts. Altogether, these results demonstrate that s-SHIP promoter expression is a new marker for neonatal basal prostate cells exhibiting stem cell properties that enables PSCs in situ identification and isolation via a single consistent parameter. Transcriptional profiling of these GFP + neonatal stem cells showed an increased expression of several components of the Wnt signaling pathway. It also identified stem cell regulators with potential applications for further analyses of normal and cancer stem cells.

Understanding normal and cancer stem cells should provide insights into the origin of prostate cancer and their mechanisms of resistance to current treatment strategies. In this study, we isolated and characterized stem-like cells present in the immortalized human prostate cell line, RWPE-1. We used a reporter system with green fluorescent protein (GFP) driven by the promoter of s-SHIP (for stem-SH2-domain-containing 5'-inositol phosphatase) whose stem cell-specific expression has been previously shown. We observed that s-SHIP-GFP-expressing RWPE-1 cells showed stem cell characteristics such as increased expression of stem cell surface markers (CD44, CD166, TROP2) and pluripotency transcription factors (Oct4, Sox2), and enhanced sphere-forming capacity and resistance to arsenite-induced cell death. Concomitant increased expression of the long noncoding RNA H19 was observed, which prompted us to investigate a putative role in stemness for this oncofetal gene. Targeted suppression of H19 with siRNA decreased Oct4 and Sox2 gene expression and colony-forming potential in RWPE-1 cells. Conversely, overexpression of H19 significantly increased gene expression of these two transcription factors and the sphere-forming capacity of RWPE-1 cells. Analysis of H19 expression in various prostate and mammary human cell lines revealed similarities with Sox2 expression, suggesting that a functional relationship may exist between H19 and Sox2. Collectively, we provide the first evidence that s-SHIP-GFP promoter reporter offers a unique marker for the enrichment of human stem-like cell populations and highlight a role in stemness for the long noncoding RNA H19.

Breast cancer is the most common cancer in women worldwide. The isolation and characterization of breast cancer stem cells (CSC) are crucial for understanding cancer biology and revealing potential therapeutic targets. One of the major issues in the study of CSC is the lack of reliable markers. A transgenic mouse model (Tg 11.5kb–GFP) was generated using the 11.5kb s-SHIP (stem-SH2-containing 5’-Inositol Phosphatase) promoter that specifically expressed enhanced green fluorescent protein (GFP) in embryonic and various tissue stem cells. In the mammary gland, previous experiments showed that GFP labels puberty cap cells and pregnancy basal alveolar bud cells, and it has been demonstrated that these mammary GFP+ cells are activated tissue stem cells. In order to determine if s-SHIP promoter expression could also mark mammary cancer stem cells, we generated a bi-transgenic mouse model by crossing Tg 11.5kb-GFP mice with Tg C3(1)/Tag mice. Tg C3(1)/Tag mice express SV40 T antigen under the regulatory control of the rat prostatic steroid binding protein C3(1) gene. In female mice, the transgene is expressed primarily in the mammary gland. Mice develop mammary hyperplasia by 3 months of age with subsequent development of mammary adenocarcinoma by 6 months of age. Here we show the presence of a rare population of GFP+ cells in mammary tumors of female bi-transgenic mice by using immunohistochemical and flow cytometry analysis. The GFP+ mammary cancer cells are also CD24+/CD49f+/CD29+. As compared to GFP- cells, GFP+ cells exhibit both a higher tumor sphere-forming potential, and a higher tumorigenicity when transplanted into SCID and FVB recipient mice. Moreover, upon subsequent transplantation, the GFP+ cells generated heterogeneous tumors that displayed properties similar to the primary tumor. Currently, we are conducting transcriptomic analysis of these GFP+ and GFP- cells in order to reveal differentially expressed genes. Altogether, these results suggest that s-SHIP promoter expression may be a new marker of mammary CSC that enables their identification and isolation via a single consistent parameter. Furthermore, our bi-transgenic C3(1)/Tag x 11.5kb-GFP mice provide a novel model for study of mammary cancer stem cells and investigation of potential therapeutic targets for breast cancer. Citation Format: Lu Tian, Chann Lagadec, Eric Adriaenssens, Emmanuel Bouchaert, Hélène Bauderlique-Le Roy, Xuefen LeBourhis, Roland P. Bourette. S-SHIP promoter expression identifies a putative cancer stem cell population in C3(1)/Tag murine mammary tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1922. doi:10.1158/1538-7445.AM2017-1922

The ability to isolate epithelial stem cells of the adult prostate (PrSCs) is crucial in terms of understanding their biology and purported role in tumor initiation. Previous isolations of PrSCs were based on subdividing total prostate epithelial cells by flow cytometry using antibodies to cell surface markers. In this study we have used a novel approach shown previously to successfully identify mammary stem cells (Bai & Rohrschneider, 2010, Genes Dev. 24:1882). This method employs the s-SHIP (stem-SH2-containing 5'-Inositol Phosphatase) promoter to tag presumptive stem cells with GFP in a transgenic mouse model, called Tg11.5kb-GFP. Here we show that in Tg11.5kb-GFP mice, epithelial GFP+ (s-SHIP-expressing) cells are present transiently during early mouse prostate development: during the first week after birth, GFP expression was present in the growing buds in 1-3 % of total prostate cells, then decreased during the second week as the solid epithelial cords canalize. During this process, luminal and basal cells differenciate, and GFP+ cells localized in the basal cell layer of the epithelium. GFP expression was then totally turned off after 3 weeks. We therefore focused our study on epithelial GFP+ cells present between day 5 and day 7 after birth in order to determine if this population represents a population of putative stem cells. Cell surface marker analyses demonstrate an antigenic profile characteristic of basal epithelial prostate cells: lineage- CD24+ Sca-1+ CD49f+ (LSC). Interestingly, other research groups have shown that LSC population contains multipotent stem cells of the adult prostate; thus, the GFP+ cell population represent a subpopulation (~15%) of this basal/stem LSC population in the neonatal prostate. Moreover, in this LSC population, GFP+ cells showed ~20 fold more in vitro sphere-forming activity than the GFP- cells. Finally transplantation assays of 103 GFP+ cells in combination with urogenital sinus mesechymal cells demonstrated the potential of this GFP+ cell population to reconstitute in vivo prostate ducts containing both basal and luminal cells in renal grafts. Altogether, these results demonstrate that s-SHIP promoter expression is a new marker for basal epithelial prostate cells exhibiting stem/progenitor cell properties. We are now performing transcriptional analysis of GFP+ and GFP- basal LSC cells in order to define gene sets that distinguish a basal cell profile from a tissue stem cell profile. Preliminary results showed interesting candidate genes including transcription factors. We anticipate that these results will lead to a better definition of adult PrSCs and will help to elucidate prostate cancer etiology. Citation Format: Guillaume Brocqueville, Hélène Bauderlique Le Roy, Larry R. Rohrschneider, Roland P. Bourette. s-SHIP promoter expression identifies a subset of murine basal prostate cells as neonatal stem cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B07.