Directed integration of new insulated lentiviral vectors to heterochromatin towards safer gene transfer to stem cells

The need for better gene transfer systems towards improved risk=benefit balance for patients remains a major challenge in the clinical translation of gene therapy (GT). We have investigated the improvement of integrating vectors safety in combining (i) new short synthetic genetic insulator elements (GIE) and (ii) directing genetic integration to heterochromatin. We have designed SIN-insulated retrovectors with two candidate GIEs and could identify a specific combination of insulator 2 repeats which translates into best functional activity, high titers and boundary effect in both gammaretro (p20) and lentivectors (DCaro4) (see Duros et al, abstract ibid). Since GIEs are believed to shield the transgenic cassette from inhibitory effects and silencing, DCaro4 has been further tested with chimeric HIV-1 derived integrases which comprise C-ter chromodomains targeting heterochromatin through either histone H3 (ML6chimera) or methylatedCpGislands (ML10). With DCaro4 only and both chimeras, a homogeneous expression is evidenced in over 20% of the cells which is sustained over time. With control lentivectors, less than 2% of cells express GFP as compared to background using a control double-mutant in both catalytic and ledgf binding-sites; in addition, a two-times increase of expression can be induced with histone deacetylase inhibitors. Our approach could significantly reduce integration into open chromatin sensitive sites in stem cells at the time of transduction, a feature which might significantly decrease subsequent genotoxicity, according to X-SCIDs patients data.Work performed with the support of EC-DG research within the FP6-Network of Excellence, CLINIGENE: LSHB-CT-2006-018933

New imaging techniques applied to Paleogene radiolaria

A simple and time efficient technique to illustrate specimens is described and demonstrated with Paleogene radiolarians. This method produces Scanning Electron Microscope (SEM) and composite focal depth Transmitted Light Microscope (TLM) images for single radiolarian specimens. We propose the use of this technique to clarify radiolarian taxonomy. This technique has distinct advantages over previously published time consuming techniques that can also require toxic materials.

Amoebae as a tool to isolate new bacterial species, to discover new virulence factors and to study the host-pathogen interactions.

Amoebae are unicellular protozoan present worldwide in several environments mainly feeding on bacteria. Some of them, the amoebae-resistant bacteria (ARBs), have evolved mechanisms to survive and replicate inside amoebal species. These mainly include legionella, mycobacteria and Chlamydia-related bacteria. Amoebae can provide a replicative niche, can act as reservoir for bacteria whereas the cystic form can protect the internalized bacteria. Moreover, the amoebae represent a Trojan horse for ARBs to infect animals. The long interaction between amoebae and bacteria has likely selected for bacterial virulence traits leading to the adaptation towards an intracellular lifestyle, and some ARBs have acquired the ability to infect mammals. This review intends to highlight the important uses of amoebae in several fields in microbiology by describing the main tools developed using amoebal cells. First, amoebae such as Acanthamoeba are used to isolate and discover new intracellular bacterial species by two main techniques: the amoebal co-culture and the amoebal enrichment. In the second part, taking Waddlia chondrophila as example, we summarize some important recent applications of amoebae to discover new bacterial virulence factors, in particular thanks to the amoebal plaque assay. Finally, the genetically tractable Dictyostelium discoideum is used as a model organism to study host-pathogen interactions, in particular with the development of several approaches to manipulate its genome that allowed the creation of a wide range of mutated strains largely shared within the Dictyostelium community.

Pharmacological inhibition of nicotinamide phosphoribosyltransferase/visfatin enzymatic activity identifies a new inflammatory pathway linked to NAD.

Nicotinamide phosphoribosyltransferase (NAMPT), also known as visfatin, is the rate-limiting enzyme in the salvage pathway of NAD biosynthesis from nicotinamide. Since its expression is upregulated during inflammation, NAMPT represents a novel clinical biomarker in acute lung injury, rheumatoid arthritis, and Crohn's disease. However, its role in disease progression remains unknown. We report here that NAMPT is a key player in inflammatory arthritis. Increased expression of NAMPT was confirmed in mice with collagen-induced arthritis, both in serum and in the arthritic paw. Importantly, a specific competitive inhibitor of NAMPT effectively reduced arthritis severity with comparable activity to etanercept, and decreased pro-inflammatory cytokine secretion in affected joints. Moreover, NAMPT inhibition reduced intracellular NAD concentration in inflammatory cells and circulating TNFalpha levels during endotoxemia in mice. In vitro pharmacological inhibition of NAMPT reduced the intracellular concentration of NAD and pro-inflammatory cytokine secretion by inflammatory cells. Thus, NAMPT links NAD metabolism to inflammatory cytokine secretion by leukocytes, and its inhibition might therefore have therapeutic efficacy in immune-mediated inflammatory disorders.

Early Jurassic to Early late Cretaceous radiolarians from the Santa Rosa accretionary complex (Northwestern Costa Rica)

In the circum-Pacific ophiolitic belts, when no other biogenic constituents are found, radiolarians have the potential to provide significant biostratigraph- ic information. The Santa Rosa Accretionary Complex, which crops out in several half-windows (Carrizal, Sitio Santa Rosa, Bahia Nancite, Playa Naranjo) along the south shores of the Santa Elena Peninsula in northwestern Costa Rica, is one of these little-known ophiolitic mélanges. It contains various oceanic assemblages of alkaline basalt, radiolarite and polymictic breccias. The radiolarian biochronology presented in this work is mainly based by correlation on the biozonations of Carter et al. (2010), Baumgartner et al. (1995b), and O'Dogherty (1994) and indicate an Early Jurassic to early Late Cretaceous (early Pliensbachian to earliest Turonian) age for the sediments associated with oceanic basalts or recovered from blocks in breccias or megabreccias. The 19 illus- trated assemblages from the Carrizal tectonic window and Sitio Santa Rosa contain in total 162 species belonging to 65 genera. The nomenclature of tecton- ic units is the one presented by (Baumgartner and Denyer, 2006). This study brings to light the Early Jurassic age of a succession of radiolarite, which was previously thought to be of Cretaceous age, intruded by alkaline basalts sills (Unit 3). The presence of Early Jurassic large reworked blocks in a polymictic megabreccia, firstly reported by De Wever et al. (1985) is confirmed (Unit 4). Therefore, the alkaline basalt associated with the radiolarites of these two units (and maybe also Units 5 and 8) could be of Jurassic age. In the Carrizal tectonic window, Middle to early Late Jurassic radiolarian chert blocks associ- ated with massive tholeitic basalts and Early Cretaceous brick-red ribbon cherts overlying pillow basalts are interpreted as fragments of a Middle Jurassic oceanic basement accreted to an Early Cretaceous oceanic Plate, in an intra-oceanic subduction context. Whereas, the knobby radiolarites and black shales of Playa Carrizal are indicative of a shallower middle Cretaceous paleoenvironment. Other remnants of this oceanic basin are found in Units 2, 6, and 7, which documented the rapid approach of the depocentre to a subduction trench during the late Early Cretaceous (Albian-Cenomanian), to possibly early Late Cretaceous (Turonian).

Electric bicycles as a new active transportation modality to promote health.

GOJANOVIC, B., J. WELKER, K. IGLESIAS, C. DAUCOURT, and G. GREMION. Electric Bicycles as a New Active Transportation Modality to Promote Health. Med. Sci. Sports Exerc., Vol. 43, No. 11, pp. 2204-2210, 2011. Electrically assisted bicycles (EAB) are an emerging transportation modality favored for environmental reasons. Some physical effort is required to activate the supporting engine, making it a potential active commuting option. Purpose: We hypothesized that using an EAB in a hilly city allows sedentary subjects to commute comfortably, while providing a sufficient effort for health-enhancing purposes. Methods: Sedentary subjects performed four different trips at a self-selected pace: walking 1.7 km uphill from the train station to the hospital (WALK), biking 5.1 km from the lower part of town to the hospital with a regular bike (BIKE), or EAB at two different power assistance settings (EAB(high), EAB(std)). HR, oxygen consumption, and need to shower were recorded. Results: Eighteen sedentary subjects (12 female, 6 male) age 36 +/- 10 yr were included, with (V) over dotO(2max) of 39.4 +/- 5.4 mL.min(-1).kg(-1). Time to complete the course was 22 (WALK), 19 (EAB(high)), 21 (EAB(std)), and 30 (BIKE) min. Mean %(V) over dotO(2max) was 59.0%, 54.9%, 65.7%, and 72.8%. Mean%HR(max) was 71.5%, 74.5%, 80.3%, and 84.0%. There was no significant difference between WALK and EAB(high), but all other comparisons were different (P < 0.05). Two subjects needed to shower after EAB(high), 3 needed to shower after WALK, 8 needed to shower after EAB(std), and all 18 needed to shower after BIKE. WALK and EAB(high) elicited 6.5 and 6.1 METs (no difference), whereas it was 7.3 and 8.2 for EAB(std) and BIKE. Conclusions: EAB is a comfortable and ecological transportation modality, helping sedentary people commute to work and meet physical activity guidelines. Subjects appreciated ease of use and mild effort needed to activate the engine support climbing hills, without the need to shower at work. EAB can be promoted in a challenging urban environment to promote physical activity and mitigate pollution issues.