Synthesis and biological evaluation of PEG-tirofiban conjugates

We have conjugated tirofiban, an antagonist of the GPIIb/IIIa integrin receptor, to PEG, and shown that these polymers effectively inhibit platelet aggregation. This inhibition decreased with the size of the polymer. Our goal was to develop new cryoprotective agents to store frozen platelets. Surprisingly, tirofiban-conjugated PEG did not exhibit any protection.     

Captive breeding of the barndoor skate (Dipturus laevis) at the Montreal Biodome,with comparison notes on two other captive‐bred skate species

In 1997, the Montreal Biodome obtained five barndoor skates (Dipturus laevis) from the waters off Boston, Massachusetts. Six years later, those specimens began reproducing, and the first egg case was collected in November 2003. Since then, 73 hatchlings have been born and raised. Egg cases were observed year round, and annual fecundity was measured for the first time: one female laid 69 eggs in 2005, 85 in 2006 and 115 in 2007. Egg incubation was longer than believed previously, ranging from 342 to 494 days. Hatching occurred throughout the year. Hatchlings averaged 193 mm total length and 128 mm disk width and weighed 32 g. They were fed krill and diced fish. All but one survived the first month. A photo identification system was useful in recognizing two groups of 10 specimens during their first year, and transponders could be inserted in the wing muscles of 1‐year‐old skates. Total lengths at birth and at age 2 were similar to the data reported from the wild, suggesting a similar growth pattern in captivity. The reproduction characteristics of the barndoor skate were compared with those of two other skate species currently bred at the Montreal Biodome, the winter skate (Leucoraja ocellata) and the thorny skate (Amblyraja radiata). Zoo Biol 27:145–153, 2008. © 2008 Wiley‐Liss, Inc.     

Emerging high-throughput approaches to analyze bioremediation of sites contaminated with hazardous and/or recalcitrant wastes

Sustainable development requires the promotion of environmental management and a constant search for new technologies to treat a wide range of aquatic and terrestrial habitats contaminated by increasing anthropogenic activities. Bioremediation, i.e. the elimination of natural or xenobiotic pollutants by living organisms, is an environmentally friendly and cost-effective alternative to physico-chemical cleanup options. However, the strategy and outcome of bioremediation in open systems or confined environments depend on a variety of physico-chemical and biological factors that need to be assessed and monitored. In particular, microorganisms are key players in bioremediation applications, yet their catabolic potential and their dynamics in situ remain poorly characterized. To perform a comprehensive assessment of the biodegradative potential of a contaminated site and efficiently monitor changes in the structure and activities of microbial communities involved in bioremediation processes, sensitive, fast and large-scale methods are needed. Over the last few years, the scientific literature has revealed the progressive emergence of genomic high-throughput technologies in environmental microbiology and biotechnology. In this review, we discuss various high--throughput techniques and their possible--or already demonstrated-application to assess biotreatment of contaminated environments.     

Drug use among youths

The levels of drug use among youths under 14 have remained extremely low for the past few years, as well as in most European countries. Yet, patterns of early drug use are often related to high-risk behaviours that require specific public policy strategies. Over the last years, the Government has set the target of reducing levels of drug use among specific groups such as young people: an awareness-raising media campaign has been launched and a specific outpatients clinics setting has been implemented for cannabis users in particular. This paper examines the relationship between health service providers and criminal justice authorities underlying the effectiveness of the current system. It underlines the need for an early screening and early interventions so as to avoid the shift from simple use to misuse or dependence.     

Anthropology at the heart of medicine

Anthropology and medicine share many concerns, but have had trouble collaborating in the past. The anthropologist has had to plead both with his colleagues and physicians to move beyond a < culturalist > vision that would confine him to the study of traditional or alternative medicines and representations of populations and the sick. The anthropologist's approach perceived as intrusive has also raised fears in the medical world. These reciprocal misunderstandings and stereotypes need to be overcome by an anthropology that studies the practices and knowledge of modern medicine as they are elaborated daily. Anthropology will dialogue with medicine without judging it. In its turn, medicine will open its sites of healing and teaching to the anthropologist. Anthropology at the heart of medicine is organized around the idea that the paths and expectations of health professionals reflect the specicifities of the local system of health. The individual dimensions of practices cannot be divorced from the functioning of structures of health and decision. Finally, like any other kind of anthropology, medical anthropology must scrutinize its own methods and ethics in a critical way.     

Diploid/polyploid syntenic shuttle mapping and haplotype-specific chromosome walking toward a rust resistance gene (Bru1) in highly polyploid sugarcane (2n approximately 12x approximately 115)

The genome of modern sugarcane cultivars is highly polyploid ( approximately 12x), aneuploid, of interspecific origin, and contains 10 Gb of DNA. Its size and complexity represent a major challenge for the isolation of agronomically important genes. Here we report on the first attempt to isolate a gene from sugarcane by map-based cloning, targeting a durable major rust resistance gene (Bru1). We describe the genomic strategies that we have developed to overcome constraints associated with high polyploidy in the successive steps of map-based cloning approaches, including diploid/polyploid syntenic shuttle mapping with two model diploid species (sorghum and rice) and haplotype-specific chromosome walking. Their applications allowed us (i) to develop a high-resolution map including markers at 0.28 and 0.14 cM on both sides and 13 markers cosegregating with Bru1 and (ii) to develop a physical map of the target haplotype that still includes two gaps at this stage due to the discovery of an insertion specific to this haplotype. These approaches will pave the way for the development of future map-based cloning approaches for sugarcane and other complex polyploid species.     

Identification of new quantitative trait Loci (other than the PRNP gene) modulating the scrapie incubation period in sheep

Although susceptibility to scrapie is largely controlled by the PRNP gene, we have searched for additional genomic regions that affect scrapie incubation time in sheep, using two half-sib families with a susceptible PRNP genotype and naturally infected by scrapie. Quantitative trait loci were detected on OAR6 and OAR18.     

PKC zeta controls DNA topoisomerase-dependent human caspase-2 pre-mRNA splicing

Caspase-2 exists as two main isoforms: the caspase-2L long isoform, which is pro-apoptotic, and the caspase-2S short isoform, which may be anti-apoptotic. Topoisomerase inhibitors drive inclusion of exon 9, specific for Casp-2S mRNA, and lower Casp-2L [corrected] mRNA and protein. With cell lines engineered to express various PKC isoforms, we demonstrate that PKC zeta, but not PKCalpha, positively regulates Casp-2S mRNA assembly triggered by topoisomerase inhibitors. In addition, exon 9 inclusion is lowered in mitosis but increased in the G1/S phase. Hence, the control of caspase-2 exon 9 inclusion by topoisomerase inhibitors depends on phosphorylation and/or dephosphorylation events, and on the cell cycle phase.     

Combination of novel green fluorescent protein mutant TSapphire and DsRed variant mOrange to set up a versatile in planta FRET-FLIM assay

Förster resonance energy transfer (FRET) measurements based on fluorescence lifetime imaging microscopy (FLIM) are increasingly being used to assess molecular conformations and associations in living systems. Reduction in the excited-state lifetime of the donor fluorophore in the presence of an appropriately positioned acceptor is taken as strong evidence of FRET. Traditionally, cyan fluorescent protein has been widely used as a donor fluorophore in FRET experiments. However, given its photolabile nature, low quantum yield, and multiexponential lifetime, cyan fluorescent protein is far from an ideal donor in FRET imaging. Here, we report the application and use of the TSapphire mutant of green fluorescent protein as an efficient donor to mOrange in FLIM-based FRET imaging in intact plant cells. Using time-correlated single photon counting-FLIM, we show that TSapphire expressed in living plant cells decays with lifetime of 2.93 ± 0.09 ns. Chimerically linked TSapphire and mOrange (with 16-amino acid linker in between) exhibit substantial energy transfer based on the reduction in the lifetime of TSapphire in the presence of the acceptor mOrange. Experiments performed with various genetically and/or biochemically known interacting plant proteins demonstrate the versatility of the FRET-FLIM system presented here in different subcellular compartments tested (cytosol, nucleus, and at plasma membrane). The better spectral overlap with red monomers, higher photostability, and monoexponential lifetime of TSapphire makes it an ideal FRET-FLIM donor to study protein-protein interactions in diverse eukaryotic systems overcoming, in particular, many technical challenges encountered (like autofluorescence of cell walls and fluorescence of pigments associated with photosynthetic apparatus) while studying plant protein dynamics and interactions.Single- and dual-color fluorescence imaging with intrinsically fluorescent proteins is increasingly being used to study the expression, targeting, colocalization, turnover, and associations of diverse proteins involved in different plant signal transduction pathways (for review, see Fricker et al., 2006). Concurrent with the use of fluorescence-based cell biology, Förster resonance energy transfer (FRET) has emerged as a convenient tool to study the dynamics of protein associations in vivo. The technique exploits the biophysical phenomenon of nonradiative energy transfer from a donor fluorophore to an appropriately positioned acceptor at a nanometer scale (1–10 nm; Jares-Erijman and Jovin, 2003). In living cells, FRET occurs when two proteins (or different domains within a single protein) fused to suitable donor and acceptor fluorophores physically interact, thus bringing the donor and the acceptor within the favorable proximity for energy transfer (Immink et al., 2002; Bhat et al., 2006). This results in a decrease in the donor''s fluorescence intensity (or quantum yield [QY]) and excited-state lifetime (Gadella et al., 1999). Furthermore, if the acceptor molecule is a fluorophore, then FRET additionally results in an increase in the acceptor''s emission intensity (sensitized emission; Shah et al., 2001; Bhat et al., 2006).However, the exploitation and use of fluorescent marker proteins to study protein trafficking and associations in plants can be problematic because plant cells contain a number of autofluorescent compounds (e.g. lignin, chlorophyll, phenols, etc.) whose emission spectra interfere with that of the most commonly used green or red fluorescent protein fluorophores and/or their spectral variants. For example, lignin fluorescence in roots, vascular tissues, and cell walls of aerial plant parts interferes with imaging at wavelengths between 490 and 620 nm, whereas the chlorophyll autofluorescence in green aerial plant parts is prevalent between 630 and 770 nm (Chapman et al., 2005). Consequently, conventional imaging of GFP and its closest spectral variants (like cyan fluorescent protein [CFP] and yellow fluorescent protein [YFP]) is most likely to be problematic in roots, whereas red-shifted intrinsic fluorescent proteins (including monomeric red fluorescent protein and recently identified spectral variants like mStrawberry and mCherry) may be hard to discriminate in chloroplast-containing aerial tissues (Chapman et al., 2005). The problems get further compounded in FRET assays because the autofluorescence arising from phenols, lignin, and chlorophyll can limit the choice of fluorophores suitable for in planta FRET assays.CFP and YFP have been widely used as a donor-acceptor pair in in planta FRET measurements (Bhat et al., 2006; Dixit et al., 2006). However, in photophysical terms, this pair is less than ideal for FRET imaging. Both have broad excitation and emission spectra with a small Stokes shift (Chapman et al., 2005). Second, QY of CFP (QY = 0.4) is relatively lower than that of YFP (QY = 0.61), and thus a significantly higher (and rather cell damaging) amount of excitation energy is needed to induce FRET (Dixit et al., 2006). Additionally, CFP displays multiexponential lifetimes with a shorter (1.3 ns) and a longer (2.6 ns) component (Becker et al., 2006). Although the deviation from the single-component decay is reasonably small (Tramier et al., 2002; Becker et al., 2006), the shorter CFP lifetime component can erroneously be interpreted as being the result of lifetime reduction due to energy transfer. At the same time, weak or transient protein associations may get masked and thus remain undetected. Whereas the parental wild-type GFP is extremely photostable and shows a monoexponential decay pattern (excited-state lifetime 3.16 ± 0.03 ns; Striker et al., 1999; Volkmer et al., 2000; Shaner et al., 2005), its close spectral overlap with YFP makes it unsuitable as a donor in GFP-YFP FRET experiments. Likewise, wild-type or enhanced GFP (or YFP) as a donor to red-shifted monomers as acceptors is suboptimal because the 488-nm (or 514-nm) laser line commonly used to excite GFP (or YFP) cross excites most of the red monomers (e.g. mOrange, mStrawberry) because of their broad excitation spectra (Zapata-Hommer and Griesbeck, 2003; Shaner et al., 2004).Recently, TSapphire (Q69M/C70P/V163A/S175G; excitation/emission 399/511 nm), a variant of the Sapphire (T203I) mutant of wild-type GFP with improved folding properties and better pH sensitivity, was described (Zapata-Hommer and Griesbeck, 2003). The T203I mutation in TSapphire (and original Sapphire as well) abolishes the 475-nm excitation peak found in the wild-type GFP (Tsien, 1998). TSapphire is efficiently excited below 410 nm, which makes it ideal for studying plant protein dynamics and interactions because, at this wavelength, there is negligible excitation of the autofluorescing chlorophyll pigments. Furthermore, TSapphire also represents a good donor to red monomer acceptors that are negligibly excited at this wavelength (Shaner et al., 2004). Using a purified Zn²⁺ sensor with TSapphire and mOrange as a donor-acceptor pair, Shaner and colleagues demonstrated the ratiometric intramolecular FRET between the two fluorophores in vitro (Shaner et al., 2004). The sensor yielded a 6-fold ratiometric increase (562/514-nm mOrange/TSapphire emission ratio) upon Zn²⁺ binding.However, currently there are no reports demonstrating the application and use of TSapphire and monomeric red-shifted fluorophores as donor-acceptor FRET pairs to probe intermolecular protein-protein interactions in vivo. In this article, we demonstrate in vivo FRET-fluorescence lifetime imaging microscopy (FLIM) between the donor TSapphire and the acceptor mOrange. We show that TSapphire expressed in living plant cells decays with a monoexponential lifetime of 2.93 ± 0.09 ns, which is in agreement with the published lifetime for its parent wild-type GFP (3.2 ns; Striker et al., 1999; Volkmer et al., 2000). Furthermore, we demonstrate intramolecular FRET-FLIM between chimerically linked TSapphire and mOrange (with a 16-amino acid linker in between). When fused to genetically known interacting proteins and expressed in intact living cells, the donor and the acceptor fluorophores show energy transfer in different subcellular compartments indicative of intermolecular protein-protein interactions. These results validate the versatility of the proposed in vivo FRET-FLIM assay based on the donor TSapphire and the acceptor mOrange, which turns out to work with both soluble and membrane proteins.