Coupling of importin beta binding peptide on plasmid DNA: transfection efficiency is increased by modification of lipoplex's physico-chemical properties

BACKGROUND: Non-viral vectors for gene transfer are less immunogenic than viral vectors but also less efficient. Significant effort has focused on enhancing non-viral gene transfer efficiency by increasing nuclear import of plasmid DNA, particularly by coupling nuclear localization peptidic sequences to plasmid DNA. RESULTS: We have coupled a 62-aminoacid peptide derived from hSRP1α importin beta binding domain, called the IBB peptide to plasmid DNA by using the heterobifunctional linker N-(4-azido-2,3,5,6 tetrafluorobenzyl)-6-maleimidyl hexanamide (TFPAM-6). When covalently coupled to plasmid DNA, IBB peptide did not increase the efficiency of cationic lipid mediated transfection. The IBB peptide was still able to interact with its nuclear import receptor, importin β, but non-specifically. However, we observed a 20-fold increase in reporter gene expression with plasmid DNA / IBB peptide complexes under conditions of inefficient transfection. In which case, IBB was associated with plasmid DNA through self assembling ionic interaction. CONCLUSIONS: The improvement of transfection activity was not due to an improved nuclear import of DNA, but rather by the modification of physicochemical properties of IBB peptide / plasmid complexes. IBB peptide increased lipoplex size and these larger complexes were more efficient for gene transfer

Habitat Use by Brood-Rearing Waterfowl in Subarctic Québec

Aerial surveys of waterfowl were conducted in subarctic Quebec in 1989 and 1990 on randomly selected 100 sq km plots. We used logistic regression for modelling relationships betweren the presence of waterfowl broods and habitat characteristics. For each species, models of habitat use were generated with one data set and tested with two others. We also compared the frequency distributions of broods of each species in different habitat types. Correct-classification rates of models varied betweren 0.53 and 0.77. Sensitivity of models generally increased when applied to validation data sets. Models showed that green-winged teals (Anas crecca) avoided lakes >501 ha and used vegetated lakes, ponds and fens or bogs more than predicted by their availability. Black scoters (Melanitta nigra) and surf scoters (M. perspicillata) were found mostly on small lakes (<10 ha) and medium lakes (10-100 ha). Black scoters were associated with the presence of ponds and lakes with sedge and grass, whereas surf scoters were not. The presence of scaup (Aythia spp.) broods was associated with the presence of vegetation. Green-winged teals were observed more often on bogs than were either scoters or scaups. Black scoters were observed more often on ponds and less often on medium-sized lakes than surf scoters and scaups. The latter differed in their use of lakes <10 ha: surf scoters, like black scoters, mostly used unvegetated lakes, while scaups and green-winged teals mostly used vegetated lakes. The accuracy and precision of logistic models may be enhanced by additional habitat variables, careful selection of sample-cell size and further investigation of the breeding biology of the surveyed species.Key words: habitat, model, logistic regression, northern Québec, black scoter, surf scoter, green-winged teal, scaup, broodDes inventaires aériens de sauvagine dans des parcelles de 100 km² sélectionnées aléatoirement ont été réalisés dans le Québec subarctique en 1989 et en 1990. La régression logistique fut utilisée pour modéliser les relations entre la présence de couvées et des paramètres d'habitat. Des modèles d'utilisation d'habitat ont été générés à l'aide d'une série de données et validés à l'aide de deux autres. Nous avons aussi comparé les distributions de fréquences des couvées des espèces étudiées dans divers types de biotopes. Les taux de classification correcte des modèles ont varié entre 0.53 et 0.77. De façon générale, la sensibilité des modèles augmentait lorsque ces derniers étaient appliqués aux données de validation. Les modèles montrent que la Sarcelle à ailes vertes (Anas crecca) a utilisé les plans d'eau avec de la végétation, les étangs et les tourbières et a évité les lacs > 501 ha. Les macreuses (Melanitta nigra, M. perspicillata) ont surtout été vues sur des lacs de petite (< 10 ha) et moyenne (10-100 ha) superficies. Les Macreuses à bec jaune se démarquaient des Macreuses à front blanc en étant associées à la présence d'étangs et de lacs avec des herbaçaies. La présence de couvées de morillons (Aythia spp.) dans les cellules était associée à celle de végétation. La Sarcelle à ailes vertes fut observée plus souvent dans les tourbières que les autres espèces. La Macreuse à bec jaune fut observée plus souvent sur des étangs et moins souvent sur des lacs de superficie moyenne que la Macreuse à front blanc et les morillons. Ces dernières espèces différaient dans leur utilisation de lacs <10 ha: les Macreuses à front blanc, comme les Macreuses à bec jaune, utilisaient surtout les lacs aux rives sans végétation alors que les morillons et les sarcelles utilisaient surtout des lacs avec végétation. La précision des modèles de régression logistique pourrait être augmentée par l'utilisation de paramètres d'habitat supplémentaires, un choix différent de la taille de l'unité de mesure et une meilleure connaissance de l'écologie des espèces cibles.Mots clés: habitat, modèle, régression logistique, Québec, Macreuse à bec jaune, Macreuse à front blanc, Sarcelle à ailes vertes, Morillons, couvé

Analysis of cellular responses of macrophages to zinc ions and zinc oxide nanoparticles: a combined targeted and proteomic approach

Two different zinc oxide nanoparticles, as well as zinc ions, are used to study the cellular responses of the RAW 264 macrophage cell line. A proteomic screen is used to provide a wide view of the molecular effects of zinc, and the most prominent results are cross-validated by targeted studies. Furthermore, the alteration of important macrophage functions (e.g. phagocytosis) by zinc is also investigated. The intracellular dissolution/uptake of zinc is also studied to further characterize zinc toxicity. Zinc oxide nanoparticles dissolve readily in the cells, leading to high intracellular zinc concentrations, mostly as protein-bound zinc. The proteomic screen reveals a rather weak response in the oxidative stress response pathway, but a strong response both in the central metabolism and in the proteasomal protein degradation pathway. Targeted experiments confirm that carbohydrate catabolism and proteasome are critical determinants of sensitivity to zinc, which also induces DNA damage. Conversely, glutathione levels and phagocytosis appear unaffected at moderately toxic zinc concentrations

Molecular responses of mouse macrophages to copper and copper oxide nanoparticles inferred from proteomic analyses

The molecular responses of macrophages to copper-based nanoparticles have been investigated via a combination of proteomic and biochemical approaches, using the RAW264.7 cell line as a model. Both metallic copper and copper oxide nanoparticles have been tested, with copper ion and zirconium oxide nanoparticles used as controls. Proteomic analysis highlighted changes in proteins implicated in oxidative stress responses (superoxide dismutases and peroxiredoxins), glutathione biosynthesis, the actomyosin cytoskeleton, and mitochondrial proteins (especially oxidative phosphorylation complex subunits). Validation studies employing functional analyses showed that the increases in glutathione biosynthesis and in mitochondrial complexes observed in the proteomic screen were critical to cell survival upon stress with copper-based nanoparticles; pharmacological inhibition of these two pathways enhanced cell vulnerability to copper-based nanoparticles, but not to copper ions. Furthermore, functional analyses using primary macrophages derived from bone marrow showed a decrease in reduced glutathione levels, a decrease in the mitochondrial transmembrane potential, and inhibition of phagocytosis and of lipopolysaccharide-induced nitric oxide production. However, only a fraction of these effects could be obtained with copper ions. In conclusion, this study showed that macrophage functions are significantly altered by copper-based nanoparticles. Also highlighted are the cellular pathways modulated by cells for survival and the exemplified cross-toxicities that can occur between copper-based nanoparticles and pharmacological agents

Deletion of Rb Accelerates Pancreatic Carcinogenesis by Oncogenic Kras and Impairs Senescence in Pre-Malignant Lesions

Rb1 encodes a cell-cycle regulator that is functionally disrupted in most human cancers. Pancreatic ductal adenocarcinomas (PDACs) have a high frequency of mutations in KRAS and INK4A/CDKN2A that might allow cells to bypass the regulatory actions of retinoblastoma (RB). To determine the role of loss of RB function in PDAC progression, we investigated the effects of Rb disruption during pancreatic malignant transformation initiated by oncogenic Kras.We generated mice with pancreas-specific disruption of Rb, in the absence or presence of oncogenic Kras, to examine the role of RB in pancreatic carcinogenesis

A study of karst hydrosystem recharge at the parcel scale, using modeling and correlation analysis - Low noise underground laboratory of Rustrel site

La caractérisation des flux d’eaux qui rechargent réellement les hydrosystèmes souterrains reste un frein à la compréhension du fonctionnement hydrogéologique des milieux souterrains. Lors d’événements pluvieux, quelle part de l’eau est évapo-transpirée ? Quelle part est temporairement stockée dans le sol ? Ces incertitudes sont particulièrement fortes dans le cas de la recharge des milieux hétérogènes tel que le karst. En général, les calculs de recharge des hydrosystèmes karstiques se basent sur une représentation simplifiée de l’évapotranspiration qui considère seulement le climat et pas le fonctionnement de la végétation. Dans cette étude, un modèle de végétation permettant de simuler les transferts d’eaux entre le sol et l’atmosphère en contexte forestier (le modèle CASTANEA), a été appliqué à une parcelle de Chêne vert. L’infiltration efficace (un indicateur de la recharge) estimé avec CASTANEA a été comparée à celle estimée par des approches classiques ainsi qu’à des séries long terme de flux d’eaux souterraines (9 années). Les résultats de cette analyse révèlent que l’infiltration efficace modélisée à partir d’un modèle de végétation comme CASTANEA est plus satisfaisante que les approches classiques ne tenant pas compte du fonctionnement de la végétation. Ce travail ouvre des perspectives intéressantes pour mieux tenir compte du fonctionnement de la végétation et de l’usage du sol sur la recharge des hydrosystèmes karstiques.Assessing the recharge of underground hydrosystems remains an obstacle to understand their hydrologeological functioning. During a rain event, which part of the rain is evapotranspired ? And how much is temporarily stored within the soil ? These questions are particularly relevant in heterogeneous media such as karst hydrosystems. Currently, the models used to compute recharge of karst hydrosystems, rely on simplistic formulations of evapotranspiration that do not account for vegetation functioning. In this study, we used the vegetation process based model CASTANEA, which is designed to compute water transfer between soil, plant and atmosphere. We computed effective infiltration (an index of recharge) with CASTANEA and with other classical approach (based on precipitation minus ETP), and for a welldocumented holm oak site in Provence. Our results provide evidences that effective infiltration computed with CASTANEA yield more satisfactory correlation with measured outflow than simulations based on the classical approach. Our results provide a promising way to improve the simulation of karst hydrosystem recharge

Resistance Evolution to Bt Crops: Predispersal Mating of European Corn Borers

Over the past decade, the high-dose refuge (HDR) strategy, aimed at delaying the evolution of pest resistance to Bacillus thuringiensis (Bt) toxins produced by transgenic crops, became mandatory in the United States and is being discussed for Europe. However, precopulatory dispersal and the mating rate between resident and immigrant individuals, two features influencing the efficiency of this strategy, have seldom been quantified in pests targeted by these toxins. We combined mark-recapture and biogeochemical marking over three breeding seasons to quantify these features directly in natural populations of Ostrinia nubilalis, a major lepidopteran corn pest. At the local scale, resident females mated regardless of males having dispersed beforehand or not, as assumed in the HDR strategy. Accordingly, 0–67% of resident females mating before dispersal did so with resident males, this percentage depending on the local proportion of resident males (0% to 67.2%). However, resident males rarely mated with immigrant females (which mostly arrived mated), the fraction of females mating before dispersal was variable and sometimes substantial (4.8% to 56.8%), and there was no evidence for male premating dispersal being higher. Hence, O. nubilalis probably mates at a more restricted spatial scale than previously assumed, a feature that may decrease the efficiency of the HDR strategy under certain circumstances, depending for example on crop rotation practices

Flight behaviour of European corn borer infected with Nosema pyrausta

The microsporidian Nosema pyrausta Paillot is a common and widespread pathogen of European corn borer, Ostrinia nubilalis (Hübner), in North America and Europe. Nosema negatively affects European corn borer longevity and fecundity. In this study, we used flight mills to examine the effects of Nosema infection on flight activity of unmated males and females at 1 and 3 days after pupal eclosion, taking the level of infection, as measured by number of Nosema spores per mg of tissue, into consideration. Nosema infection had a significant negative effect on distance, duration and speed of the longest uninterrupted flight, as well as on total distance and duration of flight of 1-day-old males, but not of 3-day-old males or females of either age. However, when insects with a light infection (\u3c15 spores/mg) were pooled with uninfected moths, significant negative effects of a moderate/heavy infection (≥15 spores/mg) were observed for most flight parameters in 1-day-old, but not 3-day-old, moths of both sexes. The magnitude of reduction was often substantial: e.g., distance and duration were sevenfold, and 3.5-fold less, respectively, in 1-day-old females with a moderate/heavy infection. Flight distance and duration were significantly negatively correlated with level of Nosema infection in 1-day-old, but not 3-day-old, moths of both sexes. The percentage of moths of either sex or age with a moderate/heavy Nosema infection flying given distances was less than that of lightly infected (\u3c15 spores/mg) moths. Among uninfected adults, forewing length was positively correlated with several measures of flight performance in males, but not females. However, some performance measures were correlated with wing length in females when infected with Nosema. Nosema infection was associated with reduced adult weight, but not forewing length, suggesting that negative effects of Nosema infection on flight performance are related to reduced energy reserves rather than smaller body size per se

A Deletion in Exon 9 of the LIPH Gene Is Responsible for the Rex Hair Coat Phenotype in Rabbits (Oryctolagus cuniculus)

The fur of common rabbits is constituted of 3 types of hair differing in length and diameter while that of rex animals is essentially made up of amazingly soft down-hair. Rex short hair coat phenotypes in rabbits were shown to be controlled by three distinct loci. We focused on the “r1” mutation which segregates at a simple autosomal-recessive locus in our rabbit strains. A positional candidate gene approach was used to identify the rex gene and the corresponding mutation. The gene was primo-localized within a 40 cM region on rabbit chromosome 14 by genome scanning families of 187 rabbits in an experimental mating scheme. Then, fine mapping refined the region to 0.5 cM (Z = 78) by genotyping an additional 359 offspring for 94 microsatellites present or newly generated within the first defined interval. Comparative mapping pointed out a candidate gene in this 700 kb region, namely LIPH (Lipase Member H). In humans, several mutations in this major gene cause alopecia, hair loss phenotypes. The rabbit gene structure was established and a deletion of a single nucleotide was found in LIPH exon 9 of rex rabbits (1362delA). This mutation results in a frameshift and introduces a premature stop codon potentially shortening the protein by 19 amino acids. The association between this deletion and the rex phenotype was complete, as determined by its presence in our rabbit families and among a panel of 60 rex and its absence in all 60 non-rex rabbits. This strongly suggests that this deletion, in a homozygous state, is responsible for the rex phenotype in rabbits

A Deletion in Exon 9 of the LIPH Gene Is Responsible for the Rex Hair Coat Phenotype in Rabbits (Oryctolagus cuniculus)

The fur of common rabbits is constituted of 3 types of hair differing in length and diameter while that of rex animals is essentially made up of amazingly soft down-hair. Rex short hair coat phenotypes in rabbits were shown to be controlled by three distinct loci. We focused on the “r1” mutation which segregates at a simple autosomal-recessive locus in our rabbit strains. A positional candidate gene approach was used to identify the rex gene and the corresponding mutation. The gene was primo-localized within a 40 cM region on rabbit chromosome 14 by genome scanning families of 187 rabbits in an experimental mating scheme. Then, fine mapping refined the region to 0.5 cM (Z = 78) by genotyping an additional 359 offspring for 94 microsatellites present or newly generated within the first defined interval. Comparative mapping pointed out a candidate gene in this 700 kb region, namely LIPH (Lipase Member H). In humans, several mutations in this major gene cause alopecia, hair loss phenotypes. The rabbit gene structure was established and a deletion of a single nucleotide was found in LIPH exon 9 of rex rabbits (1362delA). This mutation results in a frameshift and introduces a premature stop codon potentially shortening the protein by 19 amino acids. The association between this deletion and the rex phenotype was complete, as determined by its presence in our rabbit families and among a panel of 60 rex and its absence in all 60 non-rex rabbits. This strongly suggests that this deletion, in a homozygous state, is responsible for the rex phenotype in rabbits