pFAR plasmids: New Eukaryotic Expression Vectors for Gene Therapy, devoid of Antibiotic Resistance Markers

Efficient production of eukaryotic expression vectors requires the selection of plasmid-containing bacteria. To avoid the risk of dissemination of antibiotic resistance markers, we developed a new system to produce a family of plasmids Free of Antibiotic Resistance genes, called pFARs. The strategy is based on the suppression of a chromosomal nonsense mutation by a plasmid-borne function. The amber mutation was introduced into the Escherichia coli thyA gene that encodes a thymidylate synthase required for dTMP synthesis, resulting in thymidine auxotrophy. In parallel, a small plasmid vector that carries an amber suppressor t-RNA gene was entirely synthesised. The introduction of pFAR plasmids into an optimised thyA mutant restored normal growth to the auxotrophic strain, and led to an efficient production of monomeric supercoiled plasmids, as required for clinical trials. Luciferase activities measured after intramuscular injection and electrotransfer of LUC-encoding pFAR vector were similar to those obtained with a commercial vector containing the same expression cassette. Interestingly, whereas luciferase activities decreased within three weeks after intradermal electrotransfer of conventional expression vectors, sustained levels were observed with the pFAR derivative. Thus, pFAR plasmids represent a novel family of biosafe eukaryotic expression vectors, suitable for gene therapy

Behavioral Characterization of Individual Olfactory Memory Retrieval in Drosophila Melanogaster

Memory performance depends not only on effective learning and storage of information, but also on its efficient retrieval. In Drosophila, aversive olfactory conditioning generates qualitatively different forms of memory depending on the number and spacing of conditioning trials. However, it is not known how these differences are reflected at the retrieval level, in the behavior of individual flies during testing. We analyzed conditioned behaviors after one conditioning trial and after massed and spaced repeated trials. The single conditioning produces an early memory that was tested at 1.5 h. Tested at 24 h after training, the spaced and the massed protocols generate two different forms of consolidated memory, dependent, or independent of de novo protein-synthesis. We found clearly distinct patterns of locomotor activity in flies trained with either spaced or massed conditioning protocols. Spaced-trained flies exhibited immediate and dynamic choices between punished and unpunished odors during the test, whereas massed-trained flies made a delayed choice and showed earlier disappearance of the conditioned response. Flies trained with single and spaced trials responded to the punished odor by decreasing their resting time, but not massed-trained flies. These findings demonstrate that genetically and pharmacologically distinct forms of memory drive characteristically different forms of locomotor behavior during retrieval, and they may shed light on our previous observation that memory retrieval in massed-trained flies is socially facilitated. Social interactions would enhance exploratory activity, and then reduce the latency of their conditioned choice and delay its extinction

Conditional UAS-targeted repression in Drosophila

The Gal4–UAS enhancer trap system is useful for driving gene expression in various tissues. A new tool that extends Gal4 technology is described here. A fusion protein containing the Gal4 binding domain and the repression domain of the isolator suppressor of hairy wing was placed under the control of a heat shock-inducible promoter. The construct mediates the conditional repression of genes located downstream of a UAS sequence. The repressive effects of the chimeric protein on fasII gene expression were tested by western-blot analysis and in brain sections of adult Drosophila. Owing to the increasing number of Gal4 and UAS transgenic lines, this versatile system will facilitate the study of gene function

Parallel Processing of Appetitive Short- and Long-Term Memories In Drosophila

SummaryIt is broadly accepted that long-term memory (LTM) is formed sequentially after learning and short-term memory (STM) formation, but the nature of the relationship between early and late memory traces remains heavily debated [1–5]. To shed light on this issue, we used an olfactory appetitive conditioning in Drosophila, wherein starved flies learned to associate an odor with the presence of sugar [6]. We took advantage of the fact that both STM and LTM are generated after a unique conditioning cycle [7, 8] to demonstrate that appetitive LTM is able to form independently of STM. More specifically, we show that (1) STM retrieval involves output from γ neurons of the mushroom body (MB), i.e., the olfactory memory center [9, 10], whereas LTM retrieval involves output from αβ MB neurons; (2) STM information is not transferred from γ neurons to αβ neurons for LTM formation; and (3) the adenylyl cyclase RUT, which is thought to operate as a coincidence detector between the olfactory stimulus and the sugar stimulus [11–14], is required independently in γ neurons to form appetitive STM and in αβ neurons to form LTM. Taken together, these results demonstrate that appetitive short- and long-term memories are formed and processed in parallel

Two Independent Mushroom Body Output Circuits Retrieve the Six Discrete Components of Drosophila Aversive Memory

SummaryUnderstanding how the various memory components are encoded and how they interact to guide behavior requires knowledge of the underlying neural circuits. Currently, aversive olfactory memory in Drosophila is behaviorally subdivided into four discrete phases. Among these, short- and long-term memories rely, respectively, on the γ and α/β Kenyon cells (KCs), two distinct subsets of the ∼2,000 neurons in the mushroom body (MB). Whereas V2 efferent neurons retrieve memory from α/β KCs, the neurons that retrieve short-term memory are unknown. We identified a specific pair of MB efferent neurons, named M6, that retrieve memory from γ KCs. Moreover, our network analysis revealed that six discrete memory phases actually exist, three of which have been conflated in the past. At each time point, two distinct memory components separately recruit either V2 or M6 output pathways. Memory retrieval thus features a dramatic convergence from KCs to MB efferent neurons

In vitro and in vivo efficacy of edelfosine-loaded lipid nanoparticles against glioma.

Edelfosine is the prototype molecule of a family of anticancer drugs collectively known as synthetic alkyl-lysophospholipids. This drug holds promise as a selective antitumor agent, and a number of preclinical assays are in progress. In this study, we observe the accumulation of edelfosine in brain tissue after its oral administration in Compritol® and Precirol® lipid nanoparticles (LN). The high accumulation of edelfosine in brain was due to the inhibition of P-glycoprotein by Tween® 80, as verified using a P-glycoprotein drug interaction assay. Moreover, these LN were tested in vitro against the C6 glioma cell line, which was later employed to establish an in vivo xenograft mouse model of glioma. In vitro studies revealed that edelfosine-loaded LN induced an antiproliferative effect in C6 glioma cell line. In addition, in vivo oral administration of drug-loaded LN in NMRI nude mice bearing a C6 glioma xenograft tumor induced a highly significant reduction in tumor growth (p<0.01) fourteen days after the beginning of the treatment. Our results showed that Tween® 80 coated Compritol® and Precirol® LN can effectively inhibit the growth of C6 glioma cells in vitro and suggest that edelfosine-loaded LN represent an attractive option for the enhancement of antitumor activity on brain tumors in vivo

Regulation of Hedgehog Signalling Inside and Outside the Cell

The hedgehog (Hh) signalling pathway is conserved throughout metazoans and plays an important regulatory role in both embryonic development and adult homeostasis. Many levels of regulation exist that control the release, reception, and interpretation of the hedgehog signal. The fatty nature of the Shh ligand means that it tends to associate tightly with the cell membrane, and yet it is known to act as a morphogen that diffuses to elicit pattern formation. Heparan sulfate proteoglycans (HSPGs) play a major role in the regulation of Hh distribution outside the cell. Inside the cell, the primary cilium provides an important hub for processing the Hh signal in vertebrates. This review will summarise the current understanding of how the Hh pathway is regulated from ligand production, release, and diffusion, through to signal reception and intracellular transduction

Sex differences in intestinal carbohydrate metabolism promote food intake and sperm maturation

Physiology and metabolism are often sexually dimorphic, but the underlying mechanisms remain incompletely understood. Here, we use the intestine of Drosophila melanogaster to investigate how gut-derived signals contribute to sex differences in whole-body physiology. We find that carbohydrate handling is male-biased in a specific portion of the intestine. In contrast to known sexual dimorphisms in invertebrates, the sex differences in intestinal carbohydrate metabolism are extrinsically controlled by the adjacent male gonad, which activates JAK-STAT signalling in enterocytes within this intestinal portion. Sex reversal experiments establish roles for this malebiased intestinal metabolic state in controlling food intake and sperm production through gutderived citrate. Our work uncovers a male gonad-gut axis coupling diet and sperm production, and reveals that metabolic communication across organs is physiologically significant. The instructive role of citrate in inter-organ communication may be significant in more biological contexts than previously recognised