It is estimated that every year malaria infects ∼300 million people and accounts for the death of 2 million individuals. The Plasmodium parasites that cause malaria in humans are transmitted exclusively by mosquito species belonging to the Anopheles genus. The recent development of a gene transfer technology for Anopheles stephensimosquitoes, using the Minos transposable element marked with the enhanced green fluorescent protein EGFP (Catteruccia, F., Nolan, T., Loukeris, T. G., Blass, C., Savakis, C., Kafatos, F. C., and Crisanti, A. (2000) Nature 405, 959–962), provides now a powerful tool to investigate the role of mosquito molecules involved in the interaction with the malaria parasite. Such technology, when further developed with additional markers and transposable elements, will be invaluable for analyzing the biology of the vector and for developing malaria-resistant mosquitoes to be used as a tool to control malaria transmission in the field. We report here the germline transformation of A. stephensimosquitoes using a piggyBac-based transposon to drive integration of the gene encoding for the red fluorescent protein dsRED.A. stephensi embryos were injected with transformation vector pPBRED containing the dsRED marker cloned within the arms of piggyBac. Microscopic analysis of G₁ larvae revealed the presence of seven fluorescent phenotypes whose different molecular origins were confirmed by Southern blotting analysis. Sequencing of the insertion sites in two lines demonstrated that integrations had occurred at TTAA nucleotides in accordance with piggyBac-mediated transpositions.

The cylindromatosis (CYLD) gene is mutated in human tumors of skin appendages. It encodes a deubiquitylating enzyme (CYLD) that is a negative regulator of the NF-κB and JNK signaling pathways, in vitro. However, the tissue-specific function and regulation of CYLD in vivo are poorly understood. We established a genetically tractable animal model to initiate a systematic investigation of these issues by characterizing an ortholog of CYLD in Drosophila. Drosophila CYLD is broadly expressed during development and, in adult animals, is localized in the fat body, ovaries, testes, digestive tract and specific areas of the nervous system. We demonstrate that the protein product of Drosophila CYLD (CYLD), like its mammalian counterpart, is a deubiquitylating enzyme. Impairment of CYLD expression is associated with altered fat body morphology in adult flies, increased triglyceride levels and increased survival under starvation conditions. Furthermore, flies with compromised CYLD expression exhibited reduced resistance to bacterial infections. All mutant phenotypes described were reversible upon conditional expression of CYLD transgenes. Our results implicate CYLD in a broad range of functions associated with fat homeostasis and host defence in Drosophila.

Heritable RNA interference (RNAi), triggered from stably expressed transgenes with an inverted repeat (IR) configuration, is an important tool for reverse genetic studies. Here we report on the development of stable RNAi in Anopheles stephensi mosquitoes, the major vector of human malaria in Asia. Trans genic mosquitoes stably expressing a RNAi transgene, designed to produce intron‐spliced double‐stranded RNA (dsRNA) targeting the green fluorescent protein EGFP gene, were crossed to an EGFP‐expressing target line. EGFP expression was dramatically reduced at both the protein and RNA levels. The levels of gene silencing depended upon the RNAi gene copy number and its site of integration. These results demonstrate that specific RNAi‐mediated knockdown of gene function can be achieved with high efficiency in Anopheles. This will be invaluable to systematically unravel the function of Anopheles genes determining the vectorial capacity of the malaria parasite.

Primary immunodeficiencies are inborn errors of immunity that lead to life threatening conditions. These predispositions describe human immunity in natura and highlight the important function of components of the Toll-IL-1- receptor-nuclear factor kappa B (TIR-NF-κB) pathway. Since the TIR-NF-κB circuit is a conserved component of the host defence in higher animals, genetically tractable models may contribute ideas for clinical interventions. We used immunodeficient fruit flies (Drosophila melanogaster) to address questions pertaining to survival following bacterial infection. We describe here that flies lacking the NF-κB protein Relish, indispensable for countering Gram-negative bacteria, had a greatly improved survival to such infections when subject to dietary short-term starvation (STS) prior to immune challenge. STS induced the release of Nitric Oxide (NO), a potent molecule against pathogens in flies, mice and humans. Administering the NO Synthase-inhibitory arginine analog N-Nitro-L-Arginine-Methyl-Ester (L-NAME) but not its inactive enantiomer D-NAME increased once again sensitivity to infection to levels expected for relish mutants. Surprisingly, NO signalling required the NF-κB protein Dif, usually needed for responses against Gram-positive bacteria. Our results show that NO release through STS may reflect an evolutionary conserved process. Moreover, STS could be explored to address immune phenotypes related to infection and may offer ways to boost natural immunity.

Background: Glycolic acid (GA) is an effective way of reversing the signs of age and photodamage. GA enhances desquamation of the stratum corneum and induces biological responses that can help restore skin's integrity. GA can, however, cause irritation, especially when its concentration is high, and its pH is low. Thus, most commercially available products for home use contain relatively low GA concentrations and are partially neutralized to a pH around 4.Aims: The aim of this study was to determine the biological effects and relative efficacy of cosmetic formulations containing GA at concentrations ranging from 8% to 25% at pH 4 in human ex vivo skin explants.Methods: Human skin explants were topically treated with gel formulations and oil‐in‐water creams containing 8%, 10%, 15%, or 25% GA, adjusted to pH 4, daily for 5 days. The degree of desquamation, their effect on cell proliferation, and their impact upon total collagen levels were determined 24 hours later. Levels of tumor necrosis factor‐alpha (TNF‐α) were measured after days 3 and 6.Results: All formulations effectively induced desquamation in a concentration‐dependent manner. Total collagen levels were increased at all concentrations, with greatest effects at higher GA concentrations. No effect on TNF‐α expression was observed.Conclusions: These data suggest that partially neutralized GA formulations retain skin rejuvenating properties without causing irritation and inflammation and that their use can be tailored to individual needs based on the concentration of GA in the formulation.