Detection of Panulirus argus Virus 1 (PaV1) in the Caribbean spiny lobster using fluorescence in situ hybridization (FISH)

Panulirus argus Virus 1 (PaV1) is the first virus known to be pathogenic to a wild lobster. It infects the Caribbean spiny lobster P. argus from the Florida Keys, and has a predilection for juveniles. The monitoring of the virus in wild populations and study of its behavior in the laboratory require the development of reliable diagnostic tools. A sensitive and specific fluorescence in situ hybridization (FISH) assay was developed for detection of PaV1. The lower detection limit using a 110 bp DNA probe in a dot-blot hybridization for PaV1 DNA was 10 pg of cloned template PaV1 DNA and 10 ng of genomic DNA extracted from the hemolymph of diseased spiny lobster. The fluorescein (FITC)-labeled probe specifically hybridized to PaV1-infected cells in the hepatopancreas, hindgut, gills, heart, foregut, and nerve tissues. FITC staining was observed around the inner periphery of the nuclear membrane, with lighter staining in a more dispersed pattern within the nucleus. The probe did not hybridize with host tissues of uninfected spiny lobsters, nor did it cross-react with 4 other virus samples tested. This assay will facilitate our understanding of the pathogenesis of the viral disease and help in monitoring efforts directed at determining the prevalence of PaV1 in juvenile nurseries for this lobster

Detection of Panulirus Argus Virus 1 (PaV1) in the Caribbean Spiny Lobster Using Fluorescence in situ Hybridization (FISH)

Panulirus argus Virus 1 (PaV1) is the first virus known to be pathogenic to a wild lobster. It infects the Caribbean spiny lobster P. argus from the Florida Keys, and has a predilection for juveniles. The monitoring of the virus in wild populations and study of its behavior in the laboratory require the development of reliable diagnostic tools. A sensitive and specific fluorescence in situ hybridization (FISH) assay was developed for detection of PaV1. The lower detection limit using a 110 bp DNA probe in a dot-blot hybridization for PaV1 DNA was 10 pg of cloned template PaV1 DNA and 10 ng of genomic DNA extracted from the hemolymph of diseased spiny lobster. The fluorescein (FITC)-labeled probe specifically hybridized to PaV1-infected cells in the hepatopancreas, hindgut, gills, heart, foregut, and nerve tissues. FITC staining was observed around the inner periphery of the nuclear membrane, with lighter staining in a more dispersed pattern within the nucleus. The probe did not hybridize with host tissues of uninfected spiny lobsters, nor did it cross-react with 4 other virus samples tested. This assay will facilitate our understanding of the pathogenesis of the viral disease and help in monitoring efforts directed at determining the prevalence of PaV1 in juvenile nurseries for this lobster

Investigating the Antimalarial Action of 1,2,4-Trioxolanes with Fluorescent Chemical Probes

The 1,2,4-trioxolanes are a new class of synthetic peroxidic antimalarials currently in human clinical trials. The well known reactivity of the 1,2,4-trioxolane ring towards inorganic ferrous iron and ferrous iron heme is proposed to play a role in the antimalarial action of this class of compounds. We have designed structurally relevant fluorescent chemical probes to study the sub-cellular localization of 1,2,4-trioxolanes in cultured Plasmodium falciparum parasites. Microscopy experiments revealed that a probe fluorescently labeled on the adamantane ring accumulated specifically in digestive vacuole-associated neutral lipid bodies within the parasite while an isosteric, but non-peroxidic congener did not. Probes fluorescently labeled on the cyclohexane ring showed no distinct localization pattern. In their sub-cellular localization and peroxidative effects, 1,2,4-trioxolane probes behave much like artemisinin-based probes studied previously. Our results are consistent with a role for adamantane-derived carbon-centered radicals in the antimalarial action of 1,2,4-trioxolanes, as hypothesized previously on the basis of chemical reactivity studies

Accumulation of artemisinin trioxane derivatives within neutral lipids of Plasmodium falciparum malaria parasites is endoperoxide-dependent

The antimalarial trioxanes, exemplified by the naturally occurring sesquiterpene lactone artemisinin and its semi-synthetic derivatives, contain an endoperoxide pharmacophore that lends tremendous potency against Plasmodium parasites. Despite decades of research, their mechanism of action remains unresolved. A leading model of anti-plasmodial activity hypothesizes that iron-mediated cleavage of the endoperoxide bridge generates cytotoxic drug metabolites capable of damaging cellular macromolecules. To probe the malarial targets of the endoperoxide drugs, we studied the distribution of fluorescent dansyl trioxane derivatives in living, intraerythrocytic-stage Plasmodium falciparum parasites using microscopic imaging. The fluorescent trioxanes rapidly accumulated in parasitized erythrocytes, localizing within digestive vacuole-associated neutral lipid bodies of trophozoites and schizonts, and surrounding the developing merozoite membranes. Artemisinin pre-treatment significantly reduced fluorescent labeling of neutral lipid bodies, while iron chelation increased non-specific cytoplasmic localization. To further explore the effects of endoperoxides on cellular lipids, we used an oxidation-sensitive BODIPY lipid probe to show the presence of artemisinin-induced peroxyl radicals in parasite membranes. Lipid extracts from artemisinin-exposed parasites contained increased amounts of free fatty acids and a novel cholesteryl ester. The cellular accumulation patterns and effects on lipids were entirely endoperoxide-dependent, as inactive dioxolane analogs lacking the endoperoxide moiety failed to label neutral lipid bodies or induce oxidative membrane damage. In the parasite digestive vacuole, neutral lipids closely associate with heme and promote hemozoin formation. We propose that the trioxane artemisinin and its derivatives are activated by heme-iron within the neutral lipid environment where they initiate oxidation reactions that damage parasite membranes