ABSTRACT
BACKGROUND: The rise in Plasmodium falciparum resistance to dihydroartemisinin-piperaquine (DHA-PPQ) treatment has been documented in the Greater Mekong Subregion with associations with mutations in the P. falciparum chloroquine resistance transporter (pfcrt) and plasmepsin 2 (pfpm2) genes. However, it is unclear whether other genes also play a role with PPQ resistance, such as the E415G mutation in the exonuclease (pfexo) gene. The aim of this study was to investigate the role of this mutation in PPQ resistance by generating transgenic parasites expressing the pfexo-E415G mutant allele. METHODS: Transgenic parasite clones carrying the E415G mutation in PfEXO of the B5 isolate were derived by CRISPR-Cas9 gene editing and verified using PCR and gene sequencing. Polymorphisms of pfkelch-13, pfcrt, and pfexo were examined by PCR while the copy number variations of pfpm2 were examined by both relative quantitative real-time PCR and the duplication breakpoint assay. Drug sensitivity against a panel of antimalarials, the ring-stage survival assay (RSA), the PPQ survival assay (PSA), and bimodal dose-response curves were used to evaluate antimalarial susceptibility. RESULTS: The transgenic line, B5-rexo-E415G-B8, was successfully generated. The PPQ-IC90, %PPQ survival, and the bimodal dose-response clearly showed that E415G mutation in PfEXO of B5 isolate remained fully susceptible to PPQ. Furthermore, growth assays demonstrated that the engineered parasites grew slightly faster than the unmodified parental isolates whereas P. falciparum isolates harbouring pfkelch-13, pfcrt, and pfexo mutations with multiple copies of pfpm2 grew much more slowly. CONCLUSIONS: Insertion of the E415G mutation in PfEXO did not lead to increased PPQ-IC90 and %PPQ survival, suggesting that this mutation alone may not be associated with PPQ resistance, but could still be an important marker if used in conjunction with other markers for monitoring PPQ-resistant parasites. The results also highlight the importance of monitoring and evaluating suspected genetic mutations with regard to parasite fitness and resistance.
Subject(s)
Antimalarials , Malaria, Falciparum , Parasites , Quinolines , Animals , Antimalarials/pharmacology , Antimalarials/therapeutic use , Chloroquine/pharmacology , Chloroquine/therapeutic use , DNA Copy Number Variations , Drug Resistance/genetics , Exonucleases/genetics , Exonucleases/pharmacology , Exonucleases/therapeutic use , Malaria, Falciparum/parasitology , Membrane Transport Proteins/genetics , Mutation , Phosphodiesterase I/genetics , Phosphodiesterase I/pharmacology , Piperazines , Plasmodium falciparum , Point Mutation , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Quinolines/pharmacology , Quinolines/therapeutic useABSTRACT
This study investigates the scar-reducing efficacy of topical application of stratifin and acetylsalicylic acid (ASA) in a rabbit ear model. A total of five New Zealand white rabbits with four wounds per ear were examined. Either recombinant stratifin (0.002%) or ASA (0.5%) incorporated in carboxymethyl cellulose gel was topically applied on each wound at postwounding Day 5. Scars were harvested at postwounding Day 28 for histological analysis. The wounds treated with stratifin and ASA showed 82 and 73% reduction in scar volume, respectively, compared with that of untreated controls. A reduction of 57 and 41% in total tissue cellularity along with 79 and 91% reduction in infiltrated CD3+ T cells were observed in response to treatment with stratifin and ASA, respectively, compared with those of untreated controls. Wounds treated with stratifin showed a 2.8-fold increase in matrix metalloproteinase-1 expression, which resulted in a 48% decrease in collagen density compared with those of untreated controls. Qualitative wound assessment showed a reduced hypertrophic scarring in stratifin and ASA-treated wounds when compared with the controls. This study showed that topical application of either stratifin or ASA-impregnated carboxymethyl cellulose gel reduced hypertrophic scar formation following dermal injuries in a rabbit ear fibrotic model.
Subject(s)
14-3-3 Proteins/therapeutic use , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Aspirin/therapeutic use , Biomarkers, Tumor/therapeutic use , Cicatrix, Hypertrophic/prevention & control , Exonucleases/therapeutic use , Wound Healing/drug effects , 14-3-3 Proteins/pharmacology , 14-3-3 Proteins/physiology , Administration, Cutaneous , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Aspirin/pharmacology , Bandages , Biomarkers, Tumor/pharmacology , Biomarkers, Tumor/physiology , Carboxymethylcellulose Sodium/therapeutic use , Cicatrix, Hypertrophic/etiology , Cicatrix, Hypertrophic/pathology , Collagen/drug effects , Collagen/physiology , Disease Models, Animal , Drug Evaluation, Preclinical , Exonucleases/pharmacology , Exonucleases/physiology , Exoribonucleases , Fibroblasts/drug effects , Fibroblasts/physiology , Gels , Matrix Metalloproteinase 1/drug effects , Matrix Metalloproteinase 1/physiology , Rabbits , Severity of Illness IndexABSTRACT
AIDS: Presentation highlights are provided from the Nineteenth International Congress on Chemotherapy in Montreal. Some of the new data on anti-HIV therapeutics include phase I/II data on Agouron's protease inhibitor AG1343; information on the new protease inhibitor palinavir PRO2000 (a gp120/CD4 binding inhibitor); and liposomal (fat-enclosed) formulations of both foscarnet and exonuclease (a cellular enzyme that destroys viral DNA). Two additional studies from Brazil on the efficacy and safety of roxithromycin for cryptosporidiosis (the Food and Drug Administration (FDA) has not approved roxithromycin for sale in the U.S.) are discussed, as is the use of imiquimod treatment for genital warts.^ieng