Sterol methyltransferase is required for optimal mitochondrial function and virulence in Leishmania major.

Limited knowledge on the exact functions of ergostane-based sterols has hampered the application of sterol synthesis inhibitors against trypanosomatid parasites. Sterol methyltransferase (SMT) is directly involved in the synthesis of parasite-specific C24-methylated sterols, including ergosterol and 5-dehydroepisterol. While pharmacological studies hint at its potential as a drug target against trypanosomatids, direct evidence for the cellular function and essentiality of SMT is lacking. Here, we characterized the SMT knockout mutants and their complemented strains in Leishmania major, the causative agent for cutaneous leishmaniasis. Deletion of SMT alleles led to a complete loss of C24-methylated sterols, which were replaced by cholestane-based sterols. SMT-null mutants were fully viable and replicative in culture but showed increased sensitivity to sphingolipid synthesis inhibition. They were not particularly vulnerable to heat, acidic pH, nitrosative or oxidative stress, yet exhibited high mitochondrial membrane potential and increased superoxide generation indicating altered physiology of the mitochondria. Despite possessing high levels of GPI-anchored glycoconjugates, SMT-null mutants showed significantly attenuated virulence in mice. In total, our study reveals that the biosynthesis of ergostane-based sterols is crucial for the proper function of mitochondria and the proliferation of Leishmania parasites in mammals.

Isoflurane increases cell membrane fluidity significantly at clinical concentrations.

There is an on-going debate whether anesthetic drugs, such as isoflurane, can cause meaningful structural changes in cell membranes at clinical concentrations. In this study, the effects of isoflurane on lipid membrane fluidity were investigated using fluorescence anisotropy and spectroscopy. In order to get a complete picture, four very different membrane systems (erythrocyte ghosts, a 5-lipid mixture that mimics brain endothelial cell membrane, POPC/Chol, and pure DPPC) were selected for the study. In all four systems, we found that fluorescence anisotropies of DPH-PC, nile-red, and TMA-DPH decrease significantly at the isoflurane concentrations of 1 mM and 5 mM. Furthermore, the excimer/monomer (E/M) ratio of dipyrene-PC jumps immediately after the addition of isoflurane. We found that isoflurane is quite effective to loosen up highly ordered lipid domains with saturated lipids. Interestingly, 1 mM isoflurane causes a larger decrease of nile-red fluorescence anisotropy in erythrocyte ghosts than 52.2 mM of ethanol, which is three times the legal limit of blood alcohol level. Our results paint a consistent picture that isoflurane at clinical concentrations causes significant and immediate increase of membrane fluidity in a wide range of membrane systems.

Safety and Efficacy of Aminolevulinic Acid 10% Topical Gel versus Aminolevulinic Acid 20% Topical Solution Followed by Blue-light Photodynamic Therapy for the Treatment of Actinic Keratosis on the Face and Scalp: A Randomized, Double-blind Study.

Objective: Photodynamic therapy (PDT) using 10% 5-aminolevulinic acid (ALA) gel (GEL) has been shown to be highly effective for treating actinic keratosis (AK) but has only been studied using red-light activation. The goal of this study was to compare GEL and a 20% ALA solution (SOL) using blue-light activation under typical clinical conditions. Design: This double-blind, split-face study randomized subjects to GEL or SOL application to contiguous 25cm2 fields containing 4 to 8 AK lesions on either side of the face or scalp (no curettage, 1-hour incubation, no occlusion) followed by blue light exposure (1,000 seconds, 417nm, 10J/cm2). Participants: Forty adult subjects were treated on either the face (n=20) or scalp (n=20). Measurements: Primary outcomes included change in baseline AK lesions. Secondary outcomes included local skin reaction (LSR) scores and visual analog scale (VAS) pain scores. Results: Lesions treated with GEL were 97.1 percent cleared at Day 84 versus 94.9 percent for lesions treated with SOL (p<0.001 vs. baseline); additionally, 86.8 percent of areas treated with GEL and 78.9 percent of areas treated with SOL showed 100-percent clearance (p<0.001 vs. baseline). Mean VAS pain scores were minimal for the SOL and the GEL (25.4 vs. 28.5 and 16.1 vs. 19.3, respectively; p=nonsignificant). At three days after the first and second treatments, more significant LSRs were noted in areas treated with SOL, including erythema, crusting, and scaling/dryness. There were no significant adverse events observed. Conclusion: GEL was equivalent to SOL for clearing AK lesions on the face and scalp with blue-light PDT; however, SOL caused significantly more local skin reactions.