Glenn Murphy (redyacht3)
The model could reliably predict the mechanism, the half saturation constant and the Hill coefficient values. Further based on the analysis, it was observed that aggregation and oligomerization result in drastic killing action in a short range of peptide concentration owing to high Hill coefficient values. Mechanisms such as monomers binding at multiple sites with/without cooperativity result in antimicrobial activity at low half saturation constant though the killing action may not be steep. Thus, the methodology developed here can be used to develop hypothesis for studying AMP-membrane interaction mechanisms. Gene silencing mediated by double-stranded small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for a variety of diseases and, indeed, the first therapeutic siRNA was approved by the FDA in 2018. As an alternative to the traditional delivery systems for nucleic acids, peptide-based nanoparticles (PBNs) have been applied successfully for siRNA delivery. Recently, we have developed amphipathic cell-penetrating peptides (CPPs), called WRAP allowing a rapid and efficient siRNA delivery into several cell lines at low doses (20 to 50 nM). In this study, using a highly specific gene silencing system, we aimed to elucidate the cellular uptake mechanism of WRAPsiRNA nanoparticles by combining biophysical, biological, confocal and electron microscopy approaches. We demonstrated that WRAPsiRNA complexes remain fully active in the presence of chemical inhibitors of different endosomal pathways suggesting a direct cell membrane translocation mechanism. Leakage studies on lipid vesicles indicated membrane destabilization properties of the nanoparticles and this was supported by the measurement of WRAPsiRNA internalization in dynamin triple-KO cells. However, we also observed some evidences for an endocytosis-dependent cellular internalization. Indeed, nanoparticles co-localized with transferrin, siRNA silencing was inhibited by the scavenger receptor A inhibitor Poly I and nanoparticles encapsulated in vesicles were observed by electron microscopy in U87 cells. click here In conclusion, we demonstrate here that the efficiency of WRAPsiRNA nanoparticles is mainly based on the use of multiple internalization mechanisms including direct translocation as well as endocytosis-dependent pathways. BACKGROUND Oxidation of small dense low-density lipoprotein (sdLDL) and membranes is causally related to atherosclerosis. The omega-3 fatty acid (FA) eicosapentaenoic acid (EPA, 205, ω-3) significantly reduced oxidized LDL in patients with hypertriglyceridemia by unknown mechanisms. We compared EPA effects to related FAs of varying chain length and unsaturation on oxidation of sdLDL and model membranes, and on cholesterol crystal domains. We compared EPA to the FAs stearic (SA, 180), oleic (OA, 181, ω-9), linoleic (LA, 182, ω-6), alpha-linolenic (ALA, 183, ω-3), eicosanoic (EA, 200), eicosatrienoic (ETE, 203, ω-3), arachidonic (AA, 204, ω-6), docosapentaenoic (DPA, 225, ω-3), and docosahexaenoic (DHA, 226, ω-3). METHODS Human sdLDL or model membranes of cholesterol and 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine [182(cis)PC or DLPC] were preincubated with FAs followed by copper-induced oxidation. Malondialdehyde (MDA) or lipid hydroperoxides (LOOH) levels measured oxidation; small-angle X-ray diffraction assessed cholesterol domain formation. RESULTS After 40 min, EPA reduced MDA levels 70% compared to vehicle (p less then 0.001). Lesser inhibition was observed with DHA, DPA, ETE, and ALA (33%, 34%, 32%, and 16%, respectively; all p less then 0.001 versus vehicle). Similar relative FA effects were observed in model membranes where EPA more substantially inhibited cholesterol crystal domain formation. CONCLUSION We observed relationships between hydrocarbon length and unsaturation with antioxidant activity and membrane cholesterol domain formation. EPA had the most favorable molecular structure,
