Peptide Triazole Thiol Irreversibly Inactivates Metastable HIV-1 Env by Accessing Conformational Triggers Intrinsic to Virus-Cell Entry.

KR13, a peptide triazole thiol previously established to inhibit HIV-1 infection and cause virus lysis, was evaluated by flow cytometry against JRFL Env-presenting cells to characterize induced Env and membrane transformations leading to irreversible inactivation. Transiently transfected HEK293T cells were preloaded with calcein dye, treated with KR13 or its thiol-blocked analogue KR13b, fixed, and stained for gp120 (35O22), MPER (10E8), 6-helix-bundle (NC-1), immunodominant loop (50-69), and fusion peptide (VRC34.01). KR13 induced dose-dependent transformations of Env and membrane characterized by transient poration, MPER exposure, and 6-helix-bundle formation (analogous to native fusion events), but also reduced immunodominant loop and fusion peptide exposure. Using a fusion peptide mutant (V504E), we found that KR13 transformation does not require functional fusion peptide for poration. In contrast, simultaneous treatment with fusion inhibitor T20 alongside KR13 prevented membrane poration and MPER exposure, showing that these events require 6-helix-bundle formation. Based on these results, we formulated a model for PTT-induced Env transformation portraying how, in the absence of CD4/co-receptor signaling, PTT may provide alternate means of perturbing the metastable Env-membrane complex, and inducing fusion-like transformation. In turn, the results show that such transformations are intrinsic to Env and can be diverted for irreversible inactivation of the protein complex.

Cholesterols Work as a Molecular Regulator of the Antimicrobial Peptide-Membrane Interactions.

The existing cholesterols (Chols) in animal cell membranes play key roles in many fundamental cellular processes, which also promise the possibility to modulate the bioactivity of various membrane-active biomacromolecules. Here, combining dynamic giant unilamellar vesicle leakage experiments and molecular dynamics simulations, the inhibitory effect of Chols on the membrane poration activity of melittin (Mel), a typical natural antimicrobial peptide, is demonstrated. Molecular details of the Mel-Chol interactions in membrane show that, for a Chol-contained lipid membrane, Mel exposure would perturb the symmetric bilayer structure of the membrane and specifically influence the location and orientation distributions of Chol molecules to an asymmetric state between the two leaflets; moreover, the Mel-Chol interactions are significantly influenced by the membrane environment such as unsaturation degree of the lipid components. Such inhibitory effect is normally ascribed to an accumulation of Chol molecules around the membrane-bound peptide chains and formation of Chol-Mel complexes in the membrane, which hinder the further insertion of peptides into the membrane. This work clarifies the molecular interactions between membrane-active peptides and Chol-contained membranes, and suggest the possibility to develop targeted drugs due to the membrane component specificity between bacterial and animal cells.

Sodium pyruvate pre-treatment prevents cell death due to localised, damaging mechanical strains in the context of pressure ulcers.

We demonstrate sodium pyruvate (NaPy) pre-treatment as a successful approach for pressure ulcer (PU) prevention by averting their aetiological origin-cell-level damage and death by large, sustained mechanical loads. We evaluated the NaPy pre-treatment effect on permeability changes in the cell's plasma membrane (PM) following application of in vitro damaging-level strains. Fibroblasts or myoblasts, respectively, models for superficial or deep-tissue damage were grown in 0 or 1 mM NaPy, emulating typical physiological or cell culture conditions. Cells were pre-treated for 4 hours with 0 to 5 mM NaPy prior to 3-hour sustained, damaging-level loads (12% strain). PM permeability was quantified by the cell uptake of small (4 kDa), fluorescent dextran compared with unstrained control using fluorescence-activated cell sorting (FACS). Pre-treatment with 1 mM, and especially 5 mM, NaPy significantly reduces damage to PM integrity. Long-term NaPy pre-exposure can improve protective treatment, affecting fibroblasts and myoblasts differently. Pre-treating with NaPy, a natural cell metabolite, allows cells under damaging-level mechanical loads to maintain their PM integrity, that is, to avoid loss of homeostasis and inevitable, eventual cell death, by preventing initial, microscale stages of PU formation. This pre-treatment may be applied prior to planned periods of immobility, for example, planned surgery or transport, to prolong safe time in a position by preventing initial cell damage that can cascade and lead to PU formation.

Biophysical Characterization of a Nanodisc with and without BAX: An Integrative Study Using Molecular Dynamics Simulations and Cryo-EM.

B cell lymphoma-2-associated X protein (BAX) plays a pivotal role in triggering cell apoptosis by permeabilizing the mitochondrial outer membrane. Contrary to previous findings, recent electron microscopy (EM) experiments showed that BAX monomers are able to perturb phospholipid nanodiscs (NDs) by forming lipidic pores. Here, we provide structural and thermodynamic interpretation of such data using multiscale resolution molecular dynamics (MD) simulations. Our results suggest that BAX is able to disrupt the stability, lateral packing and enhance the desorption propensity of the lipids in the ND, resulting in the formation of a stable toroidal-like pore. These findings prompted to re-evaluate the previously reported cryo-EM data to generate an improved reconstruction, thereby allowing for a more accurate localization of BAX in the EM map. We conclude that the reduced stability of the BAX-embedded ND eliminates the necessity of forming active BAX oligomers for its disruption.

Ryanodine and IP3 receptor-mediated calcium signaling play a pivotal role in neurological infrared laser modulation.

Pulsed infrared (IR) laser energy has been shown to modulate neurological activity through both stimulation and inhibition of action potentials. While the mechanism(s) behind this phenomenon is (are) not completely understood, certain hypotheses suggest that the rise in temperature from IR exposure could activate temperature- or pressure-sensitive ion channels or create pores in the cellular outer membrane, allowing an influx of typically plasma-membrane-impermeant ions. Studies using fluorescent intensity-based calcium ion ([Formula: see text]) sensitive dyes show changes in [Formula: see text] levels after various IR stimulation parameters, which suggests that [Formula: see text] may originate from the external solution. However, activation of intracellular signaling pathways has also been demonstrated, indicating a more complex mechanism of increasing intracellular [Formula: see text] concentration. We quantified the [Formula: see text] mobilization in terms of influx from the external solution and efflux from intracellular organelles using Fura-2 and a high-speed ratiometric imaging system that rapidly alternates the dye excitation wavelengths. Using nonexcitable Chinese hamster ovarian ([Formula: see text]) cells and neuroblastoma-glioma (NG108) cells, we demonstrate that intracellular [Formula: see text] receptors play an important role in the IR-induced [Formula: see text], with the [Formula: see text] response augmented by ryanodine receptors in excitable cells.

Investigation of plasma induced electrical and chemical factors and their contribution processes to plasma gene transfection.

This study has been done to know what kind of factors in plasmas and processes on cells induce plasma gene transfection. We evaluated the contribution weight of three groups of the effects and processes, i.e. electrical, chemical and biochemical ones, inducing gene transfection. First, the laser produced plasma (LPP) was employed to estimate the contribution of the chemical factors. Second, liposomes were fabricated and employed to evaluate the effects of plasma irradiation on membrane under the condition without biochemical reaction. Third, the clathrin-dependent endocytosis, one of the biochemical processes was suppressed. It becomes clear that chemical factors (radicals and reactive oxygen/nitrogen species) do not work by itself alone and electrical factors (electrical current, charge and field) are essential to plasma gene transfection. It turned out the clathrin-dependent endocytosis is the process of the transfection against the 60% in all the transfected cells. The endocytosis and electrical poration are dominant in plasma gene transfection, and neither permeation through ion channels nor chemical poration is dominant processes. The simultaneous achievement of high transfection efficiency and high cell survivability is attributed to the optimization of the contribution weight among three groups of processes by controlling the weight of electrical and chemical factors.