Disruption Of Membrane Integrity Research Articles

Lipid membranes induce structural conversion from amyloid oligomers to fibrils

Formation of amyloid oligomers and fibrils underlies the pathogenesis of a number of neurodegenerative diseases such as Alzheimer’s. One mechanism of action by which Aβ aggregates cause neuronal toxicity is through interactions with cellular membranes. Aβ aggregates have been shown to disrupt membrane integrity via pore formation, membrane thinning, or lipid extraction. At the same time, lipid membranes also affect the rate of Aβ aggregation and remodel pre-formed Aβ fibrils. Here we show that Aβ42 globulomers, a type of well-characterized and stable Aβ oligomers, convert to amyloid fibrils in the presence of DOPC liposomes. Electron paramagnetic resonance studies show that the fibrils converted from Aβ42 globulomers adopt the same structure as fibrils formed directly from monomers. Our results suggest that the interactions between Aβ oligomers and cellular membranes are dynamic. By converting Aβ oligomers to fibrils, the lipid membrane can reduce the membrane-disrupting activities caused by these oligomers. Modulation of Aβ-membrane interactions as a therapeutic strategy should take into account the dynamic nature of these interactions.

Detailed analysis of liposome adsorption and its rupture on the liquid-solid interface monitored by LSPR and QCM-D integrated sensor

Liposomes have been attracted attention, because liposomes can mimic the lipid bilayer membrane of cells. Scientists apply liposomes for drug delivery systems (DDSs) and for artificial cells. Hence, the handling of liposomes is very important. In particular, it is difficult to control the adsorption of the liposome onto the target selectively and the release of the drug by its own rupture. Therefore, scientists are looking for the way to carefully monitor the behavior of liposomes from adsorption to rupture in deep.We integrated quartz crystal microbalance dissipation (QCM–D) and localized surface plasmon resonance (LSPR) into a single sensor and monitored the signals from the sensor on the adsorption and rupture of liposome. The integrated sensor detected resonance frequency and dissipation from QCM-D, and wavelength shifts from LSPR simultaneously. The integrated sensor successfully monitored conformational changes of liposomes in real time upon exposure to the surfactant, Triton X-100. The data clearly revealed liposome adsorption and deformation, and disruption of membrane integrity by normalizing three signals, resonance frequency, resonance resistance and wavelength shift. In fact, we found that normalized signals were divided into 5 steps through the experiment: liposome adsorption, liposome deformation slightly, liposome conjugated with micelles of surfactant, liposome disruption and lipid bilayer covered on the surface. These results indicate that our device provides a powerful tool to gain deeper insights into biomolecular interactions, expanding numerous applications such as monitoring of conformational changes in proteins, oligonucleotides, viruses, bacteria, vesicles, and cells.

Inactivation effects and mechanisms of plasma-activated water combined with sodium laureth sulfate (SLES) against Saccharomyces cerevisiae

The present study aimed to elucidate the antifungal effect and underlying mechanism of plasma-activated water (PAW) combined with sodium laureth sulfate (SLES) against Saccharomyces cerevisiae. S. cerevisiae, initially at 6.95 log10 colony-forming unit (CFU)/mL, decreased to an undetectable level following the synergistic treatment of PAW and SLES (0.50 mg/mL) for 20 min. After PAW treatment combined with SLES (2.5 mg/mL) for 30 min, the S. cerevisiae cells on polyethylene films also reduced to an undetectable level from the initial load of 5.84 log10 CFU/cm2. PAW + SLES treatment caused severe disruption of membrane integrity and increased lipid oxidation within the cell membrane and the intracellular reactive oxygen species levels in S. cerevisiae cells. Besides, the disruption of the mitochondrial membrane potential (∆ψm) was also observed in S. cerevisiae cells after treatment of PAW and SLES at 0.01 mg/mL for 5 min. These data suggest that the combined treatment of PAW and SLES causes oxidation injury to cell membranes and abnormal ∆ψm in S. cerevisiae, which may be eventually responsible for cell death. This study demonstrates the potential application of PAW combined with SLES as an alternative disinfection method. Key Points • PAW + SLES exhibited synergistic antifungal activity against S. cerevisiae. • PAW + SLES resulted in severe disruption of membrane integrity and permeability. • PAW + SLES induced accumulation of reactive oxygen species in S. cerevisiae cells.

Characterization of phytochemicals and validation of antioxidant and anticancer activity in some Indian polyherbal ayurvedic products

In the present comparative study, the authors studied the antioxidant and anticancer activity of commercially available polyherbal Indian Ayurvedic products namely Divya Sarvakalp Kwath (DSKK), Divya Sanjivani Vati (DSV), Kanchanar Guggulu (KG) and Shakti Drop (SD). Authors also quantified phenolic and flavonoid contents in the samples. Solid powdered samples (DSKK, DSV, and KG) were extracted in methanol and water (1:1) using cold extraction method. Spectrophotometry technique was used to quantify the phytochemicals present in test samples. DSKK showed comparatively higher content of total phenolics (247.65 ± 0.05 μgPGE/g) and flavonoid (34.66 ± 0.19 µgQE/mg). Radical scavenging, metal ion chelation and reducing potential of test products were studied using nitric oxide scavenging, DPPH, metal ion chelation, reducing power ability, and phosphomolybdate in vitro antioxidant assays at different concentration. Dose-dependent antioxidant activity was observed in all the test samples at 100–500 µg or µl/ml concentration. Anticancer efficacy of the test samples were studied in lung (A549), colon (Colo205), and breast cancer (MCF7) cell lines at different concentrations (10–100 µg or µl/ml) using MTT assay. Confocal microscopy was used to reveal the apoptotic induction, mitochondrial membrane integrity disruption and reactive oxygen species production ability of test products in cancer cells. The present study revealed that DSKK possesses comparatively better antioxidant potential and SD has potent anticancer activity against breast cancer cells.

Cell Membrane Properties can Mediate the Toxicity of Protein Misfolded Oligomers and Folded Toxins

The onset and progression of Alzheimer's disease is characterized by the aggregation of the 42-residue form of the amyloid-β peptide (Aβ42). Significant evidence indicates that the mechanism by which Aβ42 induces cell death is through the interaction of its toxic oligomeric species with cell membranes, which results in the disruption of membrane integrity and homeostatic processes within the cell. Moreover, there is evidence that Aβ42 interacts differentially with regions of membranes enriched in different cell membrane components, and that its cytotoxicity is increased as the size of the oligomers decreases and their hydrophobicity increases.

Effect of Lead and Copper on Photosynthetic Apparatus in Citrus (Citrus aurantium L.) Plants. The Role of Antioxidants in Oxidative Damage as a Response to Heavy Metal Stress

Photosynthetic changes and antioxidant activity to oxidative stress were evaluated in sour orange (Citrus aurantium L.) leaves subjected to lead (Pb), copper (Cu) and also Pb + Cu toxicity treatments, in order to elucidate the mechanisms involved in heavy metal tolerance. The simultaneous effect of Pb− and Cu on growth, concentration of malondialdehyde (MDA), hydrogen peroxide (H2O2), chlorophylls, flavonoids, carotenoids, phenolics, chlorophyll fluorescence and photosynthetic parameters were examined in leaves of Citrus aurantium L. plants. Exogenous application of Pb and Cu resulted in an increase in leaf H2O2 and lipid peroxidation (MDA). Toxicity symptoms of both Pb and Cu treated plants were stunted growth and decreased pigments concentration. Furthermore, photosynthetic activity of treated plants exhibited a significant decline. The inhibition of growth in Pb and Cu-treated plants was accompanied by oxidative stress, as indicated by the enhanced lipid peroxidation and the high H2O2 concentration. Furthermore, antioxidants in citrus plants after exposure to high Pb and Cu concentrations were significantly increased compared to control and low Pb and Cu treatments. In conclusion, this study indicates that Pb and Cu promote lipid peroxidation, disrupt membrane integrity, reduces growth and photosynthesis and inhibit mineral nutrition. Considering the potential for adverse human health effects associated with high concentrations of Pb and Cu contained in edible parts of citrus plants the study signals that it is important to conduct further research into the accessibility and uptake of the tested heavy metals in the soil and whether they pose risks to humans.

Curbing gastrointestinal infections by defensin fragment modifications without harming commensal microbiota

The occurrence and spread of multidrug-resistant pathogens, especially bacteria from the ESKAPE panel, increases the risk to succumb to untreatable infections. We developed a novel antimicrobial peptide, Pam-3, with antibacterial and antibiofilm properties to counter this threat. The peptide is based on an eight-amino acid carboxyl-terminal fragment of human β-defensin 1. Pam-3 exhibited prominent antimicrobial activity against multidrug-resistant ESKAPE pathogens and additionally eradicated already established biofilms in vitro, primarily by disrupting membrane integrity of its target cell. Importantly, prolonged exposure did not result in drug-resistance to Pam-3. In mouse models, Pam-3 selectively reduced acute intestinal Salmonella and established Citrobacter infections, without compromising the core microbiota, hence displaying an added benefit to traditional broad-spectrum antibiotics. In conclusion, our data support the development of defensin-derived antimicrobial agents as a novel approach to fight multidrug-resistant bacteria, where Pam-3 appears as a particularly promising microbiota-preserving candidate.

Membrane Damage during Ferroptosis Is Caused by Oxidation of Phospholipids Catalyzed by the Oxidoreductases POR and CYB5R1

Ferroptosis is a form of necrotic cell death caused by iron-dependent peroxidation of polyunsaturated phospholipids on cell membranes and is actively suppressed by the cellular antioxidant systems. We report here that oxidoreductases, including NADPH-cytochrome P450 reductase (POR) and NADH-cytochrome b5 reductase (CYB5R1), transfer electrons from NAD(P)H to oxygen to generate hydrogen peroxide, which subsequently reacts with iron to generate reactive hydroxyl radicals for the peroxidation of the polyunsaturated fatty acid (PUFA) chains of membrane phospholipids, thereby disrupting membrane integrity during ferroptosis. Genetic knockout of POR and CYB5R1 decreases cellular hydrogen peroxide generation, preventing lipid peroxidation and ferroptosis. Moreover, POR knockdown in mouse liver prevents ConA-induced liver damage. Ferroptosis, therefore, is a result of incidental electron transfer carried out by POR/CYB5R1 oxidoreductase and thus needs to be constitutively countered by the antioxidant systems.

Liposome‐Boosted Peroxidase‐Mimicking Nanozymes Breaking the pH Limit

Peroxidase-mimicking nanozymes such as Fe3 O4 nanoparticles are promising substitutes for natural enzymes like horseradish peroxidase. However, most such nanozymes work efficiently only in acidic conditions. In this work, the influence of various liposomes on nanozyme activity was studied. By introducing negatively charged liposomes, peroxidase-mimicking nanozymes achieved oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in neutral and even alkaline conditions, although the activity towards anionic 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was inhibited. The Fe3 O4 nanoparticles adsorbed on the liposomes without disrupting membrane integrity as confirmed by fluorescence quenching, dye leakage assays, and cryo-electron microscopy. Stabilization of the blue-colored oxidized products of TMB by electrostatic interactions was believed to be the reason for the enhanced activity. This work has introduced lipids to nanozyme research, and it also has practically important applications for using nanozymes at neutral pH, such as the detection of hydrogen peroxide and glucose.

To Kill But Not Be Killed: Controlling the Activity of Mammalian Pore-Forming Proteins.

Pore-forming proteins (PFPs) are present in all domains of life, and play an important role in host-pathogen warfare and in the elimination of cancers. They can be employed to deliver specific effectors across membranes, to disrupt membrane integrity interfering with cell homeostasis, and to lyse membranes either destroying intracellular organelles or entire cells. Considering the destructive potential of PFPs, it is perhaps not surprising that mechanisms controlling their activity are remarkably complex, especially in multicellular organisms. Mammalian PFPs discovered to date include the complement membrane attack complex (MAC), perforins, as well as gasdermins. While the primary function of perforin-1 and gasdermins is to eliminate infected or cancerous host cells, perforin-2 and MAC can target pathogens directly. Yet, all mammalian PFPs are in principle capable of generating pores in membranes of healthy host cells which—if uncontrolled—could have dire, and potentially lethal consequences. In this review, we will highlight the strategies employed to protect the host from destruction by endogenous PFPs, while enabling timely and efficient elimination of target cells.

Anionic nanoparticle-induced perturbation to phospholipid membranes affects ion channel function

Understanding the mechanisms of nanoparticle interaction with cell membranes is essential for designing materials for applications such as bioimaging and drug delivery, as well as for assessing engineered nanomaterial safety. Much attention has focused on nanoparticles that bind strongly to biological membranes or induce membrane damage, leading to adverse impacts on cells. More subtle effects on membrane function mediated via changes in biophysical properties of the phospholipid bilayer have received little study. Here, we combine electrophysiology measurements, infrared spectroscopy, and molecular dynamics simulations to obtain insight into a mode of nanoparticle-mediated modulation of membrane protein function that was previously only hinted at in prior work. Electrophysiology measurements on gramicidin A (gA) ion channels embedded in planar suspended lipid bilayers demonstrate that anionic gold nanoparticles (AuNPs) reduce channel activity and extend channel lifetimes without disrupting membrane integrity, in a manner consistent with changes in membrane mechanical properties. Vibrational spectroscopy indicates that AuNP interaction with the bilayer does not perturb the conformation of membrane-embedded gA. Molecular dynamics simulations reinforce the experimental findings, showing that anionic AuNPs do not directly interact with embedded gA channels but perturb the local properties of lipid bilayers. Our results are most consistent with a mechanism in which anionic AuNPs disrupt ion channel function in an indirect manner by altering the mechanical properties of the surrounding bilayer. Alteration of membrane mechanical properties represents a potentially important mechanism by which nanoparticles induce biological effects, as the function of many embedded membrane proteins depends on phospholipid bilayer biophysical properties.

Monoterpenoid Geraniol Improves Anti-mycobacterial Drug Efficiency by Interfering with Lipidome and Virulence of Mycobacteria.

Tuberculosis (TB) remains a global infectious disorder for which efficient therapeutics are elusive. Nature is a source of novel pharmacologically active compounds with many potential drugs being derived directly or indirectly from plants, microorganisms and marine organisms. The present study aimed to elucidate the antimycobacterial potential of Geraniol (Ger), monoterpene alcohol, against Mycobacterium smegmatis. Disrupted membrane integrity was studied by membrane permeability assay and PI uptake. Cell surface phenotypes were studied by colony morphology, sliding motility and cell sedimentation rate. Lipidome profile was demonstrated by thin-layer chromatography and liquid chromatography-electrospray ionization mass spectrometry. Amendment in iron homeostasis was assessed by using iron chelator ferrozine and ferroxidase assay while genotoxicity was estimated with EtBr and DAPI staining. Biofilm formation was measured by staining, dry mass and metabolic activity using crystal violet. Cell adherence was examined microscopically and spectrophotometrically. We found the antimycobacterial activity of Ger to be 500 μg/ml against M. smegmatis. Underlying mechanisms revealed impaired cell surface phenotypes. Lipidomics analysis exposed profound decrement of mycolic acids, phosphatidylinositol mannosides and triacylglycerides which are crucial for MTB pathogenicity. We further explored that Ger impairs iron homeostasis and leads to genotoxic stress. Moreover, Ger inhibited the potential virulence attributes such as biofilm formation and cell adherence to both polystyrene surface and epithelial cells. Finally, we have validated all the disrupted phenotypes by RT-PCR which showed good correlation with the biochemical assays. Taken together, the current study demonstrates the antimycobacterial mechanisms of Ger, which may be exploited as an effective candidate of pharmacological interest.

Physiological and Oxidative Stress Responses of Lettuce to Cleomside A: A Thiohydroximate, as a New Allelochemical from Cleome arabica L.

The inclination toward natural products have led the onset for the discovery of new bioactive metabolites that could be targeted for specific therapeutic or agronomic applications. This study aimed to isolate bioactive compounds from Cleome arabica L., and subsequently determine the unexplored mechanism of action of the newly identified compounds on Lactuca sativa L. Chemical investigation of the ethyl acetate fraction of methanolic silique extract of C. arabica afforded seven secondary metabolites belonging to different classes such as flavonoids, triterpene, and a new thiohydroximate derivative, named cleomside A. Among phytotoxic assays, the growth of lettuce was totally inhibited by cleomside A compared to the other identified compounds. This effect was associated with the increased levels of electrolyte leakage, malondialdehyde, and hydrogen peroxide indicating disruption of membrane integrity and induction of oxidative stress. Activities of the antioxidant enzymes SOD, CAT, and APX were also elevated, thereby demonstrating the enhanced generation of reactive oxygen species upon identified allelochemical exposure. Thus, the changes caused by cleomside A described herein can contribute to better understanding the allelochemical actions of thiohydroximate and the potential use of these substances in the production of natural herbicides compared to the other identified flavonoids and triterpene.

A Novel Peptide Antibiotic Produced by Streptomyces roseoflavus Strain INA-Ac-5812 With Directed Activity Against Gram-Positive Bacteria.

In this work, we report the isolation and detailed functional characterization for the new non-ribosomally synthesized antibiotic 5812-A/C, which was derived from metabolites of Streptomyces roseoflavus INA-Ac-5812. According to its chemical structure, the studied 5812-A/C preliminary is composed of a cyclic peptide part covalently bounded with an arabinose residue. N-terminal amino acid sequencing of the native peptide has identified its partial structure of Leu-Asp-Gly-Ser-Gly and consisting of a Tyr residue that is supposed to have a two-component peptide nature for the molecule studied. However, the structural analysis of the antibiotic complex derived from S. roseoflavus INA-Ac-5812 is still ongoing. The mechanism of action of 5812-A/C was assessed in comparison with its most related analog, the lipopeptide antibiotic daptomycin, given the presence in both antimicrobials of an L-kynurenine amino acid residue. The inhibitory activity of 5812-A/C against Gram-positive bacteria including methicillin-resistant strain of Staphylococcus aureus was similar to daptomycin. The mechanism of action of 5812-A/C was associated with the disruption of membrane integrity, which differs in comparison with daptomycin and is most similar to the antimicrobial membrane-disturbing peptides. However, 5812-A/C demonstrated a calcium-dependent mode of action. In addition, unlike daptomycin, 5812-A/C was able to penetrate mature biofilms and inhibit the metabolic activity of embedded S. aureus cells. At the same time, 5812-A/C has no hemolytic activity toward erythrocyte, but possessed weak cytotoxic activity represented by heterochromatin condensation in human buccal epithelium cells. The biological properties of the peptide 5812-A/C suggest its classification as a calcium-dependent antibiotic effective against a wide spectrum of Gram-positive pathogenic bacteria.

Reduction in MLKL-mediated endosomal trafficking enhances the TRAIL-DR4/5 signal to increase cancer cell death

Mixed lineage kinase domain-like (MLKL) is an essential molecule of necroptosis, a cell death process that is initiated by direct disruption of the plasma membrane. During necroptosis, MLKL is phosphorylated by receptor interacting protein kinase-3 (RIPK3 or RIP3), and then translocates to the plasma membrane to disrupt membrane integrity. Recent data suggest that MLKL also has a RIP3-indendent function in the generation of intraluminal and extracellular vesicles (EVs), as well as in myelin sheath breakdown when promoting sciatic nerve regeneration. Here we show that depletion of MLKL enhances TRAIL-induced cell death in a RIP3-independent manner. Depletion of MLKL leads to prolonged cytotoxic signals that increase TRAIL-induced cell death. Initially, TRAIL binds to DR5 at the cell surface and is endocytosed at similar rates in MLKL-expressing and MLKL-depleted cells, eventual degradation of intracellular TRAIL by the lysosome is delayed in MLKL-depleted cells, corresponding with prolonged/enhanced intracellular signals such as p-ERK and p-p38 in these cells. Colocalization of TRAIL with the marker of early endosomes, EEA1 suggests that TRAIL is accumulated in early endosomes in MLKL-depleted cells compared to MLKL-expressing cells. This indicates that depletion of MLKL reduces receptor-ligand endosomal trafficking leading to increased TRAIL-cytotoxicity. An MLKL mutant that compromises its necroptotic function and its function in the generation of EVs was sufficient to rescue MLKL deficiency, suggesting that the N-terminal structural elements necessary for these functions are not required for the function of MLKL in the intracellular trafficking associated with regulating death receptor cytotoxicity. A reduction in MLKL expression in cancer cells would therefore be expected to result in enhanced TRAIL-induced therapeutic efficacy.

Inactivation of Listeria monocytogenes and Salmonella Typhimurium in strawberry juice enriched with strawberry polyphenols.

Low molecular-weight phenolic fractions (LMPFs) were extracted from Albion (LMPF-A) and Camarosa (LMPF-C) strawberry cultivars. Their antibacterial activity against Listeria monocytogenes and Salmonella Typhimurium cocktails in vitro and in vivo was investigated using strawberry juice as a food model. This study also sought to determine their antibacterial mechanism. Quercetin was identified as a principal compound in both phenolic fractions. The minimum bactericide concentration (MBC) values were 750 and 850 μg mL-1 (LMPF-C) and 800 and 950 μg mL-1 (LMPF-A) against S.Typhimurium and L. monocytogenes, respectively. The possible antibacterial activity of the phenolic extracts could be related to the release of phosphate and potassium ions, the effect of the disruption of membrane integrity on L. monocytogenes, and the effect of the inhibition of dihydronicotinamide adenine dinucleotide (NADH) oxidase activity on S. Typhimurium. Quercetin and kaempferol were the most active compounds in producing bacterial damage. Strawberry juice supplemented with the phenolic fractions and incubated at 37, 20, and 4 °C reduced bacterial viability; moreover, after treatment with the phenolic fraction at the lowest temperature, no viable cells were detected after 7 days' incubation. Salmonella was more sensitive to the supplements than Listeria in strawberry juice. This study could form the basis for the development of natural antibacterial agents that could be included in natural juice or used by the pharmaceutical industry. © 2020 Society of Chemical Industry.

Inhibition of sigma-1 receptors substantially modulates GABA and glutamate transport in presynaptic nerve terminals

Sigma-1 receptors (Sig-1Rs) have been implicated in many neurological and psychiatric disorders and are a novel target for the treatment of such disorders. Sig-1R expression/activity deficits are linked to neurodegeneration, whereas the mechanisms mediated by Sig-1R are still unclear. Here, presynaptic [3H]GABA and L-[14C]glutamate transport was analysed in rat brain nerve terminals (synaptosomes) in the presence of the Sig-1R antagonist NE-100. NE-100 at doses of 1 and 10 μM increased the initial rate of synaptosomal [3H]GABA uptake, whereas 50 and 100 μM NE-100 decreased this rate, exerting a biphasic mode of action.Antagonists of GABAA and GABAB receptors, flumazenil and saclofen, respectively, prevented an increase in [3H]GABA uptake caused by 10 μM NE-100. L-[14C]glutamate uptake was decreased by 10–100 μM NE-100. A decrease in the uptake of both neurotransmitters mediated by NE-100 (50–100 μM) may have resulted from simultaneous antagonist-induced membrane depolarization, which was measured using the potential-sensitive fluorescent dye rhodamine 6G. The extracellular level of [3H]GABA was decreased by 1–10 μM NE-100, but that of L-[14C]glutamate remained unchanged. The tonic release of [3H]GABA measured in the presence of NO-711 was not changed by the antagonist, suggesting that NE-100 did not disrupt membrane integrity. The KCl- and FCCP-induced transporter-mediated release of L-[14C]glutamate was decreased by the antagonist; this may underlie the neuroprotective action of the antagonist in hypoxia/ischaemia. NE-100 (10–100 μM) decreased the KCl-evoked exocytotic release of [3H]GABA and L-[14C]glutamate, whereas the induction of the release of both neurotransmitters by the Ca2+ ionophore ionomycin was not affected by the antagonist; therefore, the mitigation of KCl-evoked exocytosis was associated with the NE-100-induced dysfunction of potential-dependent Ca2+ channels. Therefore, the Sig-1R antagonist can specifically act in an acute manner at the presynaptic level through the modulation of GABA and glutamate uptake, transporter-mediated release and exocytosis.

Deep-Red-Fluorescent Zinc Probe with a Membrane-Targeting Cholesterol Unit.

Organelle-targeting fluorescence probes are valuable because they can provide spatiotemporal information about the trafficking of analytes of interest. The spatiotemporal resolution can be improved by using low-energy emission signals because they are barely contaminated by autofluorescence noises. In this study, we designed and synthesized a deep-red-fluorescent zinc probe (JJ) with a membrane-targeting cholesterol unit. This zinc probe consists of a boron-azadipyrromethene (aza-BODIPY) fluorophore and a zinc receptor that is tethered to a tri(ethylene glycol)-cholesterol chain. In aqueous solutions buffered to pH 7.4, JJ exhibits weak fluorescence with a peak wavelength of 663 nm upon excitation at 622 nm. The addition of ZnCl2 elicits an approximately 5-fold enhancement of the fluorescence emission with a fluorescence dynamic range of 141000. Our electrochemical and picosecond transient photoluminescence investigations indicate that the fluorescence turn-on response is due to the zinc-induced abrogation of the formation of a nonemissive intramolecularly charge-separated species, which occurs with a driving force of 0.98 eV. The fluorescence zinc response was found to be fully reversible and to be unaffected by pH changes or the presence of biological metal ions. These properties are due to tight zinc binding with a dissociation constant of 4 pM. JJ was found to be nontoxic to HeLa cells up to submicromolar concentrations, which enables cellular imaging. Colocalization experiments were performed with organelle-specific stains and revealed that JJ is rapidly internalized into intracellular organelles, including lysosomes and endoplasmic reticula. Unexpectedly, probe internalization was found to permeabilize the cell membrane, which facilitates the influx of exogens such as zinc ions. Such permeabilization does not arise for a control probe without the tri(ethylene glycol)-cholesterol chain (JJC). Our results show that the membrane-targeting cholesterol unit can disrupt membrane integrity.

Antimicrobial and anti-inflammatory activities of short dodecapeptides derived from duck cathelicidin: Plausible mechanism of bactericidal action and endotoxin neutralization

Antimicrobial peptides (AMPs) have gained increasing attention to combat antibiotic-resistant pathogens. dCATH (duck cathelicidin) is a 20-residue avian cathelicidin with potent bactericidal activity. However, its therapeutic application is limited due to high mammalian cell cytotoxicity. To develop therapeutically useful AMPs with enhanced antimicrobial and cell-selective property, we designed a series of 12-meric (dodeca) short amphiphilic peptides based on dCATH. Among these, Trp and Lys-rich dCATH 12–4 and dCATH 12–5 exhibited higher selectivity towards bacterial cells than erythrocytes and macrophages. Additionally, these AMPs significantly reduced NO and TNF-α secretion in LPS-stimulated macrophage cells, suggesting their anti-inflammatory properties. Various fluorophore-based studies and confocal microscopic observations demonstrated that dCATH 12–4 and dCATH 12–5 could penetrate the bacterial cell membrane and accumulate in the cytoplasm, without disrupting membrane integrity. Results from the microscopic examination and gel-retardation DNA binding assay suggested that both the designed AMPs could bind with bacterial DNA, subsequently leading to cell death via arrest of DNA synthesis. Fluorescence spectroscopy and flow cytometry analysis revealed that the designed AMPs induced strong binding to LPS oligomers which resulted in dissociation of LPS aggregates, thereby preventing LPS from binding to the carrier protein lipopolysaccharide-binding protein (LBP) or alternatively to CD14 receptors of macrophage cells. Additionally, both dCATH 12–4 and dCATH 12-5 demonstrated synergistic actions with various conventional antibiotics against antibiotic resistant pathogens, thus indicating their ability as promising adjuncts to combination therapy. In summary, these findings contribute to the design of short AMPs with bactericidal and immunomodulatory properties for combating bacterial infection and sepsis.

Dimerization of Aβ40 inside dipalmitoylphosphatidylcholine bilayer and its effect on bilayer integrity: Atomistic simulation at three temperatures.

Amyloid-beta (Aβ) protein is related to Alzheimer disease (AD), and various experiments have shown that oligomers as small as dimers are cytotoxic. Recent studies have concluded that interactions of Aβ with neuronal cell membranes lead to disruption of membrane integrity and toxicity and they play a key role in the development of AD. Molecular dynamics (MD) simulations have been used to investigate Aβ in aqueous solution and membranes. We have previously studied monomeric Aβ40 embedded in dipalmitoylphosphatidylcholine (DPPC) membrane using MD simulations. Here, we explore interactions of two Aβ40 peptides in DPPC bilayer and its consequences on dimer distribution in a lipid bilayer and on the secondary structure of the peptides. We explored that N-terminals played an important role in dimeric Aβ peptide aggregations and Aβ-bilayer interactions, while C-terminals bound peptides to bilayer like anchors. We did not observe exiting of peptides in our simulations although we observed insertion of peptides into the core of bilayer in some of our simulations. So it seems that the presence of Aβ on membrane surface increases its aggregation rate, and as diffusion occurs in two dimensions, it can increase the probability of interpeptide interactions. We found that dimeric Aβ, like monomeric one, had the ability to cause structural destabilization of DPPC membrane, which in turn might ultimately lead to cell death in an in vivo system. This information could have important implications for understanding the affinity of Aβ oligomers (here dimer) for membranes and the mechanism of Aβ oligomer toxicity in AD.