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Here the hypothesis that linearized esculentin 2EM (E2EM-lin) from Glandirana emeljanovi possesses pH dependent activity is investigated. The peptide showed weak activity against Gram-negative bacteria (MLCs ≥ 75.0 μM) but potent efficacy towards Gram-positive bacteria (MLCs ≤ 6.25 μM). E2EM-lin adopted an α-helical structure in the presence of bacterial membranes that increased as pH was increased from 6 to 8 (↑ 15.5–26.9%), whilst similar increases in pH enhanced the ability of the peptide to penetrate (↑ 2.3–5.1 mN m−1) and lyse (↑ 15.1–32.5%) these membranes. Theoretical analysis predicted that this membranolytic mechanism involved a tilted segment, that increased along the α-helical long axis of E2EM-lin (1–23) in the N → C direction, with − µH > increasing overall from circa − 0.8 to − 0.3. In combination, these data showed that E2EM-lin killed bacteria via novel mechanisms that were enhanced by alkaline conditions and involved the formation of tilted and membranolytic, α-helical structure. The preference of E2EM-lin for Gram-positive bacteria over Gram-negative organisms was primarily driven by the superior ability of phosphatidylglycerol to induce α-helical structure in the peptide as compared to phosphatidylethanolamine. These data were used to generate a novel pore-forming model for the membranolytic activity of E2EM-lin, which would appear to be the first, major reported instance of pH dependent AMPs with alkaline optima using tilted structure to drive a pore-forming process. It is proposed that E2EM-lin has the potential for development to serve purposes ranging from therapeutic usage, such as chronic wound disinfection, to food preservation by killing food spoilage organisms.

Aurein 2.1, aurein 2.6 and aurein 3.1 are amphibian host defence peptides that kill bacteria via the use of lytic amphiphilic α-helical structures. The C-terminal PEGylation of these peptides led to decreased antibacterial activity (Minimum Lethal Concentration (MLCs) ↓ circa one and a half to threefold), reduced levels of amphiphilic α-helical structure in solvents (α-helicity ↓ circa 15.0%) and lower surface activity (Δπ ↓ > 1.5 mN m ). This PEGylation of aureins also led to decreased levels of amphiphilic α-helical structure in the presence of anionic membranes and zwitterionic membranes (α-helicity↓ > 10.0%) as well as reduced levels of penetration (Δπ ↓ > 3.0 mN m ) and lysis (lysis ↓ > 10.0%) of these membranes. Based on these data, it was proposed that the antibacterial action of PEGylated aureins involved the adoption of α-helical structures that promote the lysis of bacterial membranes, but with lower efficacy than their native counterparts. However, PEGylation also reduced the haemolytic activity of native aureins to negligible levels (haemolysis ↓ from circa 10% to 3% or less) and improved their relative therapeutic indices (RTIs ↑ circa three to sixfold). Based on these data, it is proposed that PEGylated aureins possess the potential for therapeutic development; for example, to combat infections due to multi-drug resistant strains of S. aureus, designated as high priority by the World Health Organization. [Abstract copyright: Copyright © 2021 Elsevier B.V. All rights reserved.]

A number of disorders and diseases are associated with conditions of high pH, and many conventional antibiotics lose their efficacy under these pH conditions, generating a need for novel antimicrobials. A potential solution to fulfill this need is Antimicrobial Peptides (AMPs) with high pH optima. This review shows that a variety of anionic and cationic AMPs with this pH dependency are produced by creatures across the eukaryotic kingdom, including rabbits, cattle, sheep, fish, crabs and frog. These AMPs exhibit activity against viruses, bacteria, and fungi that involve membrane interactions and appear to be facilitated by a variety of mechanisms that generally promote passage across membranes to attack intracellular targets, such as DNA or protein synthesisand/or membrane lysis. Some of these mechanisms are unknown, but those elucidated include the use of bacterial pores and transporters, the self-promoted uptake pathway, and established models of membrane interaction, such as the carpet mechanism, toroidal pore formation, the adoption of tilted peptide, and the SHM model. A variety of potential roles have been proposed for these AMPs, including use as antivirals, antibacterials, antifungals, adjuvants to antimicrobial therapy, biomarkers of disease, and probes for pathogenic microbes. In this review, these properties are described and discussed, emphasizing the antimicrobial mechanisms used by these AMPs and the pH dependency of these mechanisms.

We evaluate a series of thin-sheet hydrogel molecularly imprinted polymers (MIPs), using a family of acrylamide-based monomers, selective for the target protein myoglobin (Mb). The simple production of the thin-sheet MIP offers an alternative biorecognition surface that is robust, stable and uniform, and has the potential to be adapted for biosensor applications. The MIP containing the functional monomer N-hydroxymethylacrylamide (NHMAm), produced optimal specific rebinding of the target protein (Mb) with 84.9% (± 0.7) rebinding and imprinting and selectivity factors of 1.41 and 1.55, respectively. The least optimal performing MIP contained the functional monomer N,N-dimethylacrylamide (DMAm) with 67.5% (± 0.7) rebinding and imprinting and selectivity factors of 1.11 and 1.32, respectively. Hydrogen bonding effects, within a protein-MIP complex, were investigated using computational methods and Fourier transform infrared (FTIR) spectroscopy. The quantum mechanical calculations predictions of a red shift of the monomer carbonyl peak is borne-out within FTIR spectra, with three of the MIPs, acrylamide, N-(hydroxymethyl) acrylamide, and N-(hydroxyethyl) acrylamide, showing peak downshifts of 4, 11, and 8 cm−1, respectively.

In this study, PGA-co-PDL nanoparticles (NPs) encapsulating model antigen, bovine serum albumin (BSA), were prepared via double emulsion solvent evaporation. In addition, chitosan hydrochloride (CHL) was incorporated into the external phase of the emulsion solvent method, which resulted in surface adsorption onto the NPs to form hybrid cationic CHL NPs. The BSA encapsulated CHL NPs were encompassed into nanocomposite microcarriers (NCMPs) composed of l-leucine to produce CHL NPs/NCMPs via spray drying. The CHL NPs/NCMPs were investigated for in vitro aerosolization, release study, cell viability and uptake, and stability of protein structure. Hybrid cationic CHL NPs (CHL: 10 mg/mL) of particle size (480.2 ± 32.2 nm), charge (+14.2 ± 0.72 mV), and BSA loading (7.28 ± 1.3 µg/mg) were produced. The adsorption pattern was determined to follow the Freundlich model. Aerosolization of CHL NPs/NCMPs indicated fine particle fraction (FPF: 46.79 ± 11.21%) and mass median aerodynamic diameter (MMAD: 1.49 ± 0.29 µm). The BSA α-helical structure was maintained, after release from the CHL NPs/NCMPs, as indicated by circular dichroism. Furthermore, dendritic cells (DCs) and A549 cells showed good viability (≥70% at 2.5 mg/mL after 4–24 h exposure, respectively). Confocal microscopy and flow cytometry data showed hybrid cationic CHL NPs were successfully taken up by DCs within 1 h of incubation. The upregulation of CD40, CD86, and MHC-II cell surface markers indicated that the DCs were successfully activated by the hybrid cationic CHL NPs. These results suggest that the CHL NPs/NCMPs technology platform could potentially be used for the delivery of proteins to the lungs for immunostimulatory applications such as vaccines.

Linearized esculentin 2 EM (E2EM-lin) from the frog, Glandirana emeljanovi was highly active against Gram-positive bacteria (minimum lethal concentration ≤ 5.0 μM) and strongly α-helical in the presence of lipid mimics of their membranes (>55.0%). The N-terminal α-helical structure adopted by E2EM-lin showed the potential to form a membrane interactive, tilted peptide with an hydrophobicity gradient over residues 9 to 23. E2EM-lin inserted strongly into lipid mimics of membranes from Gram-positive bacteria (maximal surface pressure changes ≥5.5 mN m ), inducing increased rigidity (C ↑), thermodynamic instability (ΔG 0) and high levels of lysis (>50.0%). These effects appeared to be driven by the high anionic lipid content of membranes from Gram-positive bacteria; namely phosphatidylglycerol (PG) and cardiolipin (CL) species. The high levels of α-helicity (60.0%), interaction (maximal surface pressure change = 6.7 mN m ) and lysis (66.0%) shown by E2EM-lin with PG species was a major driver in the ability of the peptide to lyse and kill Gram-positive bacteria. E2EM-lin also showed high levels of α-helicity (62.0%) with CL species but only low levels of interaction (maximal surface pressure change = 2.9 mN m ) and lysis (21.0%) with the lipid. These combined data suggest that E2EM-lin has a specificity for killing Gram-positive bacteria that involves the formation of tilted structure and appears to be primarily driven by PG-mediated membranolysis. These structure/function relationships are used to help explain the pore forming process proposed to describe the membranolytic, antibacterial action of E2EM-lin. [Abstract copyright: Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.]

The author's final peer reviewed version can be found by following the URI link. The Publisher's final version can be found by following the DOI link. Molecularly imprinted polymers (MIPs) have potential as alternatives to antibodies in the diagnosis and treatment of disease. However, atomistic level knowledge of the prepolymerization process is limited that would facilitate rational design of more efficient MIPs. Accordingly, we have investigated using computation and experiment the protein–monomer binding interactions that may influence the desired specificity. Myoglobin was used as the target protein and five different acrylamide-based monomers were considered. Protein binding sites were predicted using SiteMap and binding free energies of monomers at each site were calculated using MM-GBSA. Statistical thermodynamic analysis and study of atomistic interactions facilitated rationalization of monomer performance in MIP rebinding studies (% rebind; imprinting factors). CD spectroscopy was used to determine monomer effects on myoglobin secondary structure, with all monomers except the smallest monomer (acrylamide) causing significant changes. A complex interplay between different protein–monomer binding effects and MIP efficacy was observed. Validation of hypotheses for key binding features was achieved by rational selection of two different comonomer MIP combinations that produced experimental results in agreement with predictions. The comonomer studies revealed that uniform, noncompetitive binding of monomers around a target protein is favorable. This study represents a step toward future rational in silico design of MIPs for proteins.

Modelin-5-CONH2 (M5-NH2) is a synthetic antimicrobial peptide, which was found to show potent activity against Bacillus subtilis (Minimum lethal concentration = 8.47 μM) and to bind strongly to membranes of the organism (Kd = 10.44 μM). The peptide adopted high levels of amphiphilic α-helical structure in the presence of these membranes (> 50 %), which led to high levels of insertion (Δπ ≥ 8.0 mN m-1). M5-NH2 showed high affinity for anionic lipid (Kd = 7.46 µM) and zwitterionic lipid (Kd = 14.7 µM), which drove insertion into membranes formed from these lipids (Δπ = 11.5 and 3.5 mN m-1, respectively) and interactions with their head group and hydrophobic core regions. Neutron diffraction studies showed that M5-NH2 inserted into B. subtilis membranes with its N-terminal residue, L16, located 5.5 Å from the membrane centre, in the acyl chain region of these membranes, and promoted a reduction in membrane thickness of circa 1.8 Å or 5 % of membrane width. Insertion into B. subtilis membranes by the peptide also promoted other effects associated with membrane thinning, including increases in membrane surface area (Cs-1 decrease) and fluidity (ΔGmix > 0 to < 0). Membrane insertion and thinning by M5-NH2 induced high levels of lysis (> 55 %) and, taken with MD simulation data, it is speculated that the antibacterial action of the peptide may involve the toroidal pore, carpet or tilted-type mechanism of membrane permeabilization.

Acoustic radiation from three commercial pest deterrents and two hair dryers were measured in an anechoic chamber. The deterrents were chosen because the frequency range at which they emit the most energy is either in the very high-frequency sound band (11.2 kHz - 17.8 kHz) or the ultrasound band (greater than 17.8 kHz). These are sources that may be heard by a subset of the general population, with the young typically having better high frequency sensitivity. A hairdryer reported to increase the frequency of the motor noise above the audible hearing range was compared with a standard hairdryer. The outputs of the deterrents are compared against six international regulations and guidelines for audible and ultrasound exposure. Multiple ambiguities in the application of these guidelines are discussed. These ambiguities could lead to a device being considered as in compliance despite unconventionally high levels. Even if a device measured here meets a guideline, actual exposures are likely to exceed those taken here and may therefore breach guidelines if the listener is closer to the device or reflections increase the exposure level.

In this study, Taguchi design was used to determine optimal parameters for the preparation of bovine serum albumin (BSA)-loaded nanoparticles (NPs) using a biodegradable polymer poly(glycerol adipate-co-ω-pentadecalactone) (PGA-co-PDL). NPs were prepared, using BSA as a model protein, by the double emulsion evaporation process followed by spray-drying from leucine to form nanocomposite microparticles (NCMPs). The effect of various parameters on NP size and BSA loading were investigated and dendritic cell (DC) uptake and toxicity. NCMPs were examined for their morphology, yield, aerosolisation, in vitro release behaviour and BSA structure. NP size was mainly affected by the polymer mass used and a small particle size ≤500 nm was achieved. High BSA (43.67 ± 2.3 μg/mg) loading was influenced by BSA concentration. The spray-drying process produced NCMPs (50% yield) with a porous corrugated surface, aerodynamic diameter 1.46 ± 141 μm, fine particle dose 45.0 ± 4.7 μg and fine particle fraction 78.57 ± 0.1%, and a cumulative BSA release of 38.77 ± 3.0% after 48 h. The primary and secondary structures were maintained as shown by sodium dodecyl sulphate poly (acrylamide) gel electrophoresis and circular dichroism. Effective uptake of NPs was seen in DCs with >85% cell viability at 5 mg/mL concentration after 4 h. These results indicate the optimal process parameters for the preparation of protein-loaded PGA-co-PDL NCMPs suitable for inhalation. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:4386–4398, 2015

Anionic antimicrobial peptides (AAMPs) with net charges ranging from -1 to -8 have been identified in frogs, toads, newts and salamanders across Africa, South America and China. Most of these peptides show antibacterial activity and a number of them are multifunctional, variously showing antifungal activity, anticancer action, neuropeptide function and the ability to potentiate conventional antibiotics. Antimicrobial mechanisms proposed for these AAMPs, include toroidal pore formation and the Shai-Huang-Matsazuki model of membrane interaction along with pH dependent amyloidogenesis and membranolysis via tilted peptide formation. The potential for therapeutic and biotechnical application of these AAMPs has been demonstrated, including the development of amyloid-based nanomaterials and antiviral agents. It is concluded that amphibian AAMPs represent an untapped potential source of biologically active agents and merit far greater research interest.

Here we report the first major example of anionic amphibian host defence peptides (HDPs) with anticancer activity. Maximin H5 is a C-terminally amidated, anionic host defence peptide (MH5N) from toads of the Bombina genus, which was shown to possess activity against the glioma cell line, T98G (EC50 = 125 μM). The peptide adopted high levels of α-helical structure (57.3%) in the presence of model cancer membranes (DMPC:DMPS in a molar ratio of 10:1). MH5N also showed a strong ability to penetrate these model membranes (Π = 10.5 mN m-1), which correlated with levels of DMPS (R2 > 0.98). Taken with the high ability of the peptide to lyse these membranes (65.7%), it is proposed that maximin H5 kills cancer cells via membranolytic mechanisms that are promoted by anionic lipid. It was also found that C-terminally deaminated maximin H5 (MH5C) exhibited lower levels of α-helical structure in the presence of cancer membrane mimics (44.8%) along with a reduced ability to penetrate these membranes (Π = 8.1 mN m-1) and induce their lysis (56.6%). These data suggested that the two terminal amide groups of native maximin H5 are required for its optimal membranolytic and anticancer activity.

PURPOSE: \ud \ud Dry powder vaccine delivery via the pulmonary route has gained significant attention as an alternate route to parenteral delivery. In this study, we investigated bovine serum albumin (BSA) adsorbed poly(glycerol adipate-co-ω-pentadecalactone), PGA-co-PDL polymeric nanoparticles (NPs) within L-leucine (L-leu) microcarriers for dry powder inhalation.\ud \ud METHODS: \ud \ud NPs were prepared by oil-in-water single emulsion-solvent evaporation and particle size optimised using Taguchi's design of experiment. BSA was adsorbed onto NPs at different ratios at room temperature. The NPs were spray-dried in aqueous suspension of L-leu (1:1.5) using a Büchi-290 mini-spray dryer. The resultant nanocomposite microparticles (NCMPs) were characterised for toxicity (MTT assay), aerosolization (Next Generation Impactor), in vitro release study and BSA was characterized using SDS-PAGE and CD respectively.\ud \ud RESULTS: \ud \ud NPs of size 128.50 ± 6.57 nm, PDI 0.07 ± 0.03 suitable for targeting lung dendritic cells were produced. BSA adsorption for 1 h resulted in 10.23 ± 1.87 μg of protein per mg of NPs. Spray-drying with L-leu resulted in NCMPs with 42.35 ± 3.17% yield. In vitro release study at 37°C showed an initial burst release of 30.15 ± 2.33% with 95.15 ± 1.08% over 48 h. Aerosolization studies indicated fine particle fraction (FPF%) dae

Globally, death due to cancers is likely to rise to over 20 million by 2030, which has created an urgent need for novel approaches to anticancer therapies such as the development of host defence peptides. Cn-AMP2 (TESYFVFSVGM), an anionic host defence peptide from green coconut water of the plant Cocos nucifera, showed anti-proliferative activity against the 1321N1 and U87MG human glioma cell lines with IC50 values of 1.25 and 1.85 mM, respectively. The membrane interactive form of the peptide was found to be an extended conformation, which primarily included β-type structures (levels > 45%) and random coil architecture (levels > 45%). On the basis of these and other data, it is suggested that the short anionic N-terminal sequence (TES) of Cn-AMP2 interacts with positively charged moieties in the cancer cell membrane. Concomitantly, the long hydrophobic C-terminal sequence (YFVFSVGM) of the peptide penetrates the membrane core region, thereby driving the translocation of Cn-AMP2 across the cancer cell membrane to attack intracellular targets and induce anti-proliferative mechanisms. This work is the first to demonstrate that anionic host defence peptides have activity against human glioblastoma, which potentially provides an untapped source of lead compounds for development as novel agents in the treatment of these and other cancers.

Fungal infections with multiple resistance to conventional antifungals are increasingly becoming a medical problem, and there is an urgent need for new antifungal compounds with novel mechanisms of action. Here, we show that aurein 2.5, a naturally occurring peptide antibiotic, displays activity against the fungal strains: Rhodotorula rubra and Schizosaccharomyces pombe (MICs 130 μM). The peptide adopted high levels of membrane-interactive α-helical structure ( 65%) in the presence of lipid membranes derived from these organisms and showed strong propensities to penetrate (π ≥ 13 mN m(-1) ) and lyse them ( 70%). Based on these data, we suggest that aurein 2.5 kills yeasts via membranolytic mechanisms and may act as a template for the development of therapeutically useful antifungal agents.

In this study, an amphibian antimicrobial peptide, aurein 2.3, was predicted to use oblique orientated α-helix formation in its mechanism of membrane destabilisation. Molecular dynamic (MD) simulations and circular dichroism (CD) experimental data suggested that aurein 2.3 exists in solution as unstructured monomers and folds to form predominantly α-helical structures in the presence of a dimyristoylphosphatidylcholine membrane. MD showed that the peptide was highly surface active, which supported monolayer data where the peptide induced surface pressure changes > 34 mN m - 1. In the presence of a lipid membrane MD simulations suggested that under hydrophobic mismatch the peptide is seen to insert via oblique orientation with a phenylalanine residue (PHE3) playing a key role in the membrane interaction. There is evidence of snorkelling leucine residues leading to further membrane disruption and supporting the high level of lysis observed using calcein release assays (76%). Simulations performed at higher peptide/lipid ratio show peptide cooperativity is key to increased efficiency leading to pore-formation. © 2012 Elsevier B.V. All rights reserved.

Antimicrobial peptides (AMPs) are potent antibiotics of the innate immune system that have been extensively investigated as a potential solution to the global problem of infectious diseases caused by pathogenic microbes. A group of AMPs that are increasingly being reported are those that utilise pH dependent antimicrobial mechanisms and here, we review research into this area. This review shows that these antimicrobial molecules are produced by a diverse spectrum of creatures, including vertebrates and invertebrates, and are primarily cationic although a number of anionic examples are known. Some of these molecules exhibit high pH optima for their antimicrobial activity but in most cases, these AMPs show activity against microbes that present low pH optima, which reflects the acidic pH generally found at their sites of action, particularly the skin. The modes of action used by these molecules are based on a number of major structure / function relationships, which include metal ion binding, changes to net charge and conformational plasticity, and primarily involve the protonation of histidine, aspartic acid and glutamic acid residues at low pH. The pH dependent activity of pore forming antimicrobial proteins involves mechanisms that generally differ fundamentally to those used by pH dependent AMPs, which can be described by the carpet, toroidal pore and barrel-stave pore models of membrane interaction. A number of pH dependent AMPs and antimicrobial proteins have been developed for medical purposes and have successfully completed clinical trials, including kappacins, LL-37, histatins and lactoferrin, along with a number of their derivatives. Major examples of the therapeutic application of these antimicrobial molecules include wound healing as well as the treatment of multiple cancers and infections due to viruses, bacteria and fungi. In general, these applications involve topical administration, such as the use of mouth washes, cream formulations and hydrogel delivery systems. Nonetheless, many pH dependent AMPs and antimicrobial proteins have yet to be fully characterized and these molecules, as a whole, represent an untapped source of novel biologically active agents that could aid fulfillment of the urgent need for alternatives to conventional antibiotics, helping to avert a return to the pre-antibiotic era.

Maximin H5 (MH5) is an amphibian antimicrobial peptide specifically targeting Staphylococcus aureus. At pH 6, the peptide showed an improved ability to penetrate (ΔΠ = 6.2 mN m(-1)) and lyse (lysis = 48%) Staphylococcus aureus membrane mimics, which incorporated physiological levels of lysylated phosphatidylglycerol (Lys-PG, 60%), compared to that at pH 7 (ΔΠ = 5.6 mN m(-1) and lysis = 40% at pH 7) where levels of Lys-PG are lower (40%). The peptide therefore appears to have optimal function at pH levels known to be optimal for the organism's growth. MH5 killed S. aureus (minimum inhibitory concentration of 90 μM) via membranolytic mechanisms that involved the stabilization of α-helical structure (approximately 45-50%) and showed similarities to the "Carpet" mechanism based on its ability to increase the rigidity (Cs(-1) = 109.94 mN m(-1)) and thermodynamic stability (ΔGmix = -3.0) of physiologically relevant S. aureus membrane mimics at pH 6. On the basis of theoretical analysis, this mechanism might involve the use of a tilted peptide structure, and efficacy was noted to vary inversely with the Lys-PG content of S. aureus membrane mimics for each pH studied (R(2) ∼ 0.97), which led to the suggestion that under biologically relevant conditions, low pH helps mediate Lys-PG-induced resistance in S. aureus to MH5 antibacterial action. The peptide showed a lack of hemolytic activity (2% hemolysis) and merits further investigation as a potential template for development as an antistaphylococcal agent in medically and biotechnically relevant areas.

Aurein 2.6-COOH and aurein 3.1-COOH were studied along with their naturally occurring C-terminally amidated analogues. Circular dichroism (CD) and molecular dynamic (MD) simulations were used to study the effects of amidation on the interaction of antimicrobial peptides (AMPs) with lipid bilayers. CD measurements and MD analysis suggested that both peptide analogues were predominantly random coil and adopted low levels of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha$$\end{document}α-helical structure in solution (

In order to gain an insight into the mechanism of antimicrobial peptide action, aurein 2.5 and modelin-5 were studied. When tested against Staphylococcus aureus, aurein 2.5 showed approximately 5-fold greater efficacy even though the higher net positive charge and higher helix stability shown by modelin-5 would have predicated modelin-5 to be the more effective antimicrobial. However, in the presence of S. aureus membrane mimics, aurein 2.5 showed greater helical content (75% helical) relative to modelin-5 (51% helical) indicative of increase in membrane association. This was supported by monolayer data showing that aurein 2.5 (6.6mNm−1) generated greater pressure changes than modelin-5 (5.3mNm−1). Peptide monolayers indicted that modelin-5 formed a helix horizontal to the plane of an asymmetric interface which would be supported by the even distribution of charge and hydrophobicity along the helical long axis and facilitate lysis by non-specific membrane binding. In contrast, a groove structure observed on the surface of aurein 2.5 was predicted to be the cause of enhanced lipid binding (Kd=75μM) relative to modelin-5 (Kd=118μM) and the balance of hydrophobicity along the aurein 2.5 long axis supported deep penetration into the membrane in a tilt formation. This oblique orientation generates greater lytic efficacy in high anionic lipid (71%) compared to modelin-5 (32%).

Cationic defence peptides show high therapeutic potential as antimicrobial and anticancer agents. Some of these peptides carry a C-terminal amide moiety which has been shown to be required for antimicrobial activity. However, whether this is a general requirement or whether C-terminal amidation is required for the anticancer activity of defence peptides is unclear. In response, this study analyses the toxicity of a series of C-terminally amidated defence peptides and their non-amidated isoforms to normal fibroblast cells, a variety of tumour cells and bacterial cells. The toxicities of these peptides to microbial and cancer cells were generally200 microM. Peptides were either unaffected by C-terminal amidation or showed up to 10-fold decreases or increases in efficacy. However, these peptides all showed toxicity to normal fibroblast cells with levels (generally150 microM) that were comparable to those of their antimicrobial and anticancer activities. In contrast to previous claims which have been based on analysis of single amidation events, the results of this study clearly show that the C-terminal amidation of defence peptides has a variable effect on their antimicrobial and anticancer efficacy and no clear effect on their selectivity for these cell types.

Aurein 2.5 (GLFDIVKKVVGAFGSL-NH(2)) is an uncharacterised antimicrobial peptide. At an air/water interface, it exhibited strong surface activity (maximal surface pressure 25mNm(-1)) and molecular areas consistent with the adoption of alpha-helical structure orientated either perpendicular (1.72nm(2)molecule(-1)) or parallel (3.6nm(2)molecule(-1)) to the interface. Aurein 2.5 was strongly antibacterial, exhibiting a minimum inhibitory concentration (MIC) of 30microM against Bacillus subtilis and Escherichia coli. The peptide induced maximal surface pressure changes of 9mNm(-1) and 5mNm(-1), respectively, in monolayers mimicking membranes of these organisms whilst compression isotherm analysis of these monolayers showed DeltaG(Mix)0, indicating destabilisation by Aurein 2.5. These combined data suggested that toxicity of the peptide to these organisms may involve membrane invasion via the use of oblique orientated alpha-helical structure. The peptide induced strong, comparable maximal surface changes in monolayers of DOPG (7.5mNm(-1)) and DOPE monolayers (6mNm(-1)) suggesting that the membrane interactions of Aurein 2.5 were driven by amphiphilicity rather than electrostatic interaction. Based on these data, it was suggested that the differing ability of Aurein 2.5 to insert into membranes of B. subtilis and E. coli was probably related to membrane-based factors such as differences in lipid packing characteristics. The peptide was active against both sessile E. coli and Staphylococcus aureus with an MIC of 125microM. The broad-spectrum antibacterial activity and non-specific modes of membrane action used by Aurein 2.5 suggested use as an anti-biofilm agent such as in the decontamination of medical devices.

VP1, a putative alpha-helical antimicrobial peptide (alpha-AMP) inhibited growth of Bacillus subtilis and Escherichia coli at 500microM. The peptide induced stable surface pressure changes in monolayers formed from B. subtilis native lipid extract (circa 4.5mNm(-1)) but transient pressure changes in corresponding E. coli monolayers (circa 1.0mNm(-1)), which led to monolayer disintegration. Synthetic lipid monolayers mimetic of the extracts were used to generate compression isotherms. Thermodynamic analysis of B. subtilis isotherms indicated membrane stabilisation by VP1 (DeltaG(Mix)0), via a mechanism dependent upon the phosphatidylglycerol to cardiolipin ratio. Corresponding analysis of E. coli isotherms indicated membrane destabilisation by the peptide (DeltaG(Mix)0). Destabilisation correlated with PE levels present and appeared to involve a mechanism resembling those used by tilted peptides. These data emphasise that structure/function analysis of alpha-AMPs must consider not only their structural characteristics but also the lipid make-up of the target microbial membrane.

It is becoming increasingly clear that plants, ranging from across the plant kingdom produce anionic host defence peptides (AHDPs) with potent activity against a wide variety of human cancers cells. In general, this activity involves membrane partitioning by AHDPs, which leads to membranolysis and / or internalization to attack intracellular targets such as DNA. Several models have been proposed to describe these events including: the toroidal pore and Shai-Matsuzaki-Huang mechanisms but, in general, the mechanisms underpinning the membrane interactions and anticancer activity of these peptides are poorly understood. Plant AHDPs with anticancer activity can be conveniently discussed with reference to two groups: cyclotides, which possess cyclic molecules stabilized by cysteine knot motifs, and other ADHPs that adopt extended and α-helical conformations. Here, we review research into the anticancer action of these two groups of peptides along with current understanding of the mechanisms underpinning this action.

When adorned with n-octyl chains azobenzene is able to disrupt a variety of calcein-loaded phospholipid liposomes. The levels of lysis observed are dependent both on the lipid headgroup and the conformation of the azobenzene compound. In all cases studied, it has been shown that the cis-conformer is more membrane-interactive than the trans-conformer, suggesting that this class of molecule could be optimised for photo-dynamic therapy applications against infectious pathogens.

Maximin H5 is an anionic antimicrobial peptide from amphibians, which carries a C-terminal amide moiety, and was found to be moderately haemolytic (20%). The α-helicity of the peptide was 42% in the presence of lipid mimics of erythrocyte membranes and was found able to penetrate (10.8 mN m(-1)) and lyse these model membranes (64 %). In contrast, the deaminated peptide exhibited lower levels of haemolysis (12%) and α-helicity (16%) along with a reduced ability to penetrate (7.8 m Nm(-1)) and lyse (55%) lipid mimics of erythrocyte membranes. Taken with molecular dynamic simulations and theoretical analysis, these data suggest that native maximin H5 primarily exerts its haemolytic action via the formation of an oblique orientated α-helical structure and tilted membrane insertion. However, the C-terminal deamination of maximin H5 induces a loss of tilted α-helical structure, which abolishes the ability of the peptide's N-terminal and C-terminal regions to H-bond and leads to a loss in haemolytic ability. Taken in combination, these observations strongly suggest that the C-terminal amide moiety carried by maximin H5 is required to stabilise the adoption of membrane interactive tilted structure by the peptide. Consistent with previous reports, these data show that the efficacy of interaction and specificity of maximin H5 for membranes can be attenuated by sequence modification and may assist in the development of variants of the peptide with the potential to serve as anti-infectives.

m-calpain is a calcium-dependent heterodimeric protease implicated in a number of pathological conditions. The activation of m-calpain appears to be modulated by membrane interaction, which has been predicted to involve oblique-orientated alpha-helix formation by a GTAMRILGGVI segment located in domain V of the protein's small subunit. Here, we have investigated this prediction. Fourier transform infrared conformational analysis showed that VP1, a peptide homolog of this segment, exhibited alpha-helicity of approximately 45% in the presence of dimyristoylphosphatidylcholine/dimyristoylphosphatidylserine (DMPS) vesicles. The level of helicity was unaffected over a 1- to 8-mM concentration range and did not alter when the anionic lipid composition of these vesicles was varied between 1% and 10% DMPS. Similar levels of alpha-helicity were observed in trifluoroethanol and the peptide appeared to adopt alpha-helical structure at an air/water interface with a molecular area of 164 A(2) at the monolayer collapse pressure. VP1 was found to penetrate dimyristoylphosphatidylcholine/DMPS monolayers, and at an initial surface pressure of 30 mN m(-1), the peptide induced surface pressure changes in these monolayers that correlated strongly with their anionic lipid content (maximal at 4 mN m(-1) in the presence of 10% DMPS). Neutron diffraction studies showed VP1 to be localized at the hydrophobic core of model palmitoyloleylphosphatidylcholine/palmitoyloleylphosphatidylserine (10:1 molar ratio) bilayer structures and, in combination, these results are consistent with the oblique membrane penetration predicted for the peptide. It would also appear that although not needed for structural stabilization anionic lipid was required for membrane penetration.

This study investigated the effects of streptozotoci n (STZ) -induced diabetes mellitus (DM) on protein and cation levels in ocular tissues (lenses, cornea, lacrimal glands and retina with sclera) of rats. Diabetic rats and their lacrimal glands weighed significantly less (p < 0.05) than age -matched controls. Diabetic animals also had significantly (p < 0.05) elevated blood glucose and significantly reduced (p < 0.05) plasma insulin compared to controls. Total protein concentrations in the cornea, lens, lacrimal gland and retina with sclera were markedly reduced compared to controls (circa 50%-90%). Diabetic cornea, lenses, lacrimal glands, and retina with sclera contained more Ca 2+ (p < 0.05) than age-matched controls (2-3 fold). Levels of Zn 2+ were also elevated (p < 0.05) in the cornea and retina with sclera of diabetic rats, as compared to control rats (2-3 fold), but were unaffected in lenses and lacrimal glands. In contrast, levels of Cu 2+ , Mg 2+ , Na + and K + were significantly reduced (p < 0.05) in all ocular tissues of diabetic rats when compared to control animals (circa 30%-70%). These results show that STZ-induced DM is generally associated with significant physiochemical changes in ocular tissues of rats with changes observed in body weight, blood glucose, and insulin levels and protein and cation concentrations compared to healthy agematched controls. Based on these data, it has been speculated that diabetes may induce changes in ocular tissues that include: higher protein turnover through increased protease activity and changes in Na + / K + channel function. It is suggested that these changes may be associated with diabetic retinopathy, diabetic cornea and sight impairment. (Int J Diabetes Metab 13:154-158, 2005)

The activity of m-calpain, a heterodimeric, Ca2+-dependent cysteine protease appears to be modulated by membrane interactions involving oblique-orientated α-helix formation by a segment, GTAMRILGGVI, in the protein's smaller subunit. Here, graphical and hydrophobic moment-based analyses predicted that this segment may form an α-helix with strong structural resemblance to the influenza virus peptide, HA2, a known oblique-orientated α-helix former. Fourier transform infrared spectroscopy showed that a peptide homologue of the GTAMRILGGVI segment, VP1, adopted low levels of α-helical structure (≈ 20%) in the presence of zwitterionic lipid and induced a minor decrease (3 °C) in the gel to liquid-crystalline phase transition temperature, TC, of the hydrocarbon chains of zwitterionic membranes, suggesting interaction with the lipid headgroup region. In contrast, VP1 adopted high levels of α-helical structure (65%) in the presence of anionic lipid, induced a large increase (10 °C) in the TC of anionic membranes, and showed high levels of anionic lipid monolayer penetration (ΔSP = 5.5 mN·m−1), suggesting deep levels of membrane penetration. VP1 showed strong haemolytic ability (LD50 = 1.45 mm), but in the presence of ionic agents, this ability, and that of VP1 to penetrate anionic lipid monolayers, was greatly reduced. In combination, our results suggest that m-calpain domain V may penetrate membranes via the adoption of an oblique-orientated α-helix and electrostatic interactions. We speculate that these interactions may involve snorkelling by an arginine residue located in the polar face of this α-helix.

Everyday we are exposed to a world of sounds that we do not hear. Many of the sounds come on the edge of our hearing range and some are even audible to a small part of the population. This presentation relates experimental measurements of sound exposures in the high-frequency regime that we are not intended to hear. There has been recent speculation about whether the regulations in the near-audible (or audible for a small population) regime are sufficient. Whether it is annoyance or some other mechanism that affects some part of the population is unknown, but this data present an idea of what exposure we receive at the edge of our hearing range.

A systematic analysis of the hypothesis of the antimicrobial peptides' (AMPs) cooperative action is performed by means of full atomistic molecular dynamics simulations accompanied by circular dichroism experiments. Several AMPs from the aurein family (2.5,2.6, 3.1), have a similar sequence in the first ten amino acids, are investigated in different environments including aqueous solution, trifluoroethanol (TFE), palmitoyloleoylphosphatidylethanolamine (POPE), and palmitoyloleoylphosphatidylglycerol (POPG) lipid bilayers. It is found that the cooperative effect is stronger in aqueous solution and weaker in TFE. Moreover, in the presence of membranes, the cooperative effect plays an important role in the peptide/lipid bilayer interaction. The action of AMPs is a competition of the hydrophobic interactions between the side chains of the peptides and the hydrophobic region of lipid molecules, as well as the intra peptide interaction. The aureins 2.5-COOH and 2.6-COOH form a hydrophobic aggregate to minimize the interaction between the hydrophobic group and the water. Once that the peptides reach the water/lipid interface the hydrophobic aggregate becomes smaller and the peptides start to penetrate into the membrane. In contrast, aurein 3.1-COOH forms only a transient aggregate which disintegrates once the peptides reached the membrane, and it shows no cooperativity in membrane penetration.

Sonodynamic antimicrobial chemotherapy (SACT) is a novel modality, which uses ultrasound to kill bacteria by the activation of molecules termed sonosensitisers (SS) to produce reactive oxygen species that are toxic to microorganism although microbial resistance to this modality has been reported. There are a growing number of SS being reported with the dual ability to be activated by both ultrasound and light, and we hypothesis that a novel antimicrobial strategy, potentially known as sonophotodynamic antimicrobial chemotherapy (SPACT), could be developed based on these agents. SPACT offers advantages over SACT and could constitute a new weapon in the fight against the growing global threat posed by microbial infections.

The protein transduction domain of the HIV-1 transactivator of transcription, Tat (Tat (48–60) ), has been shown to transport P10, a cytotoxic peptide mimic of the cyclin dependent kinase inhibitor p21WAF1/CIP1, into the nucleus of cancerous cells and induce apoptosis. Here, monolayer studies were used to investigate the membrane interactions of Tat (48–60) , P10 and the construct Tat (48–60) P10. It was found that Tat (48–60) showed no significant surface activity but that both P10 and Tat (48–60) P10, were highly surface active, inducing surface pressure changes of 9.7 and 8.9 mN m −1 , respectively, with DMPS monolayers. The comparison of Tat (48–60) P10 and P10 surface interactions would be consistent with a hypothesis that the cargo attachment influences the capacity of the Tat-protein transduction domain to mediate transport across membranes either directly or via localisation of the construct at the membrane interface.

AP1 (GEQGALAQFGEWL) was shown by theoretical analysis to be an anionic oblique-orientated alpha-helix former. The peptide exhibited a monolayer surface area of 1.42 nm(2), implying possession of alpha-helical structure at an air/water interface, and Fourier transform infrared spectroscopy (FTIR) showed the peptide to be alpha-helical (100%) in the presence of vesicle mimics of Escherichia coli membranes. FTIR lipid-phase transition analysis showed the peptide to induce large decreases in the fluidity of these E. coli membrane mimics, and Langmuir-Blodgett trough analysis found the peptide to induce large surface pressure changes in monolayer mimics of E. coli membranes (4.6 mN.m(-1)). Analysis of compression isotherms based on mixing enthalpy (DeltaH) and the Gibbs free energy of mixing (DeltaG(Mix)) predicted that these monolayers were thermodynamically stable (DeltaH and DeltaG(Mix) each negative) but were destabilized by the presence of the peptide (DeltaH and DeltaG(Mix) each positive). The peptide was found to have a minimum lethal concentration of 3 mm against E. coli and was seen to cause lysis of erythrocytes at 5 mm. In combination, these data clearly show that AP1 functions as an anionic alpha-helical antimicrobial peptide and suggest that both its tilted peptide characteristics and the composition of its target membrane are important determinants of its efficacy of action.

Pneumonia, caused by Streptococcus pneumoniae, mainly affects the immunocompromised, the very young and the old, and remains one of the leading causes of death. A steady rise in disease numbers from non-vaccine serotypes necessitates a new vaccine formulation that ideally has better antigen stability and integrity, does not require cold-chain and can be delivered non-invasively. In this study, a dry powder vaccine containing an important antigen of S. pneumoniae, pneumococcal surface protein A (PspA) that has shown cross-reactivity amongst serotypes to be delivered via the pulmonary route has been formulated. The formulation contains the antigen PspA adsorbed onto the surface of polymeric nanoparticles encapsulated in l-leucine microparticles that can be loaded into capsules and delivered via an inhaler. We have successfully synthesized particles of ∼150 nm and achieved ∼20 μg of PspA adsorption per mg of NPs. In addition, the spray-dried powders displayed a FPF of 74.31 ± 1.32% and MMAD of 1.70 ± 0.03 μm suggesting a broncho-alveolar lung deposition facilitating the uptake of the nanoparticles by dendritic cells. Also, the PspA released from the dry powders maintained antigen stability (SDS-PAGE), integrity (Circular dichroism) and activity (lactoferrin binding assay). Moreover, the released antigen also maintained its antigenicity as determined by ELISA.

The synthesis and biological evaluation of a novel pyridinium salt is reported. Initial membrane interaction with isolated phospholipid monolayers was obtained with the pyridinium salt, and two neutral analogues for comparison, and the anticancer effects of the best compound established using a cytotoxicity screening assay against glioma cells using both an established cell line and three short-term cell cultures – one of which has been largely resistant to all chemotherapeutic drugs tested to date. The results indicate that the pyridinium salt exhibits potent anticancer activity (EC50s = 9.8-312.5 μM) on all cell types, including the resistant one, for a continuous treatment of 72 hours. Microscopic examination of the treated cells using a trypan blue exclusion assay showed membrane lysis had occurred. Therefore, this letter highlights the potential for a new class of pyridinium salt to be developed as a much needed alternative treatment for glioma chemotherapy.