Hiren Patel

All-or-none membrane permeabilization by fengycin-type lipopeptides from Bacillus subtilis QST713

The fungicidal activity of Bacillus subtilis QST713 has been utilized for the highly effective and environmentally safe protection of crops against a variety of pathogens. It is based mainly on the production of cyclic lipopeptides of the fengycin (FEs), surfactin, and iturin families. The mixed population of native FEs forms micelles which solubilize individual FEs such as agrastatin 1 (AS1) that are otherwise rather insoluble on their own. Fluorescence lifetime-based calcein efflux measurements and cryo transmission electron microscopy show that these FEs show a unique scenario of membrane permeabilization. Poor miscibility of FEs with lipid probably promotes the formation of pores in 10% of the vesicles at only≈1μM free FE and in 15% of the vesicles at 10 μM. We explain why this limited, all-or-none leakage could nevertheless account for the killing of virtually all fungi whereas the same extent of graded vesicle leakage may be biologically irrelevant. Then, crystallization of AS1 and micellization of plipastatins cause a cut-off in leakage at 15% that might regulate the biological activity of FEs, protecting Bacillus and plant membranes. The fact that FE micelles solubilize only about 10 mol-% fluid lipid resembles the behavior of detergent resistance.

In Search of Aggregation Pathways of IAPP and Other Amyloidogenic Proteins: Finding Answers through NMR Spectroscopy

The deposition of aggregates of human islet amyloid peptide (hIAPP) has been correlated with the death of insulin-producing beta (β) cells in type II diabetes mellitus. The actual molecular mechanism of cell death remains unknown; however, it has been postulated that the process of aggregation and amyloid fibril growth from monomeric hIAPP is closely involved. Intermediate IAPP aggregates are highly toxic to islet cells, but lack of structural knowledge of these oligomers and complications in applying biophysical techniques to their study have been the main obstacles in designing structure-based therapeutics. Furthermore, the involvement of metal ions (Cu(2+) and Zn(2+)) associated with hIAPP has demonstrated an effect on the aggregation pathway. In the absence of well-defined targets, research attempting to attenuate amyloid-linked toxicity has been substantially slowed. Therefore, obtaining high-resolution structural insights on these intermediates through NMR techniques can provide information on preventing IAPP aggregation. In this Perspective, a review of avenues to obtain fundamental new insights into the aggregation pathway of IAPP and other amyloidogenic proteins through NMR and other techniques is presented.

Characterizing vesicle leakage by fluorescence lifetime measurements

The membrane leakage assay based on vesicles loaded with self-quenching dyes has been widely used for quantifying the activity of antibiotic peptides and other compounds that induce membrane pores or leaks. Here we show that this assay can be substantially improved if it is based on time-resolved decay curves rather than steady-state intensities only. A bi- or triexponential fit of calcein fluorescence decays allows for a parallel quantification of the free and (one or two) entrapped dye fractions and their effective local concentrations (governing the lifetime). The advantages of this technique are that it (i) allows the distinguishing of all-or-none from graded leakage for each sample, (ii) reveals the heterogeneity of graded leakage, (iii) truly quantifies the released dye rather than providing an empiric dequenching value, and (iv) is independent of many errors that may affect the intensity. We introduce the method by studying graded leakage caused by the detergent C12EO8.

Utilizing zeta potential measurements to study the effective charge, membrane partitioning, and membrane permeation of the lipopeptide surfactin.

The effective charge of membrane-active molecules such as the fungicidal lipopeptide surfactin (SF) is a crucial property governing solubility, membrane partitioning, and membrane permeability. We present zeta potential measurements of liposomes to measure the effective charge as well as membrane partitioning of SF by utilizing what we call an equi-activity analysis of several series of samples with different lipid concentrations. We observe an effective charge of -1.0 for SF at pH8.5 and insignificantly lower at pH7.4, illustrating that the effective charge may deviate strongly from the nominal value (-2 for 1 Asp, 1 Glu). The apparent partition coefficient decreases from roughly 100 to 20/mM with increasing membrane content of SF in agreement with the literature. Finally, by comparing zeta potentials measured soon after the addition of peptide to liposomes with those measured after a heat treatment to induce transmembrane equilibration of SF, we quantified the asymmetry of partitioning between the outer and inner leaflets. At very low concentration, SF binds exclusively to the outer leaflet. The onset of partial translocation to the inner leaflet occurs at about 5mol-% SF in the membrane. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Additive and Synergistic Membrane Permeabilization by Antimicrobial (Lipo)Peptides and Detergents

Certain antibiotic peptides are thought to permeabilize membranes of pathogens by effects that are also observed for simple detergents, such as membrane thinning and disordering, asymmetric bilayer expansion, toroidal pore formation, and micellization. Here we test the hypothesis that such peptides act additively with detergents when applied in parallel. Additivity is defined analogously to a fractional inhibitory concentration index of unity, and the extent and mechanism of leakage is measured by the fluorescence lifetime-based vesicle leakage assay using calcein-loaded vesicles. Good additivity was found for the concerted action of magainin 2, the fungicidal lipopeptide class of surfactins from Bacillus subtilis QST713, and the detergent octyl glucoside, respectively, with the detergent C12EO8. Synergistic or superadditive action was observed for fengycins from B. subtilis, as well as the detergent CHAPS, when combined with C12EO8. The results illustrate two mechanisms of synergistic action: First, maximal leakage requires an optimum degree of heterogeneity in the system that may be achieved by mixing a graded with an all-or-none permeabilizer. (The optimal perturbation should be focused to certain defect structures, yet not to the extent that some vesicles are not affected at all.) Second, a cosurfactant may enhance the bioavailability of a poorly soluble peptide. The results are important for understanding the concerted action of membrane-permeabilizing compounds in biology as well as for optimizing formulations of such antimicrobials for medical applications or crop protection.

Adherence to Diabetes Medication in Individuals with Schizophrenia: A Systematic Review of Rates and Determinants of Adherence.

INTRODUCTION Despite the importance of medication adherence for the effective treatment of type II diabetes mellitus (T2DM), little research has examined adherence with diabetes medication treatment in schizophrenia. The purpose of this systematic review was to: 1) evaluate rates of adherence and determinants of adherence with medication for T2DM in individuals with schizophrenia; and, where possible, 2) examine the relationship between medication adherence and glycemic control. METHODS Studies were included if they presented information on dosing regimens and adherence or compliance rates for T2DM and included samples where at least 50% of the participants were individuals with schizophrenia. RESULTS Six studies were included in this review that predominantly examined men over the age of 50 years. Studies confirmed that many individuals with schizophrenia were not adhering to their diabetes medication as adherence rates ranged from 51-85%. Two studies that compared medication adherence in individuals with and without schizophrenia found those with the mental illness had higher rates of adherence. One study reported that blood glucose control levels were not statistically different between those who did and did not adhere to their medication, indicating more research is necessary in this area. Factors that improved adherence included disease and medical service and medication-related factors. CONCLUSIONS Interventions to increase diabetes medication adherence in schizophrenia need to address disease and medical service and medication-related factors. Further research needs to examine diabetes medication adherence in women, younger individuals, and those recently diagnosed with diabetes as these individuals have been underrepresented in the literature.

Effects of Glycerol and Urea on Micellization, Membrane Partitioning and Solubilization by a Non-Ionic Surfactant

We have studied the effect of two cosolvents, urea and glycerol, on the association and interactions of a surfactant, octaethyleneglycol dodecyl ether (C12EO8) and a phospholipid (POPC). We have measured the CMC, the partition coefficient, the effective mole fractions Xesat and Xesol at the onset and completion of the membrane-to-micelle transition (membrane solubilization), and the enthalpies of transfer of surfactant by ITC. Changes in membrane order and dynamics were characterized by time-resolved fluorescence anisotropy measurements of DPH, and micelle sizes and clouding by light scattering. The cosolvents have complex effects that are not governed by the well-known ‘salting in’ or ‘salting out’ effects on the solubility alone. Instead, urea and glycerol alter also the intrinsic curvature (‘effective molecular shape’) of the detergent and the order and packing of the membrane. These curvature effects have an unusual enthalpy/entropy balance and are not additive for lipid and detergent. The results shed light on the phenomena governing lipid-detergent systems in general and have a number of implications for the use of cosolvents in membrane protein studies.

Evaluating correlations between physical activity, psychological mediators of physical activity, and negative symptoms in individuals living with psychosis and diabetes.

Objective: This study examines the relationship among physical activity, psychological mediators of physical activity, and negative symptoms in people living with psychosis and prediabetes or Type II diabetes mellitus to identify which variables should be targeted in future physical activity interventions. Method: A total of 63 individuals were recruited and filled out questionnaires with the assistance of trained research staff. Results: Spearman’s correlations showed a positive significant association between physical activity and self-efficacy. Negative significant associations were found between physical activity and negative symptoms of psychosis as well as perceived barriers of physical activity. There was no significant relationship between physical activity and perceived benefits of physical activity. Conclusion and implications for practice: Future physical activity interventions need to focus on enhancing the confidence individuals have being physically active and how barriers to activity can be recognized and managed. Interventions should be conducted alongside treatment of negative symptoms of psychosis.

Assessing Online Dietary Information for People With Schizophrenia

To the Editor: Diets high in calories and saturated fat and poor in fiber and fruit contribute to the development of obesity and chronic health conditions among people with schizophrenia (1). Providing information on healthy diets may help these individuals develop more healthful behaviors that may lead to weight maintenance or loss (2). Such information would outline the benefits of and barriers to behavior change and boost an individual’s confidence to eat healthfully.

Interactions of Cymal-6 and Lipid Vesicles

The solubilization of biological or liposomal membranes, induced by detergents and detergent-like biomolecules, is important to many technical applications and biological phenomena. In fact, the interactions of classical detergents with lipid bilayers are described and understood quite well. Recently, new types of detergents with cyclohexyl groups or branches in their hydrophobic tails have been synthesized and proposed to be superior for membrane protein studies. Cymal-6 has, for example, been used for isolating membrane proteins such as CCR5 and HIV-1 corepressors. Here we provide a rather comprehensive description of the interactions of Cymal-6 with fluid membranes of POPC. This includes the temperature-dependent phase behavior (i.e., the onset and completion of solubilization), membrane partitioning, disordering, and permeabilization as seen using ITC, time-resolved fluorescence anisotropy of DPH, dynamic light scattering, and the lifetime-based vesicle leakage assay.