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Biophysical characterization of antimicrobial peptides helps to understand the mechanistic aspects of their action. The physical behavior of the KR-12 antimicrobial peptide (e.g. orientation and changes in secondary structure), was analyzed after interactions with a Staphylococcus aureus membrane model and solid surfaces. We performed antimicrobial tests using Gram-positive S. aureus (ATCC 25923) bacteria. Moreover, Langmuir-Blodgett experiments showed that the synthetic peptide can disturb the lipidic membrane at a concentration lower than the Minimum Inhibitory Concentration, thus confirming that KR-12/lipid interactions are involved. Partially- and fully-deactivated KR-12 hybrid samples were obtained by physisorption and covalent immobilization in chitosan/silica and glyoxal-rich solid supports. The correlation of Langmuir-Blodgett data with the α-helix formation, followed by FTIR-ATR in a frozen-like state, and the antimicrobial activity showed the importance of these interactions and conformation changes on the first step action mode of this peptide. This is the first time that material science (immobilization in solid surfaces assisted by FTIR-ATR analysis in frozen-like state) and physical (Langmuir-Blodgett/Schaefer) approaches are combined for exploring mechanistic aspects of the primary action mode of the KR-12 antimicrobial peptide against S. aureus.

RESUMEN: Históricamente el oro ha sido usado debido a las especiales propiedades que posee, entre las que se pueden resaltar su resistencia a medios agresivos (por ser altamente inerte), su buena conducción térmica y eléctrica, y porque además presenta un color llamativo. Sus principales aplicaciones se han dado en áreas como la economía (en el uso de monedas), en joyería, en electrónica y en odontología. Con el fin de mejorar las propiedades que presenta el oro, se fabrican aleaciones de éste con otros metales. Dentro de las nuevas aleaciones que se están desarrollando, se encuentra el sistema de aleaciones Au-Fe. En esta investigación se pretende profundizar en el estudio de la microestructura de las aleaciones Au-Fe y Au-Fe-Co, para lo cual, se fabricaron aleaciones base oro aleadas con 25% (p/p) Fe y se han comparado a nivel de estructura y conformabilidad con la aleación Au-24.5Fe-0.5Co (% p/p), con el fin de determinar la influencia de este último aleante (el cobalto). Las aleaciones se obtuvieron en horno de inducción con atmósfera controlada de argón, utilizando crisoles conductores revestidos con silicato de zirconio. Las muestras se caracterizaron en tres diferentes procesos: as-cast con tratamiento térmico a temperaturas de 900°C a una y tres horas respectivamente y as-cast centrifugada. Los resultados de conformabilidad al finalizar el proceso son 62% de deformación (en laminador) para la aleación Au-24.5Fe-0.5Co contra el 23% para el sistema Au-25Fe para las muestras obtenidas as-cast y con tratamiento térmico a 900°C por 1 hora. Las muestras as-cast centrifugadas presentaron una deformación de 75% con fractura dúctil en Au-25Fe y de 82% sin fractura en Au-24.5Fe-0.5Co. El anterior resultado es satisfactorio para el sistema con adición de cobalto, lo cual es altamente benéfico para diversas aplicaciones, sobretodo la joyería. ABSTRACT: Since ancient times, gold has been used due to its especial properties. Among those properties are the resistance to corrosion (for being noble), its high electrical and thermal conductivity, and also its conspicuous coloration. Gold has been applied in a diversity of subjects such as economy, jewelry, electronic circuits and odontology. The fascinating properties of gold are likely to improve with the preparation of alloys; among those, the Au-Fe alloys constitute some of the most studied nowadays. In this research, we present our results concerning the microstructure of the Au-Fe and Au-Fe-Co alloys. We fabricated gold based alloys with 25% (weight) Fe and then compared their structure and formability to Au-24.5Fe-0.5Co (% weight) to determine the role of cobalt in the alloy. An induction furnace with controlled atmosphere (argon) and conductor crucibles coated with zirconium silicate was used. The samples were characterized by three processes: as-cast, with heat treatment (HT) at T=900°C (1h and 3h) and as-cast centrifuged. The formability results for the as-cast samples revealed a 23% deformation (rolling mill) for the Au-25Fe versus a 62% for the Au-24.5Fe-0.5Co alloy (HT-1h; T=900°C), whilst for the as-cast centrifuged sample revealed a 75% deformation with ductile fracture for the Au-25Fe versus an 82% without fracture for the Au-24.5Fe-0.5Co alloy. This result is particularly promissory for the applications in jewelry, showing that the addition of cobalt increases the formability of Au-Fe alloys.

Background Mag-Fluo-4 is increasingly employed for studying Ca2+ signaling in skeletal muscle; however, the lack of information on the Ca2+-Mag-Fluo-4 reaction limits its wider usage. Methods Fluorescence and isothermal titration calorimetry (ITC) experiments were performed to determine the binding stoichiometry (n) and thermodynamics (enthalpy (ΔH) and entropy (ΔS) changes), as well as the in vitro and in situ Kd of the Ca2+-Mag-Fluo-4 reaction. Rate constants (kon, koff), fluorescence maximum (Fmax), minimum (Fmin), and the dye compartmentalization were also estimated. Experiments in cells used enzymatically dissociated flexor digitorum brevis fibres of C57BL6, adult mice, loaded at room temperature for 8 min, with 6 μM Mag-Fluo-4, AM, and permeabilized with saponin or ionomycin. All measurements were done at 20 °C. Results The in vitro fluorescence assays showed a binding stoichiometry of 0.5 for the Ca2+/Mag-Fluo-4 (n = 5) reaction. ITC results (n = 3) provided ΔH and ΔS values of 2.3 (0.7) kJ/mol and 97.8 (5.9) J/mol.K, respectively. The in situ Kd was 1.652 × 105μM2(n = 58 fibres, R2 = 0.99). With an Fmax of 150.9 (8.8) A.U. (n = 8), Fmin of 0.14 (0.1) A.U. (n = 10), and ΔF of Ca2+ transients of 8.4 (2.5) A.U. (n = 10), the sarcoplasmic [Ca2+]peak reached 22.5 (7.8) μM. Compartmentalized dye amounted to only 1.1 (0.7)% (n = 10). CONCLUSIONS: Two Mag-Fluo-4 molecules coalesce around one Ca2+ ion, in an entropy-driven, very low in situ affinity reaction, making it suitable to reliably track the kinetics of rapid muscle Ca2+ transients. General significance Our results may be relevant to the quantitative study of Ca2+ kinetics in many other cell types.

The ABRI peptide is involved in the neurodegenerative disease Familial British Dementia, which has its origin in the misfolding of the peptide. Characterizing the most probable conformations of the monomer in solution can provide insights into how misfolding could occur in the steps prior to aggregation. Specifically, we analyzed the structural effects caused by the formation of a single disulfide bond, which has been reported to be important in amyloid assembly. We used all-atom molecular dynamics simulations with an enhanced sampling technique to obtain the lowest free energy conformations for two cases: the peptide with and without the disulfide bond between residues 5Cys and 22Cys. Bulk measurements on the conformations agree with experiments by elucidating ABRI as a disordered peptide. We find remarkable differences at the microscopic level between the most probable structures; with the disulfide bond the peptide is compact and α-helical, without the bond it is partially extended with slight β-bridges.

GM1 ganglioside is known to be involved in the amyloid-associated diseases and it is a crucial factor for the assembly of amyloid proteins on lipid-rafts, which are lipid structures located on the synaptic plasma membranes. Due to its slow aggregation rate, we employed salmon calcitonin (sCT) as a suitable probe representative of amyloid proteins, to study the interaction between this class of proteins and a membrane model. Here, we prepared a neuronal membrane model by depositing onto mica two Langmuir-Blodgett films in liquid-condensed phase: the outer monolayer was characterized by high content of GM1 (50%) and minority parts of cholesterol and POPC (25–25%), while the inner one by plain POPC. To deeply investigate the interaction of sCT with this model and the role-played by GM1, we prepared the outer leaflet adding sCT at a concentration such that the number of proteins equals that of GM1. Atomic Force Microscopy revealed the occurrence of two distinct kinds of flat surfaces, with globular aggregates localized exclusively on top of the highest one. To unravel the nature of the interaction, we studied by ζ-potential technique liposomes composed as the outer leaflet of the model. Results demonstrated that an electrostatic interaction sCT-GM1 occurred. Finally, to investigate the interaction thermodynamics between sCT and the outer leaflet, Langmuir films as the outer monolayer and containing increasing content of sCT were studied by compression isotherms and Brewster Angle Microscopy experiments. Based on the all body of results we propose an interaction model where GM1 plays a pivotal role., Highlights • Membrane models mimicking the occurrence of lipid-rafts and sCT, were investigated. • Atomic Force Microscopy shows sCT globular aggregates onto very flat surfaces. • ξ-potential data indicate that electrostatic interaction occurs between sCT and GM1. • Thermodynamic data of the outer leaflet reveal that sCT aggregate outside the film. • GM1, one component of the lipid-rafts, result to trigger the interaction.

RESUMEN: Se aplica la teoría del color propuesta por Heavens (1991) para el cálculo de los espesores de las películas delgadas de magnetita, Fe3 O4 , los cuales fueron obtenidos por tratamiento térmico a 250°C de una aleación de oro (Au-24.5Fe-0.5Co). Los espectros de reflectancia obtenidos de las muestras pulidas con diferentes grados de oxidación (y de color) fueron usados para calcular los índices de refracción (real) y de extinción (imaginario) del sustrato metálico (n2 , k2 ) y de la capa de magnetita (n1 =2,42, k1 ). La forma a+bλ+cλ2 se tomó del ajuste de parámetros entre las curvas experimentales y teóricas. El ajuste de los datos resultan en desviaciones entre 2 y 10%, para los espesores en el rango de 0 nm (solo metal original) y 65 nm (color azul oscuro). Por medio de un modelo matemático siguiendo la teoría de Heavens, el espesor de cada capa ha sido predicho con alta precisión, utilizando las mediciones de reflectancia espectral. En consecuencia, proponemos que mediante el uso de esta metodología se pueden obtener fácilmente los valores de los coeficientes de extinción de las especies de óxido y, además, se pueden predecir los espesores de la capa de óxido. Los valores de espesores de la magnetita encontrados en la presente investigación están dentro del intervalo reportado en la literatura para la interferencia de primer orden en los aceros, desde el amarillo claro (~46 nm) hasta el azul (~72 nm). ABSTRACT: The colour theory proposed by Heavens in 1991 has been applied to calculate the thickness of magnetite thin layers, Fe3 O4 , which were obtained by heat treatment at 250 °C in a gold alloy (Au-24.5Fe-0.5Co). The reflectance spectra obtained from the polished samples with different oxidation (and colour) degrees were used to calculate the real part of the refraction index and the (imaginary) extinction coefficients of both the metallic substrate (n2 , k2 ) and the magnetite layer (n1 =2.42, k1 ). The a+bλ+cλ2 form was taken adjusting parameters between experimental and theoretical curves. The fitting of the data resulted in deviations between 2 and 10% for thicknesses in the range of 0 nm (only substrate) and 65 nm (dark blue colour). By means of a mathematic model and following the Heavens’ theory, the thickness of each layer has been predicted with high precision, using the spectral reflectance. Consequently, we propose that by using this methodology, the values of the extinction coefficients of the oxide species can be easily obtained, and the thicknesses of the oxide layers can be predicted. The magnetite thickness values found in this study fall into the interval reported in the literature for first-order interference in steels, from light-yellow (~46 nm) to blue (~72 nm).

Since there are uncertainties about the concentration of free cytosolic Ca2+ reached in a muscle fiber during a transient elicited by electric stimulation, and about the KdCa2+ of the fast dye Magfluo-4, we performed two groups of experiments aimed at calibrating the Ca2+ transients obtained with this dye. For the fiber experiments, we used murine single skeletal muscle fibers obtained from enzymatically dissociated flexor digitorum brevis, loaded with 8-10 μM of the dye in its acetoxymethyl ester form, during 40 min at 21-22°C, to determine Fmax, Fmin and dye compartmentalization. The cells were permeabilized with saponine and exposed to 50 mM of free calcium to intracellularly saturate the dye. Compartmentalization was assessed using digitonin and triton-X100. For our conditions, Fmax and Fmin were 160±14 (n=9, mean±SEM) and 3.4±0.7 (n=6). Dye compartmentalization was found to be less than 10% (n=1). For the in vitro experiments, KdCa2+ (20°C) values of 21.6 μM, 27.8 μM and 29.1 μM were obtained in presence of 0, 1 and 2 mM free Mg2+, respectively. To calculate Ca2+ concentration from the fluorescence signals, we used the methodologies proposed by Grynkiewicz et al. (1985) and by Caputo et al. (1994). Preliminary results, taking into account our data or some values from the literature (Baylor et al. 2009), show single Ca2+ transient's peak between 2 and 7 μM (n=4). We are now calculating the kinetics of Ca2+ transients and the quantitative changes induced by modulating Ca2+ release and sequestration mechanisms in different fiber types. In conclusion, we have calculated the peak Ca2+ concentration after single stimulation in fibers loaded with Magfluo-4. Our preliminary results are comparable to those obtained with other fast Ca2+ dyes, such as Magfura-5, but lower than those reported with Magfura-2.