Mussel adhesive protein inspired coatings: a versatile method to fabricate silica films on various surfaces

A simple and versatile biomimetic strategy for the fabrication of silica films on a variety of substrates including gold, polystyrene and silicon wafers was developed using nanogram amounts per cm 2 of silicatein. The strategy exploits the adhesive property of 3,4-dihydroxyphenylalanine (DOPA) and a decapeptide (Ala-Lys-Pro-Ser-Tyr-DHP-Hyp-Thr-DOPA-Lys), important components of mussel adhesive proteins, to modify the surface of substrates. DOPA molecules polymerize to poly(DOPA) and the decapeptide forms thin films on gold substrates at pH 8.5, rendering the substrate compatible for silicatein immobilization. Nearly 50 ng cm 2 of silicatein is immobilized on poly(DOPA) and decapeptide coated surfaces where these polymer films act as "cushion" to protect the active structure and maintain the activity of the largely chemically adsorbed silicatein at ca. 95% of that experienced in solution. Uniform silica films of thickness 130-140 nm and roughness 12-14.5 nm were fabricated on coated gold surfaces. Evidence to show that this method is also applicable for the fabrication of uniform silica films on polystyrene and silicon substrates over multiple length scales in an economical way is also presented

Direct evidence of ZnO morphology modification via the selective adsorption of ZnO-binding peptides

Biomolecule-mediated ZnO synthesis has great potential for the tailoring of ZnO morphology for specific application in biosensors, window materials for display and solar cells, dye-sensitized solar cells (DSSCs), biomedical materials, and photocatalysts due to its specificity and multi-functionality. In this contribution, the effect of a ZnO-binding peptide (ZnO-BP, G-12: GLHVMHKVAPPR) and its GGGC-tagged derivative (GT-16: GLHVMHKVAPPRGGGC) on the growth of ZnO crystals expressing morphologies dependent on the relative growth rates of (0001) and (10 (1) over bar0) planes of ZnO have been studied. The amount of peptide adsorbed was determined by a depletion method using oriented ZnO films grown by Atomic Layer Deposition (ALD), while the adsorption behavior of G-12 and GT-16 was investigated using XPS and a computational approach. Direct evidence was obtained to show that (i) both the ZnO-BP identified by phage display and its GGGC derivative (GT-16) are able to bind to ZnO and modify crystal growth in a molecule and concentration dependent fashion, (ii) plane selectivity for interaction with the (0001) versus the (10 (1) over bar0) crystal planes is greater for GT-16 than G-12; and (iii) specific peptide residues interact with the crystal surface albeit in the presence of charge compensating anions. To our knowledge, this is the first study to provide unambiguous and direct quantitative experimental evidence of the modification of ZnO morphology via (selective and nonselective) adsorption-growth inhibition mechanisms mediated by a ZnO-BP identified from phage display libraries

Thermodynamic study of interactions between ZnO and ZnO binding peptides using isothermal titration calorimetry

Whilst material specific peptide binding sequences have been identified using a combination of combinato-rial methods and computational modelling tools, a deep molecular level understanding of the fundamental principles through which these interactions occur and in some instances modify the morphology of inorganic materials is far from being fully realized. Understanding the thermodynamic changes that occur during peptide-inorganic interactions and correlating these to structural modifications of the inorganic materials could be the key to achieving and mastering con-trol over material formation processes. This study is a detailed investigation applying isothermal titration calorimetry (ITC) to directly probe thermodynamic changes that occur during interaction of ZnO binding peptides (ZnO-BPs) and ZnO. The ZnO-BPs used are reported sequences G-12 (GLHVMHKVAPPR), GT-16 (GLHVMHKVAPPR-GGGC) and alanine mutants of G-12 (G-12A6, G-12A11 and G-12A12) whose interaction with ZnO during solution synthesis studies have been extensively investigated. The interactions of the ZnO-BPs with ZnO yielded biphasic isotherms comprising both an endo-thermic and an exothermic event. Qualitative differences were observed in the isothermal profiles of the different pep-tides and ZnO particles studied. Measured ΔG values were between -6 and -8.5 kcal/mol and high adsorption affinity val-ues indicated the occurrence of favourable ZnO-BP-ZnO interactions. ITC has great potential in its use to understand peptide-inorganic interactions and with continued development, the knowledge gained may be instrumental for simplifi-cation of selection processes of organic molecules for the advancement of material synthesis and design

Moulage in high-fidelity simulation-A chest wall burn escharotomy model for visual realism and as an educational tool

Introduction: There is a paucity of literature pertaining to the role and techniques of moulage for creating high-fidelity medical simulations. As part of an Intensive Care Crisis Event Management Course, simulation of an extensive torso burn was desired. The aim of the moulage was to enhance the realism of the scenario but additionally to enable a chest wall escharotomy to be performed

The S.T.A.B. Trial-Standardized testing of artificial blood: A comparative study of various products that may be used as artificial blood for high fidelity simulation training in the critical care setting

Aim: In the current climate of medical education, there is an ever-increasing demand for and emphasis on simulation as both a teaching and training tool. The objective of our study was to compare the realism and practicality of a number of artificial blood products that could be used for high-fidelity simulation. Method: A literature and internet search was performed and 15 artificial blood products were identified from a variety of sources. One product was excluded due to its potential toxicity risks. Five observers, blinded to the products, performed two assessments on each product using an evaluation tool with 14 predefined criteria including color, consistency, clotting, and staining potential to manikin skin and clothing. Each criterion was rated using a five-point Likert scale. The products were left for 24 hours, both refrigerated and at room temperature, and then reassessed. Statistical analysis was performed to identify the most suitable products, and both inter- and intra-rater variability were examined. Results: Three products scored consistently well with all five assessors, with one product in particular scoring well in almost every criterion. This highest-rated product had a mean rating of 3.6 of 5.0 (95% posterior Interval 3.4-3.7). Inter-rater variability was minor with average ratings varying from 3.0 to 3.4 between the highest and lowest scorer. Intrarater variability was negligible with good agreement between first and second rating as per weighted kappa scores (K = 0.67). Conclusion: The most realistic and practical form of artificial blood identified was a commercial product called KD151 Flowing Blood Syrup. It was found to be not only realistic in appearance but practical in terms of storage and stain removal

Dynamic interplay between human myoblasts and 3D fibrin-based matrix

International audienceAnchorage of muscle cells to the extracellular matrix is crucial for a range of fundamental biological processes including migration, survival and differentiation. Three-dimensional (3D) culture has been proposed to provide a more physiological in vitro model of muscle growth and differentiation than routine 2D cultures. However, muscle cell adhesion and cell-matrix interplay of engineered muscle tissue remain to be determined. We have characterized cell-matrix interactions in 3D muscle culture and analyzed their consequences on cell differentiation. Human myoblasts were embedded in a fibrin matrix cast between two posts, cultured until confluence, and then induced to differentiate. Myoblasts in 3D aligned along the longitudinal axis of the gel. They displayed actin stress fibers evenly distributed around the nucleus and a cortical mesh of thin actin filaments. Adhesion sites in 3D were smaller in size than in rigid 2D culture but expression of adhesion site proteins, including alpha5 integrin and vinculin, was higher in 3D compared with 2D (p,0.05). Myoblasts and myotubes in 3D exhibited thicker and ellipsoid nuclei instead of the thin disk-like shape of the nuclei in 2D (p,0.001). Differentiation kinetics were faster in 3D as demonstrated by higher mRNA concentrations of alpha-actinin and myosin. More important, the elastic modulus of engineered muscle tissues increased significantly from 3.5+/-0.8 to 7.4+/-4.7 kPa during proliferation (p,0.05) and reached 12.2+/-6.0 kPa during differentiation (p,0.05), thus attesting the increase of matrix stiffness during proliferation and differentiation of the myocytes. In conclusion, we reported modulations of the adhesion complexes, the actin cytoskeleton and nuclear shape in 3D compared with routine 2D muscle culture. These findings point to complex interactions between muscle cells and the surrounding matrix with dynamic regulation of the cell-matrix stiffness

Boxplot analysis of elastic modulus (E) of the human engineered muscle tissues during the course of the culture.

<p>E was measured by AFM at the onset of gel polymerization (D0), after 24 h and 48 h of 3D culture in proliferative medium (D1 and D2) and 7 days after the switch from proliferative to differentiation media (D10). At D0, there was no significant difference in E values between gels without cells and gels with cells. E significantly increased during the proliferative period and after myotube differentiation. ** p<0.05.</p

Striation patterns in human engineered muscle tissues (A) and in 2D conditions (B).

<p>Immunofluorescence with anti α-actinin (a,d,g,j) and anti-myosin heavy chain (b,e,h,k) 4 and 7 days after the onset of myotube differentiation. Scale bar 10 µm. Panels c,f,i,l: Transmission electron microscopy 4 and 7 days after the onset of myotube differentiation. Single arrow: Z body; Double arrow: thick filament. *: Cytoplasmic membrane; N: nucleus. Scale bar: 1 µm. Insert: close up view of Z bodies; scale bar: 100 nm.</p

Characterization of the 3D fibrin constructs.

<p>A–B: Cell distribution within the fibrin matrix after 1 day (day 1: D1) and 10 days (D10) of 3D culture. Toluidine blue staining of cryostat sections of constructs was analyzed by light microscopy (scale bar = 100 µm). C–D: Cell morphology was analyzed just after gel polymerization (C) (D0) and after 24 hours (D) (D1). Living myoblasts were stained with green fluorescent calcein and visualized by confocal fluorescent microscopy (calcein appeared in green). Fibrin, in red, was visualized by confocal reflectance microscopy. Myoblast alignment was observed as early as day 1. E–F: α-tubulin immunofluorescence visualized by confocal fluorescent microscopy (in red) confirmed the alignment of the myoblasts (E) and myotubes (F) along the gel axis (arrow). Nuclei were stained with DAPI (in blue) (scale bar = 10 µm). G–H: The length of the human engineered muscle tissue greatly reduced overtime due to compaction of the construct. 3D constructs soon after the gel polymerization (G) and 10 days of 3D culture (H). In absence of cells, fibrin gels did not exhibit any compaction.</p