Adhesive Properties of Adsorbed Layers of Two Recombinant Mussel Foot Proteins with Different Levels of DOPA and Tyrosine.

Using a surface forces apparatus and an atomic force microscope, we characterized the adhesive properties of adsorbed layers of two recombinant variants of Perna viridis foot protein 5 (PVFP-5), the main surface-binding protein in the adhesive plaque of the Asian green mussel. In one variant, all tyrosine residues were modified into 3,4-dihydroxy-l-phenylalanine (DOPA) during expression using a residue-specific incorporation strategy. DOPA is a key molecular moiety underlying underwater mussel adhesion. In the other variant, all tyrosine residues were preserved. The layer was adsorbed on a mica substrate and pressed against an uncoated surface. While DOPA produced a stronger adhesion than tyrosine in contact with the nanoscopic Si3N4 probe of the atomic force microscope, the two variants produced comparable adhesion on the curved macroscopic mica surfaces of the surface forces apparatus. These findings show that the presence of DOPA is not a sufficient condition to generate strong underwater adhesion. Surface chemistry and contact geometry affect the strength and abundance of protein-surface bonds created during adsorption and surface contact. Importantly, the adsorbed protein layer has a random and dynamic polymer-network structure that should be optimized to transmit the tensile stress generated during surface separation to DOPA surface bonds rather than other weaker bonds.

Gi Protein Modulation of the Potassium Channel TASK-2 Mediates Vesicle Osmotic Swelling to Facilitate the Fusion of Aquaporin-2 Water Channel Containing Vesicles.

Vesicle fusion is a fundamental cell biological process similar from yeasts to humans. For secretory vesicles, swelling is considered a step required for the expulsion of intravesicular content. Here this concept is revisited providing evidence that it may instead represent a general mechanism. We report the first example that non-secretory vesicles, committed to insert the Aquaporin-2 water channel into the plasma membrane, swell and this phenomenon is required for fusion to plasma membrane. Through an interdisciplinary approach, using atomic force microscope (AFM), a fluorescence-based assay of vesicle volume changes and NMR spectroscopy to measure water self-diffusion coefficient, we provide evidence that Gi protein modulation of potassium channel TASK-2 localized in AQP2 vesicles, is required for vesicle swelling. Estimated intravesicular K⁺ concentration in AQP2 vesicles, as measured by inductively coupled plasma mass spectrometry, was 5.3 mM, demonstrating the existence of an inwardly K⁺ chemical gradient likely generating an osmotic gradient causing vesicle swelling upon TASK-2 gating. Of note, abrogation of K⁺ gradient significantly impaired fusion between vesicles and plasma membrane. We conclude that vesicle swelling is a potentially important prerequisite for vesicle fusion to the plasma membrane and may be required also for other non-secretory vesicles, depicting a general mechanism for vesicle fusion.

Staphylococcus aureus clumping factor A is a force-sensitive molecular switch that activates bacterial adhesion.

Clumping factor A (ClfA), a cell-wall-anchored protein from Staphylococcus aureus, is a virulence factor in various infections and facilitates the colonization of protein-coated biomaterials. ClfA promotes bacterial adhesion to the blood plasma protein fibrinogen (Fg) via molecular forces that have not been studied so far. A unique, yet poorly understood, feature of ClfA is its ability to favor adhesion to Fg at high shear stress. Unraveling the strength and dynamics of the ClfA-Fg interaction would help us better understand how S. aureus colonizes implanted devices and withstands physiological shear stress. By means of single-molecule experiments, we show that ClfA behaves as a force-sensitive molecular switch that potentiates staphylococcal adhesion under mechanical stress. The bond between ClfA and immobilized Fg is weak (∼0.1 nN) at low tensile force, but is dramatically enhanced (∼1.5 nN) by mechanical tension, as observed with catch bonds. Strong bonds, but not weak ones, are inhibited by a peptide mimicking the C-terminal segment of the Fg γ-chain. These results point to a model whereby ClfA interacts with Fg via two distinct binding sites, the adhesive function of which is regulated by mechanical tension. This force-activated mechanism is of biological significance because it explains at the molecular level the ability of ClfA to promote bacterial attachment under high physiological shear stress.

Non-invasive optical method for real-time assessment of intracorneal riboflavin concentration and efficacy of corneal cross-linking.

Keratoconus is the primary cause of corneal transplantation in young adults worldwide. Riboflavin/UV-A corneal cross-linking may effectively halt the progression of keratoconus if an adequate amount of riboflavin enriches the corneal stroma and is photo-oxidated by UV-A light for generating additional cross-linking bonds between stromal proteins and strengthening the biomechanics of the weakened cornea. Here we reported an UV-A theranostic prototype device for performing corneal cross-linking with the ability to assess corneal intrastromal concentration of riboflavin and to estimate treatment efficacy in real time. Seventeen human donor corneas were treated according to the conventional riboflavin/UV-A corneal cross-linking protocol. Ten of these tissues were probed with atomic force microscopy in order to correlate the intrastromal riboflavin concentration recorded during treatment with the increase in elastic modulus of the anterior corneal stroma. The intrastromal riboflavin concentration and its consumption during UV-A irradiation of the cornea were highly significantly correlated (R = 0.79; P = .03) with the treatment-induced stromal stiffening effect. The present study showed an ophthalmic device that provided an innovative, non-invasive, real-time monitoring solution for estimating corneal cross-linking treatment efficacy on a personalized basis.

Biomechanical Strengthening of the Human Cornea Induced by Nanoplatform-Based Transepithelial Riboflavin/UV-A Corneal Cross-Linking.

Purpose: The purpose of this study was to investigate the biomechanical stiffening effect induced by nanoplatform-based transepithelial riboflavin/UV-A cross-linking protocol using atomic force microscopy (AFM). Methods: Twelve eye bank donor human sclerocorneal tissues were investigated using a commercial atomic force microscope operated in force spectroscopy mode. Four specimens underwent transepithelial corneal cross-linking using a hypotonic solution of 0.1% riboflavin with biodegradable polymeric nanoparticles of 2-hydroxypropyl-ß-cyclodextrin plus enhancers (trometamol and ethylenediaminetetraacetic acid) and UV-A irradiation with a 10 mW/cm2 device for 9 minutes. After treatment, the corneal epithelium was removed using the Amoils brush, and the Young's modulus of the most anterior stroma was quantified as a function of scan rate by AFM. The results were compared with those collected from four specimens that underwent conventional riboflavin/UV-A corneal cross-linking and four untreated specimens. Results: The average Young's modulus of the most anterior stroma after the nanoplatform-based transepithelial and conventional riboflavin/UV-A corneal cross-linking treatments was 2.5 times (P < 0.001) and 1.7 times (P < 0.001) greater than untreated controls respectively. The anterior stromal stiffness was significantly different between the two corneal cross-linking procedures (P < 0.001). The indentation depth decreased after corneal cross-linking treatments, ranging from an average of 2.4 ± 0.3 µm in untreated samples to an average of 1.2 ± 0.1 µm and 1.8 ± 0.1 µm after nanoplatform-based transepithelial and conventional cross-linking, respectively. Conclusions: The present nanotechnology-based transepithelial riboflavin/UV-A corneal cross-linking was effective to improve the biomechanical strength of the most anterior stroma of the human cornea.

Multiscale Investigation of the Depth-Dependent Mechanical Anisotropy of the Human Corneal Stroma.

PURPOSE: To investigate the depth-dependent mechanical anisotropy of the human corneal stroma at the tissue (stroma) and molecular (collagen) level by using atomic force microscopy (AFM). METHODS: Eleven human donor corneas were dissected at different stromal depths by using a microkeratome. Mechanical measurements were performed in 15% dextran on the surface of the exposed stroma of each sample by using a custom-built AFM in force spectroscopy mode using both microspherical (38-µm diameter) and nanoconical (10-nm radius of curvature) indenters at 2-µm/s and 15-µm/s indentation rates. Young's modulus was determined by fitting force curve data using the Hertz and Hertz-Sneddon models for a spherical and a conical indenter, respectively. The depth-dependent anisotropy of stromal elasticity was correlated with images of the corneal stroma acquired by two-photon microscopy. RESULTS: The force curves were obtained at stromal depths ranging from 59 to 218 µm. At the tissue level, Young's modulus (ES) showed a steep decrease at approximately 140-µm stromal depth (from 0.8 MPa to 0.3 MPa; P = 0.03) and then was stable in the posterior stroma. At the molecular level, Young's modulus (EC) was significantly greater than at the tissue level; EC decreased nonlinearly with increasing stromal depth from 3.9 to 2.6 MPa (P = 0.04). The variation of microstructure through the thickness correlated highly with a nonconstant profile of the mechanical properties in the stroma. CONCLUSIONS: The corneal stroma exhibits unique anisotropic elastic behavior at the tissue and molecular levels. This knowledge may benefit modeling of corneal behavior and help in the development of biomimetic materials.

Ferric Ions Inhibit the Amyloid Fibrillation of ß-Lactoglobulin at High Temperature.

The energetics of amyloid fibrillar aggregation of ß-lactoglobulin (ßLG) following incubation at high temperature and acid pH was studied by differential scanning calorimetry in the presence of Cu(2+) or Fe(3+) cations, and without any metal. Cu(2+) and metal-free protein solutions showed a distinct exothermic response that disappeared almost completely when the Fe(3+) molar concentration was ten times greater than the ßLG concentration. Thioflavin T fluorescence studies in solution and atomic force microscopy analysis of the deposit left on flat mica substrates by heat-incubated ßLG solutions correlated the absence of exothermic response of Fe(3+)-ßLG solutions with a lack of fibril production. In contrast, abundant fibril deposits were observed for Cu(2+)-ßLG solutions, with a rich polymorphism of multistrand fibrillar structures. Electron paramagnetic resonance revealed that Fe(3+) permanently binds to ßLG in the aggregate state whereas Cu(2+) plays a catalytic role without binding to the protein. We propose that Fe(3+) inhibits fibril production after binding to a key region of the protein sequence, possibly interfering with the nucleation step of the fibrillation process and opening a nonfibrillar aggregation pathway. These findings suggest that transition metal ions can be utilized to effectively modulate protein self-assembly into a variety of structures with distinct morphologies at the nanoscale level.

Understanding of the viscoelastic response of the human corneal stroma induced by riboflavin/UV-a cross-linking at the nano level.

PURPOSE: To investigate the viscoelastic changes of the human cornea induced by riboflavin/UV-A cross-linking using Atomic Force Microscopy (AFM) at the nano level. METHODS: Seven eye bank donor corneas were investigated, after gently removing the epithelium, using a commercial AFM in the force spectroscopy mode. Silicon cantilevers with tip radius of 10 nm and spring elastic constants between 26- and 86-N/m were used to probe the viscoelastic properties of the anterior stroma up to 3 µm indentation depth. Five specimens were tested before and after riboflavin/UV-A cross-linking; the other two specimens were chemically cross-linked using glutaraldehyde 2.5% solution and used as controls. The Young's modulus (E) and the hysteresis (H) of the corneal stroma were quantified as a function of the application load and scan rate. RESULTS: The Young's modulus increased by a mean of 1.1-1.5 times after riboflavin/UV-A cross-linking (P<0.05). A higher increase of E, by a mean of 1.5-2.6 times, was found in chemically cross-linked specimens using glutaraldehyde 2.5% (P<0.05). The hysteresis decreased, by a mean of 0.9-1.5 times, in all specimens after riboflavin/UV-A cross-linking (P<0.05). A substantial decrease of H, ranging between 2.6 and 3.5 times with respect to baseline values, was observed in glutaraldehyde-treated corneas (P<0.05). CONCLUSIONS: The present study provides the first evidence that riboflavin/UV-A cross-linking induces changes of the viscoelastic properties of the cornea at the scale of stromal molecular interactions.