Nanocomposite gold-silk nanofibers.

Cell-biomaterial interactions can be controlled by modifying the surface chemistry or nanotopography of the material, to induce cell proliferation and differentiation if desired. Here we combine both approaches in forming silk nanofibers (SNFs) containing gold nanoparticles (AuNPs) and subsequently chemically modifying the fibers. Silk fibroin mixed with gold seed nanoparticles was electrospun to form SNFs doped with gold seed nanoparticles (SNF(seed)). Following gold reduction, there was a 2-fold increase in particle diameter confirmed by the appearance of a strong absorption peak at 525 nm. AuNPs were dispersed throughout the AuNP-doped silk nanofibers (SNFs(Au)). The Young's modulus of the SNFs(Au) was almost 70% higher than that of SNFs. SNFs(Au) were modified with the arginine-glycine-aspartic acid (RGD) peptide. Human mesenchymal stem cells that were cultured on RGD-modified SNF(Au) had a more than 2-fold larger cell area compared to the cells cultured on bare SNFs; SNF(Au) also increased cell size. This approach may be used to alter the cell-material interface in tissue engineering and other applications.

Antimicrobial protegrin-1 forms amyloid-like fibrils with rapid kinetics suggesting a functional link.

Protegrin-1 (PG-1) is an 18 residues long, cysteine-rich ß-sheet antimicrobial peptide (AMP). PG-1 induces strong cytotoxic activities on cell membrane and acts as a potent antibiotic agent. Earlier we reported that its cytotoxicity is mediated by its channel-forming ability. In this study, we have examined the amyloidogenic fibril formation properties of PG-1 in comparison with a well-defined amyloid, the amyloid-ß (Aß(1-42)) peptide. We have used atomic force microscopy (AFM) and thioflavin-T staining to investigate the kinetics of PG-1 fibrils growth and molecular dynamics simulations to elucidate the underlying mechanism. AFM images of PG-1 on a highly hydrophilic surface (mica) show fibrils with morphological similarities to Aß(1-42) fibrils. Real-time AFM imaging of fibril growth suggests that PG-1 fibril growth follows a relatively fast kinetics compared to the Aß(1-42) fibrils. The AFM results are in close agreement with results from thioflavin-T staining data. Furthermore, the results indicate that PG-1 forms fibrils in solution. Significantly, in contrast, we do not detect fibrillar structures of PG-1 on an anionic lipid bilayer 2-dioleoyl-sn-glycero-3-phospho-L-serine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; only small PG-1 oligomers can be observed. Molecular dynamics simulations are able to identify the presence of these small oligomers on the membrane bilayer. Thus, our current results show that cytotoxic AMP PG-1 is amyloidogenic and capable of forming fibrils. Overall, comparing ß-rich AMPs and amyloids such as Aß, in addition to cytotoxicity and amyloidogenicity, they share a common structural motif, and are channel forming. These combined properties support a functional relationship between amyloidogenic peptides and ß-sheet-rich cytolytic AMPs, suggesting that amyloids channels may have an antimicrobial function.

Antimicrobial protegrin-1 forms ion channels: molecular dynamic simulation, atomic force microscopy, and electrical conductance studies.

Antimicrobial peptides (AMPs) are an emerging class of antibiotics for controlling health effects of antibiotic-resistant microbial strains. Protegrin-1 (PG-1) is a model antibiotic among beta-sheet AMPs. Antibiotic activity of AMPs involves cell membrane damage, yet their membrane interactions, their 3D membrane-associated structures and the mechanism underlying their ability to disrupt cell membrane are poorly understood. Using complementary approaches, including molecular dynamics simulations, atomic force microscopy (AFM) imaging, and planar lipid bilayer reconstitution, we provide computational and experimental evidence that PG-1, a beta-hairpin peptide, forms ion channels. Simulations indicate that PG-1 forms channel-like structures with loosely attached subunits when reconstituted in anionic lipid bilayers. AFM images show the presence of channel-like structures when PG-1 is reconstituted in dioleoylphosphatidylserine/palmitoyloleoyl phosphatidylethanolamine bilayers or added to preformed bilayers. Planar lipid bilayer electrical recordings show multiple single channel conductances that are consistent with the heterogeneous oligomeric channel structures seen in AFM images. PG-1 channel formation seems to be lipid-dependent: PG-1 does not easily show ion channel electrical activity in phosphatidylcholine membranes, but readily shows channel activity in membranes rich in phosphatidylethanolamine or phosphatidylserine. The combined results support a model wherein the beta-hairpin PG-1 peptide acts as an antibiotic by altering cell ionic homeostasis through ion channel formation in cell membranes.

Neuronal elasticity as measured by atomic force microscopy.

A cell's form and function is determined to a great extent by its cellular membrane and the underlying cytoskeleton. Understanding changes in the cellular membrane and cytoskeleton can provide insight into aging and disease of the cell. The atomic force microscope (AFM) allows unparalled resolution for the imaging of these cellular components and the ability to probe their mechanical properties. This report describes our progress toward the use of AFM as a tool in neuroscience applications. Elasticity measurements are reported on living chick embryo dorsal root ganglion and sympathetic neurons in vitro. The neuronal cellular body and growth cones regions are examined for variations in cellular maturity. In addition, cellular changes due to exposure to various environmental conditions and neurotoxins are investigated. This report includes data obtained on different AFM systems, using various AFM techniques and thus also provides knowledge of AFM instruments and methodology.

K3 fragment of amyloidogenic beta(2)-microglobulin forms ion channels: implication for dialysis related amyloidosis.

Beta(2)-microglobulin (beta(2)m) amyloid deposits are linked to dialysis-related amyloidosis (DRA) in hemodialysis patients. The mechanism by which beta(2)m causes DRA is not understood. It is also unclear whether only the full-length beta(2)m induces pathophysiology or if proteolytic fragments are sufficient for inducing this effect. Ser20-Lys41 (K3) is a digestion fragment of full-length beta(2)m. Solid state NMR (ssNMR) combined with X-ray diffraction and atomic force microscopy (AFM) revealed the characteristic oligomeric amyloid conformation of the U-turn beta-strand-turn-beta-strand motif stacked in parallel and stabilized by intermolecular interactions also shown by Abeta(9-40)/Abeta(17-42) and the CA150 WW domain. Here we use the K3 U-turn atomic coordinates and molecular dynamic (MD) simulations to model K3 channels in the membrane. Consistent with previous AFM imaging of other amyloids that show channel-like structures in the membrane, in the simulations K3 also forms ion channels with 3-6 loosely attached mobile subunits. We carry out AFM, single channel electrical recording, and fluorescence imaging experiments. AFM images display 3D ion channel topography with shapes, morphologies, and dimensions consistent with the theoretical model. Electrical conductance measurements indicate multiple single channel conductances, suggesting that various K3 oligomer sizes can constitute the channel structure. Fluorescence measurements in kidney cells show channel-mediated cell calcium uptake. These results suggest that the beta(2)m-induced DRA can be mediated by ion channels formed by its K3 fragment. Because the beta-strand-turn-beta-strand motif appears to be a universal amyloid feature, its ability to form ion channels further suggests that the motif may play a generic role in toxicity.

Holographic optical coherence imaging of rat osteogenic sarcoma tumor spheroids.

Holographic optical coherence imaging is a full-frame variant of coherence-domain imaging. An optoelectronic semiconductor holographic film functions as a coherence filter placed before a conventional digital video camera that passes coherent (structure-bearing) light to the camera during holographic readout while preferentially rejecting scattered light. The data are acquired as a succession of en face images at increasing depth inside the sample in a fly-through acquisition. The samples of living tissue were rat osteogenic sarcoma multicellular tumor spheroids that were grown from a single osteoblast cell line in a bioreactor. Tumor spheroids are nearly spherical and have radial symmetry, presenting a simple geometry for analysis. The tumors investigated ranged in diameter from several hundred micrometers to over 1 mm. Holographic features from the tumors were observed in reflection to depths of 500-600 microm with a total tissue path length of approximately 14 mean free paths. The volumetric data from the tumor spheroids reveal heterogeneous structure, presumably caused by necrosis and microcalcifications characteristic of some human avascular tumors.