Nanoscale assembly in biological systems: from neuronal cytoskeletal proteins to curvature stabilizing lipids

The review will describe experiments inspired by the rich variety of bundles and networks of interacting microtubules (MT), neurofilaments, and filamentous-actin in neurons where the nature of the interactions, structures, and structure-function correlations remain poorly understood. We describe how three-dimensional (3D) MT bundles and 2D MT bundles may assemble, in cell free systems in the presence of counter-ions, revealing structures not predicted by polyelectrolyte theories. Interestingly, experiments reveal that the neuronal protein tau, an abundant MT-associated-protein in axons, modulates the MT diameter providing insight for the control of geometric parameters in bio- nanotechnology. In another set of experiments we describe lipid-protein-nanotubes, and lipid nano-tubes and rods, resulting from membrane shape evolution processes involving protein templates and curvature stabilizing lipids. Similar membrane shape changes, occurring in cells for the purpose of specific functions, are induced by interactions between membranes and proteins. The biological materials systems described have applications in bio-nanotechnology.     

Implementation of targeted interventions to control Escherichia coli O157:H7 in a commercial abattoir

The objective of this study was to define locations on the carcass with highest contamination of E. coli O157 throughout the harvest process and implement targeted interventions to reduce or eliminate contamination. To establish a pathogen baseline, samples were collected at the foreshank, hindshank, inside round, neck and midline area and evaluated for E. coli O157:H7 presence. Environmental samples were also collected in the harvest area and the fabrication area of the facility. E. coli O157:H7 prevalence was highest on the foreshank, hindshank and inside rounds in the baseline study and steam vacuums/cones were implemented as an intervention in these specific areas on the harvest floor. At pre-evisceration, foreshank prevalence of E. coli O157:H7 was significantly (P<0.05) reduced from 21.7% to 3.1% after the application of steam interventions. At the final rail, foreshank prevalence in the baseline study was 4.2% while no E. coli O157:H7 was detected post-intervention implementation. E. coli O157:H7 on hindshanks and inside rounds was significantly reduced after intervention implementation from 24.2 to 11.5% and 37.5 to 16.7%, respectively at the final rail. Pathogen contamination of environmental samples collected in fabrication declined from 6.7% to 0.7% after slaughter interventions were implemented. Data indicate the identifying areas of contamination on the carcass and implementing interventions can significantly reduce E. coli O157 on the carcasses and in the fabrication environment.     

Fabrication of Multifaceted Micropatterned Surfaces with Laser Scanning Lithography

The implementation of engineered surfaces presenting micrometer‐sized patterns of cell adhesive ligands against a biologically inert background has led to numerous discoveries in fundamental cell biology. While existing surface patterning strategies allow patterning of a single ligand, it is still challenging to fabricate surfaces displaying multiple patterned ligands. To address this issue we implemented laser scanning lithography (LSL), a laser‐based thermal desorption technique, to fabricate multifaceted, micropatterned surfaces that display independent arrays of subcellular‐sized patterns of multiple adhesive ligands with each ligand confined to its own array. We demonstrate that LSL is a highly versatile “maskless” surface patterning strategy that provides the ability to create patterns with features ranging from 460 nm to 100 μm, topography ranging from ‐1 to 17 nm, and to fabricate both stepwise and smooth ligand surface density gradients. As validation for their use in cell studies, surfaces presenting orthogonally interwoven arrays of 1 μm × 8 μm elliptical patterns of Gly‐Arg‐Gly‐Asp‐terminated alkanethiol self‐assembled monolayers and human plasma fibronectin are produced. Human umbilical vein endothelial cells cultured on these multifaceted surfaces form adhesion sites to both ligands simultaneously and utilize both ligands for lamella formation during migration. The ability to create multifaceted, patterned surfaces with tight control over pattern size, spacing, and topography provides a platform to simultaneously investigate the complex interactions of extracellular matrix geometry, biochemistry, and topography on cell adhesion and downstream cell behavior.     

A Distortion-Sensitive Seam Carving Algorithm for Content-Aware Image Resizing

As displays become less expensive and are incorporated into more and more devices, there has been an increased focus on image resizing techniques to fill an image to an arbitrary screen size. Traditional methods such as cropping or resampling can introduce undesirable losses in information or distortion in perception. Recently, content-aware image retargeting methods have been proposed (Avidan and Shamir, ACM Trans Graphics 26(3), 2007; Guo et al., IEEE Trans Multimedia 11(5):856–867, 2009; Shamir and Avidan, Commun ACM 52(1), 2009; Simakov et al. 2008; Wolf et al. 2007), which produce exceptional results. In particular, seam carving, proposed by Avidan and Shamir, has gained attention as an effective solution. However, there are many cases where it can fail. In this paper we propose a distortion-sensitive seam carving algorithm for content-aware image resizing that improves edge preservation and decreases aliasing artifacts. In the proposed approach, we use local gradient information along with a thresholding technique to guide the seam selection process and provide a mechanism to halt seam carving when further processing would introduce unacceptable visual distortion in the resized image. Furthermore, anti-aliasing filter is used to reduce the aliasing artifacts caused by seam removal. Experiments have demonstrated superior performance over the current seam carving methods.     

Synchrotron infrared microspectroscopy detecting the evolution of Huntington's disease neuropathology and suggesting unique correlates of dysfunction in white versus gray brain matter

Huntington's disease (HD), caused by a mutation of the corresponding gene encoding the protein huntingtin (htt), is characterized by progressive deterioration of cognitive and motor functions, paralleled by extensive loss of striatal neurons. At the cellular level, pathogenesis involves an early and prolonged period of neuronal dysfunction followed by neuronal death. Understanding the molecular events driving these deleterious processes is critical to the successful development of therapies to slow down or halt the progression of the disease. Here, we examined biochemical processes in a HD ex vivo rat model, as well as in a HD model for cultured neurons using synchrotron-assisted Fourier transform infrared microspectroscopy (S-FTIRM). The model, based on lentiviral-mediated delivery of a fragment of the HD gene, expresses a mutant htt fragment in one brain hemisphere and a wild-type htt fragment in the control hemisphere. S-FTIRM allowed for high spatial resolution and distinction between spectral features occurring in gray and white matter. We measured a higher content of β-sheet protein in the striatal gray matter exposed to mutant htt as early as 4 weeks following the initiation of mutant htt exposure. In contrast, white matter tracts did not exhibit any changes in protein structure but surprisingly showed reduced content of unsaturated lipids and a significant increase in spectral features associated with phosphorylation. The former is reminiscent of changes consistent with a myelination deficiency, while the latter is characteristic of early pro-apoptotic events. These findings point to the utility of the label-free FTIRM method to follow mutant htt's β-sheet-rich transformation in striatal neurons ex vivo, provide further evidence for mutant htt amyloidogenesis in vivo, and demonstrate novel chemical features indicative of white matter changes in HD. Parallel studies in cultured neurons expressing the same htt fragments showed similar changes.     

Photothermal nanoblade for large cargo delivery into mammalian cells

It is difficult to achieve controlled cutting of elastic, mechanically fragile, and rapidly resealing mammalian cell membranes. Here, we report a photothermal nanoblade that utilizes a metallic nanostructure to harvest short laser pulse energy and convert it into a highly localized explosive vapor bubble, which rapidly punctures a lightly contacting cell membrane via high-speed fluidic flows and induced transient shear stress. The cavitation bubble pattern is controlled by the metallic structure configuration and laser pulse duration and energy. Integration of the metallic nanostructure with a micropipet, the nanoblade generates a micrometer-sized membrane access port for delivering highly concentrated cargo (5 × 10(8) live bacteria/mL) with high efficiency (46%) and cell viability (>90%) into mammalian cells. Additional biologic and inanimate cargo over 3-orders of magnitude in size including DNA, RNA, 200 nm polystyrene beads, to 2 μm bacteria have also been delivered into multiple mammalian cell types. Overall, the photothermal nanoblade is a new approach for delivering difficult cargo into mammalian cells.     

Characterization of strong fiber Bragg gratings using an applied thermal chirp and iterative algorithm

Coupling coefficients of various grating types and strengths are calculated from measurements of the complex reflectivity using an applied thermal chirp and optical frequency domain reflectometry (OFDR). The complex reflectivity is then utilized by a layer peeling algorithm to determine the coupling coefficient of the thermally chirped grating. A guess of the temperature profile enables the coupling coefficient of the unchirped grating to be estimated. An iterative algorithm is then used to converge on the exact coupling coefficient, employing an error minimization method applied to the reflectivity spectra. This technique removes the need for a reference grating while preserving the spatial resolution obtained with the initial OFDR measurement. Successful reconstruction of gratings with integrated |κ|L ~ 9.0 are demonstrated with a spatial resolution of less than 100 μm.     

MHz-rate nitric oxide planar laser-induced fluorescence imaging in a Mach 10 hypersonic wind tunnel

Nitric oxide planar laser-induced fluorescence (NO PLIF) imaging at repetition rates as high as 1 MHz is demonstrated in the NASA Langley 31 in. Mach 10 hypersonic wind tunnel. Approximately 200 time-correlated image sequences of between 10 and 20 individual frames were obtained over eight days of wind tunnel testing spanning two entries in March and September of 2009. The image sequences presented were obtained from the boundary layer of a 20° flat plate model, in which transition was induced using a variety of different shaped protuberances, including a cylinder and a triangle. The high-speed image sequences captured a variety of laminar and transitional flow phenomena, ranging from mostly laminar flow, typically at a lower Reynolds number and/or in the near wall region of the model, to highly transitional flow in which the temporal evolution and progression of characteristic streak instabilities and/or corkscrew-shaped vortices could be clearly identified.     

Development of a fiber-optic laser delivery system capable of delivering 213 and 266 nm pulsed Nd:YAG laser radiation for tissue ablation in a fluid environment

Ultraviolet (UV) lasers have the capability to precisely remove tissue via ablation; however, due to strong absorption of the applicable portion the UV spectrum, their surgical use is currently limited to extraocular applications at the air/tissue boundary. Here we report the development and characterization of a fiber-optic laser delivery system capable of outputting high-fluence UV laser pulses to internal tissue surfaces. The system has been developed with a view to intraocular surgical applications and has been demonstrated to ablate ocular tissue at the fluid/tissue boundary. The fifth (213?nm) and fourth(266?nm) harmonics of a Nd:YAG laser were launched into optical fibers using a hollow glass taper to concentrate the beam. Standard and modified silica/silica optical fibers were used, all commercially available. The available energy and fluence as a function of optical fiber length was evaluated and maximized. The maximum fluence available to ablate tissue was affected by the wavelength dependence of the fiber transmission; this maximum fluence was greater for 266?nm pulses (8.4?J/cm2) than for 213?nm pulses (1.4?J/cm2). The type of silica/silica optical fiber used did not affect the transmission efficiency of 266?nm pulses, but transmission of 213?nm pulses was significantly greater through modified silica/silica optical fiber. The optical fiber transmission efficiency of 213?nm pulses decreased as a function of number of pulses transmitted, whereas the transmission efficiency of 266?nm radiation was unchanged. Single pulses have been used to ablate fresh porcine ocular tissue. In summary, we report a method for delivering the fifth (213?nm) and fourth (266?nm) harmonics of a Nd:YAG laser to the surface of immersed tissue, the reliability and stability of the system has been characterized, and proof of concept via tissue ablation of porcine ocular tissue demonstrates the potential for the intraocular surgical application of this technique.