Cellular and molecular responses of smooth muscle cells to surface nanotopography

Recent studies have demonstrated that surface nano-topography affect cell responses and activities. However, the molecular mechanism of the nano-structures on cellular behavior is yet to be determined. To bridge this gap, the present study was aimed to investigate the cellular and molecular responses of smooth muscle cells (SMCs) to surface nano-topography in vitro using nano-porous alumina membranes with different sizes (200 nm- and 20 nm-pores). Cellular responses such as cell adhesion, morphology, and proliferation were assessed using scanning electron microscopy (SEM), hematoxylin and eosin (HE) staining, and cell counting. The molecular cell responses were also investigated using cDNA microarrays. Results from these studies showed an unchanged response in cell adhesion, an alteration in cell morphology, and an increase in cell proliferation for cells grown on 200 nm-pore surfaces than on 20 nm-pore surfaces. In addition, exposure of SMCs to larger nano-pores induced the expression of various genes involved in cell cycle, DNA replication, cell proliferation, and signaling transduction pathways. These findings demonstrated that cellular responses of SMCs are dependent on the underlying nano-topography, and thereby suggesting nano-dimensional surface is one of the most important considerations to design of the next generation of medical devices and tissue engineering scaffolds.     

In situ applications of a new diver-operated motorized microsensor profiler

Microsensors are powerful tools for microenvironment studies, however their use has often been restricted to laboratory applications due to the lack of adequate equipment for in situ deployments. Here we report on new features, construction details, and examples of applications of an improved diver-operated motorized microsensor profiler for underwater field operation to a water depth of 25 m. The new motorized profiler has a final precision of 5 microm, and can accommodate amperometric Clark-type microsensors for oxygen and hydrogen sulfide, potentiometric microsensors (e.g., for pH, Ca2+), and fiber-optic irradiance microsensors. The profiler is interfaced by a logger with a signal display, and has pushbuttons for underwater operation. The system can be pre-programmed to autonomous operation or interactively operated by divers. Internal batteries supply power for up to 24 h of measurements and 36 h of data storage (max. 64 million data points). Two flexible stands were developed for deployment on uneven or fragile surfaces, such as coral reefs. Three experimental pilot studies are presented, where (1) the oxygen distribution in a sand ripple was 3-D-mapped, (2) the microenvironment of sediment accumulated on a stony coral was studied, and (3) oxygen dynamics during an experimental sedimentation were investigated. This system allows SCUBA divers to perform a wide array of in situ measurements, with deployment precision and duration similar to those possible in the laboratory.     

Droplet-based pyrosequencing using digital microfluidics

The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned on a printed-circuit board was used to control the formation, transportation, merging, mixing, and splitting of submicroliter-sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, deoxyribonucleotide triphosphates (dNTPs), and DNA templates. The DNA templates were anchored to magnetic beads which enabled them to be thoroughly washed between nucleotide additions. Reagents and protocols were optimized to maximize signal over background, linearity of response, cycle efficiency, and wash efficiency. As an initial demonstration of feasibility, a portion of a 229 bp Candida parapsilosis template was sequenced using both a de novo protocol and a resequencing protocol. The resequencing protocol generated over 60 bp of sequence with 100% sequence accuracy based on raw pyrogram levels. Excellent linearity was observed for all of the homopolymers (two, three, or four nucleotides) contained in the C. parapsilosis sequence. With improvements in microfluidic design it is expected that longer reads, higher throughput, and improved process integration (i.e., "sample-to-sequence" capability) could eventually be achieved using this low-cost platform.     

Use of periplasmic target protein capture for phage display engineering of tight-binding protein-protein interactions

Phage display is a powerful tool to study and engineer protein and peptide interactions. It is not without its limitations, however, such as the requirement for target protein purification and immobilization in a correctly folded state. A protein capture method is described here that allows enrichment of tight-binding protein variants in vivo thereby eliminating the need for target protein purification and immobilization. The linkage of genotype to phenotype is achieved by placing both receptor and ligand encoding genes on the same plasmid. This allows the isolation of the tight-binding ligand-receptor pair complexes after their association in the bacterial periplasm. The interaction between the TEM-1-β-lactamase fused to the gene 3 coat protein displayed on the surface of M13 bacteriophage and the β-lactamse inhibitory protein (BLIP) expressed in soluble form with a signal sequence to export it to the periplasm was used as a model system to test the method. The system was experimentally validated using a previously characterized collection of BLIP alanine mutants with a range of binding affinities for TEM-1 β-lactamase and by isolating tight-binding variants from a library of mutants randomized at residue position Tyr50 in BLIP which contacts β-lactamase.     

An evaluation of the predictive accuracy of wake effects models for offshore wind farms

Wake losses are perceived as one of the largest uncertainties in energy production estimates (EPEs) for new offshore wind projects. In recent years, significant effort has been invested to improve the accuracy of wake models. However, it is still common for a standard wake loss uncertainty of 50% to be assumed in EPEs for new offshore wind farms. This paper presents a body of evidence to support reducing that assumed uncertainty. It benchmarks the performance of four commonly used wake models against production data from five offshore wind farms. Three levels of evidence are presented to substantiate the performance of the models:

• Case studies, i.e. efficiencies of specific turbines under specific wind conditions;
• Array efficiencies for the wind farm as a whole for relatively large bins of wind speed and direction; and
• Validation wake loss, which corresponds to the overall wake loss within the proportion of the annual energy production where validation is possible.

The most important result for predicting annual energy production is the validation wake loss. The other levels of evidence demonstrate that this result is not unduly reliant on cancellation of errors between wind speed and/or wind direction bins. All of the root‐mean‐squared errors in validation wake loss are substantially lower than the 50% uncertainty commonly assumed in EPEs; indeed, even the maximum errors are below 25%. It is therefore concluded that there is a good body of evidence to support reducing this assumed uncertainty substantially, to a proposed level of 25%. Copyright © 2015 John Wiley & Sons, Ltd.     

Biodegradable zinc-based materials with a polymer coating designed for biomedical applications

Over the last decades, biodegradable metals have gained popularity for biomedical applications due to their ability to assist in tissue healing. These materials degrade in vivo, while the corrosion products formed are either absorbed or excreted by the body, and no further surgical intervention is required for removal. Intensive research has been carried out mainly on degradable biomaterials based on Mg and Fe. In recent years, zinc-based degradable biomaterials have been explored by the biomedical community for their intrinsic physiological relevance, desirable biocompatibility, intermediate degradation rate, tuneable mechanical properties and pro-regeneration properties. Since pure Zn does not exhibit sufficient mechanical properties for orthopedic applications, various Zn alloys with better properties are being developed. In this work, the combined effect of minor Fe addition to Zn and a polyethyleneglycol (PEG) coating on the surface morphology, degradation, cytotoxicity and mechanical properties of Zn-based materials was studied. There are several studies regarding the influence of the production of Zn alloys, but the effect of polymer coating on the properties of Zn-based materials has not been reported yet. A positive effect of Fe addition and polymer coating on the degradation rate and mechanical properties was observed. However, a reduction in biocompatibility was also detected.     

Variable-temperature Fourier transform near-infrared (FT-NIR) Imaging spectroscopy of the diffusion process of butanol(OD) into polyamide 11

Time-resolved Fourier transform near-infrared (FT-NIR) spectroscopic imaging was applied to the diffusion process of butanol(OD) into polyamide 11 (PA11) with a novel sheet-structured variable-temperature-controlled sample holder in order to demonstrate the significant differences of diffusion rate below and above the glass transition temperature of PA11. The diffusant butanol(OD) was chosen for two reasons: (1) it allows the diffusion front to be monitored by the intensity decrease of a NH-specific absorption band of PA11 due to the NH/ND isotope exchange and (2) under the measurement conditions the diffusion of butanol(OD) into PA11 takes place in an experimentally manageable time frame. Apart from the in situ visualization of the diffusion front in the time-resolved FT-NIR images, the type of diffusion and the diffusion coefficient of butanol(OD) into PA11 have been determined.     

Synthesis and characterization of silicone-modified polyester as a clearcoat for automotive pre-coated metals

Four types of silicone-modified polyester resins were synthesized for cleanable characteristics with silicone intermediate, which has a long chain, to extend the polymer chains of the resins. These resins were formulated to make polyester/melamine heat-cured coatings to control the formability. The characteristics, viscoelastic behavior and flexibility of the resins were measured by DMA and tensile test. The contact angle measurement can be measured by the water repellence of the coating surface, which is a standard method to evaluate cleanable characteristics. The surface free energy was calculated by the contact angle measurement, and the surface analysis of each cured coating was evaluated using an XPS. Silicone-modified polyester coatings were coated on the cold rolled steel sheets to verify their formability, using a deep drawing test. Results showed that the storage modulus decreased, and the glass transition temperature shifted to a lower temperature with increasing contents of silicone intermediate. So, silicone intermediate provides lower stiffness and higher softness to polyester coating. To analyze the formability, we calculated F_U (the forming coefficient based on strain energy) and F_? (the forming coefficient based on strain). When F_U and F_? are both larger than 1, the polyester coatings have good formability. CSiPE-3 and CSiPE-5 had good formability. Also, CSiPE-5, which had the highest amount of silicone intermediate, had 93.5° of water contact angle, and had 26.5 mN/m of surface free energy and had 5.5 N/25 mm of the peel strength. So, it is implied that silicone intermediate can give a low surface energy and peel strength to polyester coatings. From those tests, the polyester/melamine coating of CSiPE-5 that had 0.5 mol of silicone intermediate had good formability and low peel strength, which are semi-removable characteristics. So, it would be an appropriate coating as a clearcoat for automotive pre-coated metals.     

Intracellular Delivery of Doxorubicin by Iron Oxide-Based Nano-Constructs Increases Clonogenic Inactivation of Ionizing Radiation in HeLa Cells

In this study, we determined the potential of polyethylene glycol-encapsulated iron oxide nanoparticles (IONP_CO) for the intracellular delivery of the chemotherapeutic doxorubicin (IONP_DOX) to enhance the cytotoxic effects of ionizing radiation. The biological effects of IONP and X-ray irradiation (50 kV and 6 MV) were determined in HeLa cells using the colony formation assay (CFA) and detection of γH2AX foci. Data are presented as mean ± SEM. IONP were efficiently internalized by HeLa cells. IONP_CO radiomodulating effect was dependent on nanoparticle concentration and photon energy. IONP_CO did not radiosensitize HeLa cells with 6 MV X-rays, yet moderately enhanced cellular radiosensitivity to 50 kV X-rays (DMF_SF0.1 = 1.13 ± 0.05 (p = 0.01)). IONP_DOX did enhance the cytotoxicity of 6 MV X-rays (DMF_SF0.1 = 1.3 ± 0.1; p = 0.0005). IONP treatment significantly increased γH2AX foci induction without irradiation. Treatment of HeLa cells with IONP_CO resulted in a radiosensitizing effect for low-energy X-rays, while exposure to IONP_DOX induced radiosensitization compared to IONP_CO in cells irradiated with 6 MV X-rays. The effect did not correlate with the induction of γH2AX foci. Given these results, IONP are promising candidates for the controlled delivery of DOX to enhance the cytotoxic effects of ionizing radiation.