AQP3 Increases Intercellular Cohesion in NSCLC A549 Cell Spheroids through Exploratory Cell Protrusions

Tumor cell aggregation is critical for cell survival following the loss of extracellular matrix attachment and dissemination. However, the underlying mechanotransduction of clustering solitary tumor cells is poorly understood, especially in non-small cell lung cancers (NSCLC). Here, we examined whether cell surface protrusions played an important role in facilitating the physical contact between floating cells detached from a substrate. We employed poly-2-hydroxyethyl methacrylate-based 3D culture methods to mimic in vivo tumor cell cluster formation. The suprastructural analysis of human NSCLC A549 cell spheroids showed that finger-like protrusions clung together via the actin cytoskeleton. Time-lapse holotomography demonstrated that the finger-like protrusions of free-floating cells in 3D culture displayed exploratory coalescence. Global gene expression analysis demonstrated that the genes in the organic hydroxyl transport were particularly enriched in the A549 cell spheroids. Particularly, the knockdown of the water channel aquaporin 3 gene (AQP3) impaired multicellular aggregate formation in 3D culture through the rearrangement of the actomyosin cytoskeleton. Moreover, the cells with reduced levels of AQP3 decreased their transmigration. Overall, these data indicate that cell detachment-upregulated AQP3 contributes to cell surface protrusions through actomyosin cytoskeleton remodeling, causing the aggressive aggregation of free-floating cells dependent on the property of the substratum and collective metastasis.     

Magnetically Guidable Proteinaceous Adhesive Microbots for Targeted Locoregional Therapeutics Delivery in the Highly Dynamic Environment of the Esophagus

The esophagus is a tubular-shaped muscular organ where swallowed fluids and muscular contractions constitute a highly dynamic environment. The turbulent, coordinated processes that occur through the oropharyngeal conduit can often compromise targeted administration of therapeutic drugs to a lesion, significantly reducing therapeutic efficacy. Here, magnetically guidable drug vehicles capable of strongly adhering to target sites using a bioengineered mussel adhesive protein (MAP) to achieve localized delivery of therapeutic drugs against the hydrodynamic physiological conditions are proposed. A suite of highly uniform microparticles embedded with iron oxide (IO) nanoparticles (MAP@IO MPs) is microfluidically fabricated using the genipin-mediated covalent cross-linking of bioengineered MAP. The MAP@IO MPs are successfully targeted to a specific region and prolongedly retained in the tubular-structured passageway. In particular, orally administered MAP@IO MPs are effectively captured in the esophagus in vivo in a magnetically guidable manner. Moreover, doxorubicin (DOX)-loaded MAP@IO MPs exhibit a sustainable DOX release profile, effective anticancer therapeutic activity, and excellent biocompatibility. Thus, the magnetically guidable locomotion and robust underwater adhesive properties of the proteinaceous soft microbots can provide an intelligent modular approach for targeted locoregional therapeutics delivery to a specific lesion site in dynamic fluid-associated tubular organs such as the esophagus.     

Optimization of Pathogen Capture in Flowing Fluids with Magnetic Nanoparticles

Magnetic nanoparticles have been employed to capture pathogens for many biological applications; however, optimal particle sizes have been determined empirically in specific capturing protocols. Here, a theoretical model that simulates capture of bacteria is described and used to calculate bacterial collision frequencies and magnetophoretic properties for a range of particle sizes. The model predicts that particles with a diameter of 460 nm should produce optimal separation of bacteria in buffer flowing at 1 L h⁻¹. Validating the predictive power of the model, Staphylococcus aureus is separated from buffer and blood flowing through magnetic capture devices using six different sizes of magnetic particles. Experimental magnetic separation in buffer conditions confirms that particles with a diameter closest to the predicted optimal particle size provide the most effective capture. Modeling the capturing process in plasma and blood by introducing empirical constants (c_e), which integrate the interfering effects of biological components on the binding kinetics of magnetic beads to bacteria, smaller beads with 50 nm diameters are predicted that exhibit maximum magnetic separation of bacteria from blood and experimentally validated this trend. The predictive power of the model suggests its utility for the future design of magnetic separation for diagnostic and therapeutic applications.     

Solid phase crystallization of amorphous silicon on glass by thin film heater for thin film transistor (TFT) application

A new process for solid phase crystallization (SPC) of amorphous silicon (a-Si) using thin film heater is reported. With this localized Ti silicide thin film heater, we successfully crystallized 500 Å-thick a-Si in a few minutes without any thermal deformation of glass substrate. The size of crystallized silicon grain was abnormally big (30-40 μm). Polycrystalline thin film transistors (TFT) fabricated using this unique thin film heater showed better mobility than those of conventional ones by furnace annealing.     

Development of geometrically exact new shell elements based on general curvilinear co‐ordinates

In the present study first‐order shear deformable shell finite elements based on general curvilinear co‐ordinates are proposed. For the development of the present shell elements, a partial mixed variational functional with independently assumed strains is provided in order to avoid the severe locking troubles known as transverse shear and membrane lockings. Bubble functions are included in the shape function of displacement to improve the performance of the developed element. The proposed assumed strain four‐ and nine‐node elements based on the general tensor shell theory provide an efficient linkage framework for shell surface modelling and finite element analysis. In the several benchmark problems, the present shell elements with exact geometric representations demonstrate their performance compared to previously reported results. Copyright © 2002 John Wiley & Sons, Ltd.     

Enhanced thermal stability of a sputtered titanium-nitride film as a diffusion barrier for capacitor-bottom electrodes

The effect of a thin RuO_x layer formed on the Ru/TiN/doped poly-Si/Si stack structure was compared with that on the RuO_x/TiN/doped poly-Si/Si stack structure over the post-deposition annealing temperature ranges of 450–600°C. The Ru/TiN/poly-Si/Si contact system exhibited linear behavior at forward bias with a small increase in the total resistance up to 600°C. The RuO_x/TiN/poly-Si/Si contact system exhibited nonlinear characteristics under forward bias at 450°C, which is attributed to no formation of a thin RuO_x layer at the RuO_x surface and porous-amorphous microstructure. In the former case, the addition of oxygen at the surface layer of the Ru film by pre-annealing leads to the formation of a thin RuO_x layer and chemically strong Ru-O bonds. This results from the retardation of oxygen diffusion caused by the discontinuity of diffusion paths. In particular, the RuO_x layer in a nonstoichiometric state is changed to the RuO₂-crystalline phase in a stoichiometric state after post-deposition annealing; this phase can act as an oxygen-capture layer. Therefore, it appears that the electrical properties of the Ru/TiN/poly-Si/Si contact system are better than those of the RuO_x/TiN/poly-Si/Si contact system.     

Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.     

Phacoemulsification with a bevel-down phaco tip: phaco-drill

We present a technique for in situ lens nucleus emulsification using low phaco power and high vacuum, a continuous curvilinear capsulorhexis, and hydrodelineation. Emulsification is done with the phaco tip slanted down 30 or 45 degrees. Cutting and aspiration do not cause an undesirable energy loss. This technique can be combined with the nuclear chopping or divide and conquer methods because of its ability to drill and hold the nucleus. Posterior capsular rupture is prevented because the separated epinucleus acts as a barrier between the nucleus and the cortex. The low power used minimizes the energy transfer to the corneal endothelium. This technique is particularly useful in eyes with brunescent cataract.