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Filamin A (FLNa) belongs to an actin-binding protein family in binding and cross-linking actin filaments into a three-dimensional structure. However, little attention has been given to its mechanobiological role in cancer cells. Here, we quantitatively investigated the role of FLNa by analyzing the following parameters in negative control (NC) and FLNa-knockdown (KD) U87 glioma cells using submicron pillars (900 nm diameter and 2 μm height): traction force (TF), rigidity sensing ability, cell aspect ratio, migration speed, and invasiveness. During the initial phase of cell adhesion (

The skin experiences constant physical stimuli, such as stretching. Exposure to excessive physical stimuli stresses the skin and can accelerate aging. In this study, we applied a method that allowed human fibroblasts and keratinocytes to be perfused with media to form 3D skin equivalents that were then uniaxially 10%-stretched for 12 h per day (at either 0.01 or 0.05 Hz) for up to 7 days to form wrinkled skin-on-a-chip (WSOC). There was more wrinkling seen in skin equivalents under 0.01 Hz uniaxial stretching than there was for non-stretched skin equivalents. At 0.05 Hz, the stratum corneum almost disappeared from the skin equivalents, indicating that stretching was harmful for the epidermis. At both frequencies, the production of collagen and related proteins in the skin equivalents, such as fibronectin 10 and keratin, decreased more than those in the non-stretched equivalents, indicating that the dermis also suffered from the repeated tensile stress. These results suggest that WSOCs can be used to examine skin aging and as an in vitro tool to evaluate the efficacy of anti-wrinkle cosmetics and medicines.

In-plane electrical conduction in sp2-hybridized boron nitride (sp2-BN) is presented to explore a huge potential of sp2-BN as an active material for electronics and ultraviolet optoelectronics. Systematic investigation on temperature-dependent current-voltage ( I- V) characteristics of a few-layer sp2-BN grown by metal-organic vapor-phase epitaxy reveals two types of predominant conduction mechanisms that are Ohmic conduction at the low bias region and space-charge-limited conduction at the high bias region. From the temperature-dependent I- V characteristics, two shallow traps with activation energies of approximately 25 and 185 meV are observed. On the basis of the near-edge X-ray absorption fine-structure spectroscopy, boron-boron (B-B) homoelemental bonding which can be related to grain boundary and nitrogen vacancy (VN) are proposed as the origin of the shallow traps mediating the in-plane conduction in the sp2-BN layer. In addition, a drastic enhancement in the electrical conductivity is observed with the increasing amount of VN that acts as a donor, implying that controlled generation of VN can be an alternative and better approach for the n-type doping of the sp2-BN film rather than ineffective conventional substitutional doping methods.

The structural evolution of h-BN powder by bead-milling is investigated. A unique initial stage in h-BN milling is discovered in which the x-ray diffraction (XRD) out-of-plane (0 0 2) and (0 0 4) peak intensities increase distinctly up to 3 and 4.8 times respectively, corresponding to an effective c-axis ordering of the h-BN particles. The structural evolution of h-BN due to milling is correlated to the change in the dominant milling mechanism from out-of-plane cleaving to in-plane cutting. An abnormal contraction of out-of-plane (0 0 2) interplanar spacing up to 0.044 A due to bead-milling is reported, which could be attributed to the introduction of oxygen functional groups (B-O and N-O) on the h-BN powder.

A few-layer hexagonal boron nitride (h-BN) films with wafer-scale thickness uniformity were grown by using a multiwafer metal–organic chemical vapor deposition (MOCVD) system at relatively low temperature of 1050 °C under various reactor pressures. The effect of the reactor pressure on the structural properties of the h-BN films was systematically investigated by various spectroscopic and microscopic analysis tools including near-edge X-ray absorption fine structure spectroscopy and transmission electron microscopy. We found that the defects in the MOCVD-grown h-BN films such as nitrogen vacancies and grain boundaries were strongly affected by the reactor pressure, which was elucidated by pressure-dependent change of Gibbs free energy for the nuclei formation and reaction rates. Based on our experimental observations, the growth strategies were discussed for realization of high-quality, multiwafer-scale uniformity h-BN films grown by MOCVD at temperature of 1050 °C.

Influenza A viruses are often present in environmental and clinical samples at concentrations below the limit of detection (LOD) of molecular diagnostics. Here we report an integrated microfluidic preconcentration and nucleic amplification system (&mu, FPNAS) which enables both preconcentration of influenza A virus H1N1 (H1N1) and amplification of its viral RNA, thereby lowering LOD for H1N1. H1N1 virus particles were first magnetically preconcentrated using magnetic nanoparticles conjugated with an antibody specific for the virus. Their isolated RNA was amplified to cDNA through thermocycling in a trapezoidal chamber of the &mu, FPNAS. A detection limit as low as 100 TCID50 (50% tissue culture infective dose) in saliva can be obtained within 2 hours. These results suggest that the LOD of molecular diagnostics for virus can be lowered by systematically combining immunomagnetic separation and reverse transcriptase-polymerase chain reaction (RT-PCR) in one microfluidic device.

Molecular diagnostics for sepsis is still a challenge due to the presence of compounds that interfere with gene amplification and bacteria at concentrations lower than the limit of detection (LOD). Here, we report on the development of a 3D printed modular microfluidic device (3Dpm&mu, FD) that preconcentrates bacteria of interest in whole blood and purifies their genomic DNA (gDNA). It is composed of a W-shaped microchannel and a conical microchamber. Bacteria of interest are magnetically captured from blood in the device with antibody conjugated magnetic nanoparticles (Ab-MNPs) at 5 mL/min in the W-shaped microchannel, while purified gDNA of the preconcentrated bacteria is obtained with magnetic silica beads (MSBs) at 2 mL/min in the conical microchamber. The conical microchamber was designed to be connected to the microchannel after the capturing process using a 3D-printed rotary valve to minimize the exposure of the MSBs to interfering compounds in blood. The pretreatment process of spiked blood (2.5 mL) can be effectively completed within about 50 min. With the 3Dpm&mu, FD, the LOD for the target microorganism Escherichia coli O157:H7 measured by both polymerase chain reaction (PCR) with electrophoresis and quantitative PCR was 10 colony forming unit (CFU) per mL of whole blood. The results suggest that our method lowers the LOD of molecular diagnostics for pathogens in blood by providing bacterial gDNA at high purity and concentration.

Polymicrobial biofilms play important roles in oral and systemic infections. The oral plaque bacteriumStreptococcus gordoniiis known to attach to the hyphal cell wall of the fungusCandida albicansto form corn-cob like structures in biofilms. However, the role ofC. albicansin formation of polymicrobial biofilms is not completely understood. The objective of this study was to determine the role ofC. albicanstranscription factors in regulation of polymicrobial biofilms and antibiotic tolerance ofS. gordonii. The proteins secreted byC. albicansandS. gordoniiin mixed planktonic cultures were determined using mass spectrometry. Antibiotic tolerance ofS. gordoniito ampicillin and erythromycin was determined in mixed cultures and mixed biofilms withC. albicans. Additionally, biofilm formation ofS. gordoniiwithC. albicansknock-out mutants of 45 transcription factors that affect cell wall integrity, filamentous growth and biofilm formation was determined. Furthermore, these mutants were also screened for antibiotic tolerance in mixed biofilms withS. gordonii. Analysis of secreted proteomes resulted in the identification of proteins being secreted exclusively in mixed cultures. Antibiotic testing showed thatS. gordoniihad significantly increased survival in mixed planktonic cultures with antibiotics as compared to single cultures.C. albicansmutants of transcription factors Sfl2, Brg1, Leu3, Cas5, Cta4, Tec1, Tup1, Rim101 and Efg1 were significantly affected in mixed biofilm formation. Also mixed biofilms ofS. gordoniiwith mutants ofC. albicanstranscription factors, Tec1 and Sfl2, had significantly reduced antibiotic tolerance as compared to control cultures. Our data indicates thatC. albicansmay have an important role in mixed biofilm formation as well as antibiotic tolerance ofS. gordoniiin polymicrobial biofilms.C. albicansmay play a facilitating role than being just an innocent bystander in oral biofilms and infections.

Various types of microfluidic systems have been developed to detect bacterial pathogens. However, most of these require enrichment steps that take at least several hours when detecting bacteria that are present with a low number of cells and, in addition, fabrication requires complicated assembly steps. In this study, we report the development of 3D microfluidic magnetic preconcentrator (3DμFMP) made of plastic via 3D printing without the need for any assembly. 3DμFMP could selectively preconcentrate enterohemorrhagic Escherichia coli O157:H7 in 100mL by a factor of 700 within 1h using antibody-conjugated magnetic nanoparticles (Ab-MNPs). With the combined use of an ATP luminometer, as low as 10 E. coli O157:H7 CFU (colony forming unit)/mL could be detected in blood. These results demonstrate the feasibility of 3DμFMP as a preconcentrator to improve the detection limit of existing bacterial detection systems.

Fibroblast activation is a crucial step in tumor growth and metastatic progression. Activated fibroblasts remodel the extracellular matrix (ECM) in primary tumor and metastatic microenvironments, exerting both pro- and antitumorigenic effects. However, the intrinsic mechanisms that regulate the activation of fibroblasts are not well-defined. The signaling axis comprising the calcium-activated Ser/Thr phosphatase calcineurin (CN), and its downstream target nuclear factor of activated T cells, has been implicated in endothelial (EC) and immune cell activation, but its role in fibroblasts is not known. Here, we demonstrate that deletion of CN in fibroblasts in vitro altered fibroblast morphology and function consistent with an activated phenotype relative to wild-type fibroblasts. CN-null fibroblasts had a greater migratory capacity, increased collagen secretion and remodeling, and promoted more robust EC activation in vitro. ECM generated by CN-null fibroblasts contained more collagen with greater alignment of fibrillar collagen compared with wild-type fibroblast-derived matrix. These differences in matrix composition and organization imposed distinct changes in morphology and cytoskeletal architecture of both fibroblasts and tumor cells. Consistent with this in vitro phenotype, mice with stromal CN deletion had a greater incidence and larger lung metastases. Our data suggest that CN signaling contributes to the maintenance of fibroblast homeostasis and that loss of CN is sufficient to promote fibroblast activation. Significance: Calcineurin signaling is a key pathway underlying fibroblast homeostasis that could be targeted to potentially prevent fibroblast activation in distant metastatic sites.

This study was conducted to investigate the effects of elevated water temperature (WT) on biochemical and immunological factors in the hemolymph of the abalones, Haliotis discus hannai and H. discus discus. The abalone were exposed to various WT; 20, 22, 24, 26 and for 4 days. In the control and , total-protein (TP), glucose and calcium (Ca) in hemolymph of H. discus discus were higher than the values in H. discus hannai. The values of magnesium (Mg), alkaline phosphatase (ALP) and lysozyme in H. discus hannai were similar to the H. discus discus in the control. There were no significant alterations in TP, glucose and Mg levels of hemolymph in H. discus hannai and H. discus discus by WT increases. The values of Ca, ALP and lysozyme were increased in H. discus hannai exposed to the high temperature (26 and ) compared to control, while the values in H. discus discus were not significant difference between the WT groups. The phenoloxidase (PO) activity was increased in hemolymph of H. discus hannai exposed to high temperature () compared to the control (P

Morphology and histochemistry of gill of the abalone, Haliotis discus hannai were described using light microscopy and scanning electron microscopy (SEM). The abalone has bipectinate gill of protobranch. The cilia and microvilli were well-developed on the free surface of the gill filaments. The epithelial layer of gill filament was simple and composed columnar epithelia and mucous cells. Result of AB-PAS (pH 2.5 and 1.0) and AF-AB (pH 2.5) reaction showed that mucous cells contained mainly acidic carboxylated mucosubstances.

Few layer hexagonal boron nitride (h-BN) films were grown on 2-inch sapphire substrates by using metal-organic chemical vapor deposition (MOCVD) with two different carrier gases, hydrogen (H-2) and nitrogen (N-2). Structural, optical and electrical properties of the MOCVD-grown h-BN films were systematically investigated by various spectroscopic analyses and electrical conduction measurement. Based on the experimental findings including narrower X-ray photoelectron spectra, reduced intensity of the shoulder peaks in near edge X-ray absorption fine structure spectra, and decreased electrical conduction by more than three orders of magnitude when H-2 carrier gas is employed, it was concluded that H-2 has an advantage over N-2 as the carrier gas for MOCVD growth of h-BN which is attributed to the healing of crystalline defects by etching and regrowth processes occurring under the pulsed source-injection mode. (C) 2017 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

The beta-turn is the most common type of nonrepetitive structure in globular proteins, comprising ~25% of all residues; however, a detailed understanding of effects of specific residues upon beta-turn stability and conformation is lacking. Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold and contains a total of five beta-hairpin structures (antiparallel beta-sheets connected by a reverse turn). beta-Turns related by the characteristic threefold structural symmetry of this superfold exhibit different primary structures, and in some cases, different secondary structures. As such, they represent a useful system with which to study the role that turn sequences play in determining structure, stability, and folding of the protein. Two turns related by the threefold structural symmetry, the beta4/beta5 and beta8/beta9 turns, were subjected to both sequence-swapping and poly-glycine substitution mutations, and the effects upon stability, folding, and structure were investigated. In the wild-type protein these turns are of identical length, but exhibit different conformations. These conformations were observed to be retained during sequence-swapping and glycine substitution mutagenesis. The results indicate that the beta-turn structure at these positions is not determined by the turn sequence. Structural analysis suggests that residues flanking the turn are a primary structural determinant of the conformation within the turn.

Specific residues in a polypeptide may be key contributors to the stability and foldability of the unique native structure. Identification and prediction of such residues is, therefore, an important area of investigation in solving the protein folding problem. Atypical main-chain conformations can help identify strains within a folded protein, and by inference, positions where unique amino acids may have a naturally high frequency of occurrence due to favorable contributions to stability and folding. Non-Gly residues located near the left-handed alpha-helical region (L-alpha) of the Ramachandran plot are a potential indicator of structural strain. Although many investigators have studied mutations at such positions, no consistent energetic or kinetic contributions to stability or folding have been elucidated. Here we report a study of the effects of Gly, Ala and Asn substitutions found within the L-alpha region at a characteristic position in defined beta-hairpin turns within human acidic fibroblast growth factor, and demonstrate consistent effects upon stability and folding kinetics. The thermodynamic and kinetic data are compared to available data for similar mutations in other proteins, with excellent agreement. The results have identified that Gly at the i+3 position within a subset of beta-hairpin turns is a key contributor towards increasing the rate of folding to the native state of the polypeptide while leaving the rate of unfolding largely unchanged.

The palladium-catalyzed cross-coupling of hypervalent iodonium salts with organobismuth(V) derivatives was accomplished with PdCl2 (5 mol%) in CH3CN at room temperature.

The palladium-catalyzed cross-coupling of hypervalent iodonium salts with organobismuth(V) derivatives was accomplished with PdCl2 (5 mol%) in CH3CN at room temperature.

The unique cation exchange chromatography (CEX) charge variant profile of mAb1 is characterized by a combination of mass spectrometry, limited Lys-C digestion followed by CEX separation and structural analysis. During CEX method development, mAb1 showed several unexpected phenomena, including a unique profile containing two main species (acidic 2 and main) and significant instability during stability studies of the main species. Reduced Lys-C peptide mapping identified a small difference in one of the heavy chain peptides (H4) in acidic 2 and further mass analysis identified this difference as Asn55 deamidation. However, the amount of Asn55 deamidation in acidic 2 could account for only half of the species present in this peak. Lys-C limited digest followed by CEX separated several unique peaks in the acidic peak 2 including two pre Fab peaks (LCC1 and LCC2). Whole protein mass analysis suggested that both LCC1 and LCC2 were potentially deamidated species. Subsequent peptide mapping with MS/MS determined that LCC1 contained isoAsp55 and LCC2 contained Asp55. Combining LCC1 and LCC2 CEX peak areas could account for nearly all of the species present in acidic peak 2. Subsequent detailed sequence analysis combined with molecular modeling identified Asn55 and its surrounding residues are responsible for the different CEX behavior and instability of mAb1 following forced degradation at high pH. Overall, the combinatorial approach used in this study proved to be a powerful tool to understand the unique charge variant and stability profile of a monoclonal antibody.

An alternative core packing group, involving a set of five positions, has been introduced into human acidic FGF-1. This alternative group was designed so as to constrain the primary structure within the core region to the same threefold symmetry present in the tertiary structure of the protein fold (the beta-trefoil superfold). The alternative core is essentially indistinguishable from the WT core with regard to structure, stability, and folding kinetics. The results show that the beta-trefoil superfold is compatible with a threefold symmetric constraint on the core region, as might be the case if the superfold arose as a result of gene duplication/fusion events. Furthermore, this new core arrangement can form the basis of a structural "building block" that can greatly simplify the de novo design of beta-trefoil proteins by using symmetric structural complementarity. Remaining asymmetry within the core appears to be related to asymmetry in the tertiary structure associated with receptor and heparin binding functionality of the growth factor.

Human acidic fibroblast growth factor (FGF-1) has a beta-trefoil structure, one of the fundamental protein superfolds. The X-ray crystal structures of wild-type and various mutant forms of FGF-1 have been solved in five different space groups: C2, C222(1), P2(1) (four molecules/asu), P2(1) (three molecules/asu), and P2(1)2(1)2(1). These structures reveal two characteristically different conformations for the beta8/beta9 beta-hairpin comprising residue positions 90-94. This region in the wild-type FGF-1 structure (P2(1), four molecules/asu), a his-tagged His93-->Gly mutant (P2(1), three molecules/asu) and a his-tagged Asn106-->Gly mutant (P2(1)2(1)2(1)) adopts a 3:5 beta-hairpin known as a type I (1-4) G1 beta-bulge (containing a type I turn). However, a his-tagged form of wild-type FGF-1 (C222(1)) and a his-tagged Leu44-->Phe mutant (C2) adopt a 3:3 beta-hairpin (containing a type I' turn) for this same region. A feature that distinguishes these two types of beta-hairpin structures is the number and location of side chain positions with eclipsed C(beta) and main-chain carbonyl oxygen groups (Psi is equivalent to +60 degrees). The effects of glycine mutations upon stability, at positions within the hairpin, have been used to identify the most likely structure in solution. Type I' turns in the structural data bank are quite rare, and a survey of these turns reveals that a large percentage exhibit crystal contacts within 3.0 A. This suggests that many of the type I' turns in X-ray structures may be adopted due to crystal packing effects.