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In previous studies, the biological characteristics of the fungus Cladosporium phlei and its genetic manipulation by transformation were assessed to improve production of the fungal pigment, phleichrome, which is a fungal perylenequinone that plays an important role in the production of a photodynamic therapeutic agent. However, the low production of this metabolite by the wild-type strain has limited its application. Thus, we attempted to clone and characterize the genes that encode polyketide synthases (PKS), which are responsible for the synthesis of fungal pigments such as perylenequinones including phleichrome, elsinochrome and cercosporin. Thus, we performed genomic DNA PCR using 11 different combinations of degenerate primers targeting conserved domains including β-ketoacyl synthase and acyltransferase domains. Sequence comparison of the PCR amplicons revealed a high homology to known PKSs, and four different PKS genes showing a high similarity to three representative types of PKS genes were amplified. To obtain full-length PKS genes, an ordered gene library of a phleichrome-producing C. phlei strain (ATCC 36193) was constructed in a fosmid vector and 4800 clones were analyzed using a simple pyramidal arrangement system. This hierarchical clustering method combines the efficiency of PCR with enhanced specificity. Among the three representative types of PKSs, two reducing, one partially reducing, and one non-reducing PKS were identified. These genes were subsequently cloned, sequenced, and characterized. Biological characterization of these genes to determine their roles in phleichrome production is underway, with the ultimate aim of engineering this pathway to overproduce the desired substance.

Background Tissue factor (TF), a primary initiator of blood coagulation, also plays a pivotal role in angiogenesis. TF expression in the airways is associated with asthma, a disease characterized in part by subepithelial angiogenesis. Objectives To determine potential sources of TF and the mechanisms of its availability in the lung microenvironment. Methods Normal human bronchial epithelial cells grown in air-liquid interface culture were subjected to a compressive stress of 30 cm H 2 O; this is comparable to that generated in the airway epithelium during bronchoconstriction in asthma. Conditioned media and cells were harvested to measure TF mRNA and TF protein. We also tested bronchoalveolar lavage fluid and airway biopsies from asthmatic patients and healthy controls for TF. Results TF mRNA was upregulated 2.2-fold after 3 hours of stress compared with unstressed cells. Intracellular and secreted TF proteins were enhanced 1.6-fold and more than 50-fold, respectively, compared with those of control cells after the onset of compression. The amount of TF in the bronchoalveolar lavage fluid from patients with asthma was found at mean concentrations that were 5 times greater than those of healthy controls. Immunohistochemical staining of endobronchial biopsies identified epithelial localization of TF with increased expression in asthma. Exosomes isolated from the conditioned media of normal human bronchial epithelial cells and the bronchoalveolar lavage fluid of asthmatic subjects by ultracentrifugation contained TF. Conclusions Our in vitro and in vivo studies show that mechanically stressed bronchial epithelial cells are a source of secreted TF and that exosomes are potentially a key carrier of the TF signal.

Bronchospasm induced in non-asthmatic human subjects can be easily reversed by a deep inspiration (DI) whereas bronchospasm that occurs spontaneously in asthmatic subjects cannot. This physiological effect of a DI has been attributed to the manner in which a DI causes airway smooth muscle (ASM) cells to stretch, but underlying molecular mechanisms-and their failure in asthma-remain obscure. Using cells and tissues from wild type and zyxin-/- mice we report responses to a transient stretch of physiologic magnitude and duration. At the level of the cytoskeleton, zyxin facilitated repair at sites of stress fiber fragmentation. At the level of the isolated ASM cell, zyxin facilitated recovery of contractile force. Finally, at the level of the small airway embedded with a precision cut lung slice, zyxin slowed airway dilation. Thus, at each level zyxin stabilized ASM structure and contractile properties at current muscle length. Furthermore, when we examined tissue samples from humans who died as the result of an asthma attack, we found increased accumulation of zyxin compared with non-asthmatics and asthmatics who died of other causes. Together, these data suggest a biophysical role for zyxin in fatal asthma.

Collective cellular migration within the epithelial layer impacts upon development, wound healing and cancer invasion, but remains poorly understood. Prevailing conceptual frameworks tend to focus on the isolated role of each particular underlying factor – taken one at a time or at most a few at a time – and thus might not be tailored to describe a cellular collective that embodies a wide palette of physical and molecular interactions that are both strong and complex. To bridge this gap, we shift the spotlight to the emerging concept of cell jamming, which points to only a small set of parameters that govern when a cellular collective might jam and rigidify like a solid, or instead unjam and flow like a fluid. As gateways to cellular migration, the unjamming transition (UJT) and the epithelial-to-mesenchymal transition (EMT) share certain superficial similarities, but their congruence – or lack thereof – remains unclear. In this Commentary, we discuss aspects of cell jamming, its established role in human epithelial cell layers derived from the airways of non-asthmatic and asthmatic donors, and its speculative but emerging roles in development and cancer cell invasion.

Rationale: We have previously showed that primary human bronchial epithelial (HBE) cells in air-liquid interface (ALI) culture undergo a transition from a fluid-like unjammed to a solid-like jammed state during maturation. This transition is delayed in HBE cells from asthmatic donors compared to cells from non-asthmatic donors. We tested the hypothesis that this delay is recapitulated by priming non-asthmatic cells with transforming growth factor-beta (TGF-β), a fibrotic mediator elevated in asthmatic airways. Methods: HBE cells were incubated with vehicle or TGF-β during expansion, then plated on transwells without further treatment. We quantified cell motions during maturation in ALI, and measured cell-substrate tractions and intercellular stresses exerted by these cells on soft polyacrylamide gels. We detected vimentin and E-cadherin proteins by western blot. Results: Control, unprimed HBE cells were highly mobile and unjammed at day 3 of ALI culture, but were immobile and jammed by day 7. However, TGF-β primed HBE cells remained unjammed until day 10. Compared to unprimed cells, primed cells exhibited significantly higher tractions and intercellular stresses by 1.7- and 1.5-fold, respectively. In TGF-β primed cells, the level of vimentin protein was substantially higher compared to control cells, but the level of E-cadherin protein was the same. Conclusion: Here we show that priming human airway epithelial cells with TGF-β causes a delay in the jamming transition during maturation in ALI, accompanied by increased monolayer physical forces. These findings suggest that TGF-β promotes a dysmature epithelial phenotype that delays HBE cell jamming in asthma.

As the activation of autophagy contributes to the efficacy of many anticancer therapies, deciphering the precise role of autophagy in cancer therapy is critical. Here, we report that the dual mTORC1/2 inhibitors PP242 and OSI-027 decreased cell viability but did not induce apoptosis in the non-small cell lung cancer (NSCLC) cell lines H460 and A549. PP242 induced autophagy in NSCLC cells as demonstrated by the formation of massive vacuoles and acidic vesicular organelles and the accumulation of LC3-II. JNK was activated by PP242, and PP242-induced autophagy was blocked by inhibiting JNK pathway with SP600125 or JNK siRNA, suggesting that JNK activation is required for the mTORC1/2 inhibitor-mediated induction of autophagy in NSCLC cells. Inhibiting JNK or autophagy increased the sensitivity of H460 cells to mTORC1/2 inhibitors, indicating that JNK or autophagy promoted survival in NSCLC cells treated with mTORC1/2 inhibitors. Together, these data suggest that combining mTORC1/2 inhibitors with inhibitors of JNK or autophagy might be an effective approach for improving therapeutic outcomes in NSCLC.

Chestnut blight fungus, Cryphonectria parasitica , and its hypovirus present a useful model system for investigating the mechanisms of hypoviral infection. To identify gene products associated with fungal pathogenicity and hypoviral regulation, we attempted a proteomic analysis of the virus-free EP155/2 strain and its isogenic virus-infected UEP1 strain in response to tannic acid (TA), which is abundant in the bark of chestnut trees. In this study, pretreatment of mycelia grown on TA-supplemented media was developed for proteomic analysis. Approximately 704 proteins from the mycelia of the EP155/2 strain were reproducibly present in 3 independent extractions. Among these, 111 and 79 spots were found to be responsive to hypovirus infection and TA supplementation, respectively. The TA-grown UEP1 strain yielded 28 spots showing an expression pattern different from that of untreated UEP1. Thirty protein spots showing considerable differences in spot density were selected for further analysis. Hybrid tandem LC-MS/MS spectrometry of the 30 selected protein spots revealed that 29 were identified while 1 was unidentified. Among the identified 29 proteins, 15 were metabolic enzymes; 5 were stress-related, of which 4 were heat-shock proteins and 1 was glutathione S-transferase; 5 were signaling and cellular process-related proteins; 2 were structural proteins; and 2 matched proteins of hypothetical genes.

See paper for full list of authors; The Korea Rare Isotope Accelerator, currently referred to as KoRIA, is briefly presented. The KoRIA facility is aimed to enable cutting-edge sciences in a wide range of fields. It consists of a 70 kW isotope separator on-line (ISOL) facility driven by a 70 MeV, 1 mA proton cyclotron and a 400 kW in-flight fragmentation (IFF) facility. The ISOL facility uses a superconducting (SC) linac for post-acceleration of rare isotopes up to about 18 MeV/u, while the SC linac of IFF facility is capable of accelerating uranium beams up to 200 MeV/u, 8 pμA and proton beams up to 600 MeV, 660 μA. Overall features of the KoRIA facility are presented with a focus on the accelerator design.

A coexpression strategy in Saccharomyces cerevisiae using episomal and integrative vectors for the Escherichia coli heat-labile enterotoxin B subunit (LTB) and a fusion protein of an ApxIIA toxin epitope produced by Actinobacillus pleuropneumoniae coupled to LTB, respectively, was adapted for the hetero-oligomerization of LTB and the LTB fusion construct. Enzyme-linked immunosorbent assay (ELISA) with GM1 ganglioside indicated that the LTB fusion construct, along with LTB, was oligomerized to make the functional heteropentameric form, which can bind to receptors on the mucosal epithelium. The antigen-specific antibody titer of mice orally administered antigen was increased when using recombinant yeast coexpressing the pentameric form instead of recombinant yeast expressing either the LTB fusion form or antigen alone. Better protection against challenge infection with A. pleuropneumoniae was also observed for coexpression in recombinant yeast compared with others. The present study clearly indicated that the coexpression strategy enabled the LTB fusion construct to participate in the pentameric formation, resulting in an improved induction of systemic and mucosal immune responses.

SUMMARY The gene CpSte11 of Cryphonectria parasitica, which encodes a yeast Ste11 homologue, was cloned and characterized. Gene replacement analysis revealed a high frequency of CpSte11 null mutants. When compared with the wild-type parent strain, CpSte11 null mutants showed no difference in terms of growth rate or pigmentation. However, CpSte11 null mutants showed a marked decrease in both the number and size of stromal pustules on chestnut twigs. The virulence test showed that, in comparison with those of the wild-type and virus-infected hypovirulent strains, CpSte11 null mutants produced necrotic areas of intermediate size. Disruption of the CpSte11 gene also resulted in defects in female fertility. Down-regulation of transcripts for the mating pheromone precursor gene, Mf2/2, and mating response transcription factors, such as cpst12 and pro1, was observed in CpSte11 null mutants. The down-regulation of Mf2/2, cpst12 and pro1 was also observed in the mutant phenotype of Cpmk2, a mating response Fus3-like mitogen-activated protein kinase (MAPK) gene, but not in the mutant of Cpmk1, a high-osmolarity glycerol Hog1-like MAPK gene. These results indicate that the cloned CpSte11 gene is functionally involved in the mating response pathway and acts through downstream targets, including Cpmk2, cpst12, pro1 and Mf2/2. However, the characteristics of the CpSte11 null mutant were fully phenocopied only in the cpst12 null mutant, but not in other studied null mutants of components of the putative mating response pathway.

UV-mutagenesis was performed to obtain mutant strains that demonstrate altered production of phleichrome, a secondary metabolite of Cladosporium phlei. Among fifty mutants selected, based on the increased area and intensity of the purple pigment surrounding the colonies, the strain M0035 showed the highest production of phleichrome, more than seven fold over wild type. Plate cultures of the M0035 strain resulted in a total of 592 mg phleichrome consisting of 146 mg and 446 mg from the mycelia and agar media, respectively. The M0035 strain displayed a growth rate and a mycelial mass comparable to the parental strain but had significantly reduced asexual sporulation.

Interphasic chromatin condenses into the chromosomes in order to facilitate the correct segregation of genetic information. It has been previously reported that the phosphorylation and methylation of the N-terminal tail of histone H3 are responsible for chromosome condensation. In this study, we demonstrate that the deacetylation and methylation of histone H3 lysine 9 (H3K9) are required for proper chromosome condensation. We confirmed that H3K9ac levels were reduced, whereas H3K9me3 levels were increased in mitotic cells, via immunofluorescence and Western blot analysis. Nocodazole treatment induced G2/M arrest but co-treatment with TSA, an HDAC inhibitor, delayed cell cycle progression. However, the HMTase inhibitor, AdoX, had no effect on nocodazole-induced G2/M arrest, thereby indicating that sequential modifications of H3K9 are required for proper chromosome condensation. The expression of SUV39H1 and SETDB1, H3K9me3-responsible HMTases, are specifically increased along with H3K9me3 in nocodazole-arrested buoyant cells, which suggests that the increased expression of those proteins is an important step in chromosome condensa-tion. H3K9me3 was highly concentrated in the vertical chromosomal axis during prophase and prometaphase. Collectively, the results of this study indicate that sequential modifications at H3K9 are associated with correct chromosome condensation, and that H3K9me3 may be relevant to the condensation of chromosome length.

Colloidal templating and pulsed laser deposition were combined to fabricate arrays of ordered two-dimensional macroporous ZnO–NiO thin films. The underlying principles involved in the formation of colloidal templates were investigated using Fourier transformations and radial distribution functions. The macroporous thin film consists of hollow half-spheres of nanocrystalline wurtzite ZnO and rock salt NiO. The electrical properties of macroporous ZnO–NiO thin films were investigated by impedance and DC current–voltage measurements. The electrical properties are controlled by the interfaces between oxide grains. The potential applications such as gas sensors and optics are discussed.

Multi-layered random network structure of titanium dioxides (TiO 2 ) nanofibers was fabricated by calcining electrospun TiO 2 /PVP hybrid fibers. Their structural, electrical and gas sensing properties were investigated in a calcination temperature range of 400–800 °C. TiO 2 nanofibers are polycrystalline with anatase structure below 600 °C and a mixture of anatase and rutile structures at 800 °C. The diameter of TiO 2 nanofibers ranged from 350 nm to 500 nm, and the grain size increased from 8 nm to 45 nm with the increase in calcining temperature. The activation energies were in the range of 0.50–0.57 eV. The TiO 2 nanofibers calcined at 600 °C showed the gas response upon exposure to CO concentration even below 1 ppm.

This work presents a new route to enhance the sensitivity of nanocrystalline TiO 2 fibers based gas sensors. Pure and Pd-doped TiO 2 nanofiber mats were synthesized by electrospinning and subsequent calcination. Highly porous fibrillar morphology was observed in the resultant nanocrystalline TiO 2 fibers. In addition to anatase TiO 2 crystallites, tiny (∼2 nm) PdO crystallites were observed in the Pd-doped TiO 2 fibers. The sensors using Pd-doped TiO 2 fibers showed promising gas sensing characteristics, such as low operation temperature (180 °C) and sufficient gas response ( R / R o = 38 to 2.1 ppm NO 2 ). The techniques outlined here offer new means for preparing novel metal-oxide nanofibers with catalytic dopants.

To enhance the sensitivity of a metal oxide-based gas sensor, we introduce a novel electrical method that relies jointly on the use of impedance and an applied DC bias. We used polycrystalline ZnO–NiO composite thin films prepared by pulsed layer deposition as CO gas sensor material. The applied DC bias lowers the interface barrier between oxide grains and thereby enhances the sensitivity. Our approach enhances the sensitivity to CO gas by approximately 200% more than a conventional DC method. The enhancement effect is more pronounced when the CO concentration is lowered. This method offers an efficient characterization tool for modulating the sensitivity of gas sensors using semiconducting metal oxides.

The transfer slab system is a structural system that transfers the loads from the upper shear wall structure to the lower columns. This is a costly system due to a very thick slab, and the relatively high cost can be mitigated by introducing voids in the slab. However, this system of flat plate containing voids is vulnerable to brittle failure caused by punching shear in vicinity of slab-column connection. Thus, the punching shear capacity of the void system is very important. However, the current code doesn`t provide a clear design provision for the strength of slabs with a void section. In this study, experimental study was conducted to investigate the punching shear strength of the void slab system. The shear strength of the specimens was predicted by current code and previous researches. In result, the punching shear strength of the void system is determined as the least value calculated at critical section located a distance d/2 from the face of the column and the center of the void section using the effective area at critical section.

In the version of this Article originally published, the Supplementary Movies were linked to the wrong descriptions. These have now been corrected. Additionally, the authors would like to note that co-authors James P. Butler and Jeffrey J. Fredberg contributed equally to this Article; this change has now been made.

SnO2–ZnO hybrid nanofibers were fabricated by combining the electrospinning and the pulsed laser deposition methods. After calcining at 600 °C, the nanocrystalline SnO2 coated ZnO nanofibers with a random network structure were obtained. The fiber diameter and the size of SnO2 deposit were 55–80 nm and 10–15 nm, respectively. SnO2–ZnO hybrid nanofibers-based sensor exhibited very high gas response to NO2 concentration as low as 400 ppb level at 200 °C.

It is well known that PR/SET family members participate in transcriptional regulation via chromatin remodeling. PRDM10 might play an essential role in gene expression, but no such evidence has been observed so far. To assess PRDM10 expression at various stages of mouse development, we performed immunohistochemistry using available PRDM10 antibody. Embryos were obtained from three distinct developmental stages. At E8.5, PRDM10 expression was concentrated in the mesodermal and neural crest populations. As embryogenesis proceeded further to E13.5, PRMD10 expression was mainly in mesoderm-derived tissues such as somites and neural crest-derived populations such as the facial skeleton. This expression pattern was consistently maintained to the fetal growth period E16.5 and adult mouse, suggesting that PRDM10 may function in tissue differentiation. Our study revealed that PRDM10 might be a transcriptional regulator for normal tissue differentiation during mouse embryonic development. [BMB reports 2010; 43(1): 29-33].

ZnO nanofibers were electro-spun from a solution containing poly 4-vinyl phenol and Zn acetate dihydrate. The calcination process of the ZnO/PVP composite nanofibers brought forth a random network of polycrystalline wurtzite ZnO nanofibers of 30 nm to 70 nm in diameter. The electrical properties of the ZnO nanofibers were governed by the grain boundaries. To investigate possible applications of the ZnO nanofibers, their CO and NO 2 gas sensing responses are demonstrated. In particular, the SnO2-deposited ZnO nanofibers exhibit a remarkable gas sensing response to NO2 gas as low as 400 ppb. Oxide nanofibers emerge as a new proposition for oxide-based gas sensors.

Effective beam width model(EBWM) has been used for analysis of post-tensioned(PT) flat plate slab frames under lateral loads. The accuracy of this model in predicting lateral drifts and unbalanced moments strongly depends on the estimated effective stiffness of PT flat plate slabs. As moments on the slab due to lateral loads increases, cracks occur which leads to stiffness reduction in slabs. For analyzing PT flat plate slab structure under lateral loads with good precision, reduction in slab stiffness has to be accurately estimated for EBWM. For this purpose, this study collected test results of PT flat plate system conducted by former researches. And this study reduced the width of slab so that the stiffness of the EBWM converged into the lateral stiffness of each test specimens by trial and error. By conducting nonlinear regression analysis using the stiffness ratio of the reduced width of slab to the effective width of EBWM with respect to the level of slab moments, an equation for calculating stiffness reduction factor for slab is proposed. For verifying the accuracy of the proposed equation, this study compared with the test result of the PT flat plate frame. It is shown that the EBWM with the proposed equation predicts the actual stiffness of the PT specimen which varied according to the level of applied moment.

Colloidal templating and pulsed laser deposition (PLD) have been combined to fabricate arrays of ordered two-dimensional hollow ZnO cells. Based on a spin-coating method, a facile method has been developed to distribute colloidal bead in an ordered two-dimensional fashion between the fingers of inter-digital transducers. The underlying principle involved in the spin-coating method has been analyzed by obtaining Fourier transformation patterns from the arrangements of beads. The surfaces of oxide shells consist of protruding nano-crystallites, which have a wurtzite structure of ZnO. Their electrical properties were measured as a function of annealing temperature. The change in the electrical properties upon grain growth has been attributed to alteration in the fraction site availability for defect formation at the grain boundaries.

Genetic diversity of the chrysovirus within the four fungal strains was analyzed by comparing the full-length sequences of cloned chrysoviral genes encoding the RNA-dependent RNA polymerase (RdRp) and capsid protein (CP). Because the morphological characteristics of four chrysovirus-infected Cryphonectria spp. strains were different, strain identification was conducted via sequence comparison of the internal transcribed spacers (ITSs) of the fungal rRNA gene. Phylogenic analysis of the ITS regions revealed that the four strains were closely clustered with the reference strain of Cryphonectria nitschkei, while they were more distantly related to other common Cryphonectria species, indicating that they were likely C. nitschkei. Sequence comparison among chrysoviruses from Korean C. nitschkei strains revealed that similarities of the RdRp and CP genes ranged from 98% to 100% and from 95% to 100%, respectively, at the protein level. Their corresponding nucleotide sequences showed 97% to 100% and 84% to 100% identities, respectively. Compared to RdRp, the CP gene had more divergence, suggesting the presence of genes possessing different evolutionary rates within the chrysovirus genome. Sequence comparisons with other known chrysoviruses showed that the four Korean chrysoviruses were clustered together at the next lineage level. Discovering why two strains (bsl31 and bsl32) containing identical ITS sequences and chrysoviruses display different phenotypes should prove interesting.

Colloidal templating and pulsed laser deposition (PLD) have been combined to fabricate arrays of ordered two-dimensional hollow ZnO shells. Templates were formed by spin-coating colloidal solutions containing monodispersed polymethylmethacrylate (PMMA) beads. The optimum condition for forming templates of ordered two-dimensional geometry was deduced by calculating the radial distribution functions. On templates ZnO films were deposited by a PLD method. Upon annealing ZnO films crystallize into a wurtzite structure. The surface morphology of hollow ZnO shells consists of protruding columnar nano-crystallites with no distinct ZnO texture. The electrical properties were measured as a function of annealing temperature. The electrical conductivity increases with increase in annealing temperature. The activation energy was estimated to be 0.7 eV. The change in the electrical properties upon grain growth has been attributed to alteration in the fraction site availability for defect formation at the grain boundaries.

ZnO–NiO mixed oxide nanofibers have been successfully produced from a solution containing poly 4-vinyl phenol and Zn and Ni acetic acids by an electrospinning method. The calcination process of the ZnO–NiO/PVP composite nanofibers brings forth polycrystalline two-phase ZnO–NiO nanofibers of 30–50 nm in diameters. ZnO and NiO crystallize into wurtzite and rock salt structures, respectively. The electrical conductivity of ZnO–NiO nanofibers increases with an increase in temperature, which has been attributed to the thermal emission of carrier from the energy state of the grain boundaries. The activation was estimated to be 0.5 eV. The capacitance of ZnO–NiO nanofibers upon NO x gas exposure was observed to decrease. The feasibility of producing ZnO–NiO mixed oxide nanofibers opens a new door for capacitive gas sensors.

Colloidal solutions containing polymer beads of 230 nm in diameter have been spin-coated to investigate the effect of shear on the arrangements of beads. Based on Fourier transformations and radial distribution functions analyses, a diagram, which shows the arrangement of beads as a function of rpm, has been constructed. We find that, in a certain range of spin-coating speed, the shear stress has an effect of inducing ordering into the colloidal system. Phenomenologically, in the ordering induced range, the Reynold number is understood as having the attribute of the supercooling.

The effect of Al2O3 on the electrical properties of ZnO-Pr6O11-based ceramics is investigated in this work. The average grain size of ZnO increased as the Al2O3 content increased from 10.3 to 13.5 μm. It was found that a sample doped with Al2O3 of 0.005 mol% showed the highest nonlinear current–voltage characteristics with a nonlinear exponent of 43.8 and a leakage current of 0.66 μA. When the Al2O3 content was increased, the donor concentration was increased from 0.51×1018/cm3 to 1.59×1018/cm3, but the barrier height was decreased from 1.01 to 0.87 eV. The best electrical stability against aging stress was obtained by doping Al2O3 of 0.001 mol%.

Mucus hypersecretion is a major pathophysiologic feature of chronic bronchitis. Although mucus functions as a barrier and a facilitator of mucociliary clearance, persistent mucus hypersecretion results in airway obstruction and compromised clearance of inhaled bacteria and particles from the airways of patients with chronic obstructive pulmonary disease. Treatment of mucus hypersecretion is a major therapeutic target; however, mechanisms of mucus hypersecretion remain unknown. Herein, we present evidence that human neutrophil elastase (HNE), a pathophysiologically relevant stimulant of mucus hypersecretion in the airways of patients with chronic bronchitis, provokes release of mucin (the glycoprotein component of mucus) by human airway epithelial cells in vitro. Signaling molecules involved in HNE-induced mucin hypersecretion include protein kinase C, specifically the delta isoform, and the myristoylated alanine-rich C kinase substrate protein. These molecules represent potential therapeutic targets for r...

As do all things in biology, cell mechanosensation, adhesion and migration begin at the scale of the molecule. Collections of molecules assemble to comprise microscale objects such as adhesions, organelles and cells. And collections of cells in turn assemble to comprise macroscale tissues. From the points of view of mechanism and causality, events at the molecular scale are seen most often as being the most upstream and, therefore, the most fundamental and the most important. In certain collective systems, by contrast, events at many scales of length conspire to make contributions of equal importance, and even interact directly and strongly across disparate scales. Here we highlight recent examples in cellular mechanosensing and collective cellular migration where physics at some scale bigger than the cell but smaller than the tissue – the mesoscale – becomes the missing link that is required to tie together findings that might otherwise seem counterintuitive or even unpredictable. These examples, taken together, establish that the phenotypes and the underlying physics of collective cellular migration are far richer than previously anticipated.

Asthma is characterized by chronic inflammation, airway hyperresponsiveness, and progressive airway remodeling. The airway epithelium is known to play a critical role in the initiation and perpetuation of these processes. Here, we review how excessive epithelial stress generated by bronchoconstriction is sufficient to induce airway remodeling, even in the absence of inflammatory cells.

Despite excellent initial clinical responses of non-small cell lung cancer (NSCLC) patients to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), many patients eventually develop resistance. According to a recent report, vacuolar H+ ATPase (vATPase) is overexpressed and is associated with chemotherapy drug resistance in NSCLC. We investigated the combined effects of EGFR TKIs and vATPase inhibitors and their underlying mechanisms in the regulation of NSCLC cell death. We found that combined treatment with EGFR TKIs (erlotinib, gefitinib, or lapatinib) and vATPase inhibitors (bafilomycin A1 or concanamycin A) enhanced synergistic cell death compared to treatments with each drug alone. Treatment with bafilomycin A1 or concanamycin A led to the induction of Bnip3 expression in an Hif-1α dependent manner. Knock-down of Hif-1α or Bnip3 by siRNA further enhanced cell death induced by bafilomycin A1, suggesting that Hif-1α/Bnip3 induction promoted resistance to cell death induced by the vATPase inhibitors. EGFR TKIs suppressed Hif-1α and Bnip3 expression induced by the vATPase inhibitors, suggesting that they enhanced the sensitivity of the cells to these inhibitors by decreasing Hif-1α/Bnip3 expression. Taken together, we conclude that EGFR TKIs enhance the sensitivity of NSCLC cells to vATPase inhibitors by decreasing Hif-1α/Bnip3 expression. We suggest that combined treatment with EGFR TKIs and vATPase inhibitors is potentially effective for the treatment of NSCLC.

A disposable cyclic voltammetry (CV) tag is printed on a plastic film by integrating wireless power transmitter, polarized triangle wave generator, electrochemical cell and signage through a scalable gravure printing method. By proximity of 13.56 MHz RF reader, the printed CV tag generates 320 mHz of triangular sweep wave from +500 mV to −500 mV which enable to scan a printed electrochemical cell in the CV tag. By simply dropping any specimen solution on the electrochemical cell in the CV tag, the presence of solutes in the solution can be detected and shown on the signage of the CV tag in five sec. 10 mM of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) was used as a standard solute to prove the working concept of fully printed disposable wireless CV tag. Within five seconds, we can wirelessly diagnose the presence of TMPD in the solution using the CV tag in the proximity of the 13.56 MHz RF reader. This fully printed and wirelessly operated flexible CV tag is the first of its kind and marks the path for the utilization of inexpensive and disposable wireless electrochemical sensor systems for initial diagnose hazardous chemicals and biological molecules to improve public hygiene and health.

The presence of mucus obstruction and neutrophil-predominant inflammation in several lung disorders, such as cystic fibrosis, suggests a relationship between neutrophils and excess mucus production. Mechanisms of human neutrophil elastase (HNE)-induced mucin secretion by well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air/liquid interface culture were investigated. HNE increased mucin secretion in a concentration-dependent manner, with maximal stimulation (more than twofold) occurring within a short (15 minutes) time period. Mucins MUC 5 AC and MUC 5 B, but not MUC 2, were released in response to HNE. Stimulation of mucin secretion required partial elastase enzymatic activity and did not appear to involve a soluble product released by the cells. HNE-stimulated secretion involved activation of protein kinase C (PKC), as HNE exposure rapidly provoked PKC enzymatic activity that was attenuated by the general PKC inhibitors calphostin C and bisindoylmaleimide I. Of the different isoforms, PKCalpha, delta, zeta, lambda, iota, and epsilon were constitutively expressed in NHBE cells while PKCbeta, eta, and mu were PMA-inducible. PKCdelta was the only isoform to translocate from cytoplasm to membrane in response to HNE. Inhibition of PKCdelta attenuated HNE-mediated mucin secretion. The results suggest HNE stimulation of mucin release by human airway epithelial cells involves intracellular activation of PKC, specifically the delta isoform.

Piperlongumine, a natural alkaloid isolated from the long pepper, selectively increases reactive oxygen species production and apoptotic cell death in cancer cells but not in normal cells. However, the molecular mechanism underlying piperlongumine-induced selective killing of cancer cells remains unclear. In the present study, we observed that human breast cancer MCF-7 cells are sensitive to piperlongumine-induced apoptosis relative to human MCF-10A breast epithelial cells. Interestingly, this opposing effect of piperlongumine appears to be mediated by heme oxygenase-1 (HO-1). Piperlongumine upregulated HO-1 expression through the activation of nuclear factor-erythroid-2-related factor-2 (Nrf2) signaling in both MCF-7 and MCF-10A cells. However, knockdown of HO-1 expression and pharmacological inhibition of its activity abolished the ability of piperlongumine to induce apoptosis in MCF-7 cells, whereas those promoted apoptosis in MCF-10A cells, indicating that HO-1 has anti-tumor functions in cancer cells but cytoprotective functions in normal cells. Moreover, it was found that piperlongumine-induced Nrf2 activation, HO-1 expression and cancer cell apoptosis are not dependent on the generation of reactive oxygen species. Instead, piperlongumine, which bears electrophilic α,β-unsaturated carbonyl groups, appears to inactivate Kelch-like ECH-associated protein-1 (Keap1) through thiol modification, thereby activating the Nrf2/HO-1 pathway and subsequently upregulating HO-1 expression, which accounts for piperlongumine-induced apoptosis in cancer cells. Taken together, these findings suggest that direct interaction of piperlongumine with Keap1 leads to the upregulation of Nrf2-mediated HO-1 expression, and HO-1 determines the differential response of breast normal cells and cancer cells to piperlongumine.

For the first time, fully gravure printed wireless cyclic voltammetry (CV) tag is reported as a platform for the disposable wireless sensor tags by utilizing Ag nano-particle based conducting ink, BaTiO 3 nano-particle based dielectric ink and single-walled carbon nanotube based semiconducting ink. The printed CV tag contains four functional units for the disposable wireless sensor tag: the rectenna to generate polarized DC volt through coupled 13.56 MHz AC, triangle wave generator to scan electrochemical cell by ±500 mV at lower frequency (< 500 mHz), amplifier to enhance the output signal and signage to indicate detected level of specimens. Those printed four units will be used as a platform to further develop disposable wireless sensor tags by simply replacing sensor unit.

Extraction of high-quality RNA is a crucial step in gene expression profiling. To achieve optimal RNA extraction from frozen blood, the performance of three RNA extraction kits- TRI reagent, PAXgene blood RNA system (PAXgene) and NucleoSpin RNA blood kit (NucleoSpin)- was evaluated. Fifteen blood specimens collected in tubes containing potassium ethylenediaminetetraacetic acid (EDTA) and stored at −80°C for approximately 5 years were randomly selected. The yield and purity of RNA, RIN (RNA integrity number) values and cycle threshold (Ct) values were assessed. Mean RNA yields with TRI reagent, PAXgene and NucleoSpin were 15.6 ± 8.7 μg/ml, 3.1 ± 1.7 μg/ml and 9.0 ± 5.5 μg/ml, respectively. Mean A260/280 ratios of RNA for the three kits were 1.7 ± 0.1, 2.0 ± 0.1, and 2.0 ± 0.0, and mean RIN values recorded as 3.2 ± 0.8, 6.0 ± 1.1, and 6.4 ± 0.9, respectively. The Ct values of housekeeping genes, 18S rRNA, β-actin, RPLP0 and HPRT1, were as follows: TRI reagent (19.2 ± 1.6, 30.6 ± 1.8, 29.9 ± 1.4 and 36.3 ± 1.3), PAXgene 16.6 ± 1.4, 26.4 ± 1.3, 28.2 ± 1.8 and 33.8 ± 1.1), and NucleoSpin (16.3 ± 1.5, 27.2 ± 1.3, 27.0 ± 1.6 and 32.9 ± 1.6). RNA yield using TRI reagent was 1.7 times higher than that with NucleoSpin and 5 times higher than that with PAXgene. However, the purity and integrity of TRI-extracted RNA was lower than that extracted with PAXgene and NucleoSpin. Moreover, the Ct values of housekeeping genes after extraction with TRI reagent were approximately 1.7-3.8 times higher than those obtained with PAXgene and NucleoSpin. The PAXgene and NucleoSpin kits produced similar results in terms of RNA purity and integrity and subsequent gene amplification. However, RNA yields from NucleoSpin were 2.9-fold higher, compared to PAXgene. Based on these findings, we conclude that NucleoSpin is the most effective kit for extraction of abundant and high-quality RNA from frozen blood.

The PI3K/Akt/mTOR axis in lung cancer is frequently activated and implicated in tumorigenesis. Specific targeting of this pathway is therefore an attractive therapeutic approach for lung cancer. However, non-small cell lung cancer cells are resistant to BEZ235, a dual inhibitor of PI3K and mTOR. Interestingly, blockage of Stat3 with a selective inhibitor, S3I-201, or siRNA dramatically sensitized the BEZ235-induced cell death, as evident from increased PARP cleavage. Furthermore, inhibition of Stat3 led to enhancement of cell death induced by LY294002, a PI3K inhibitor. Treatment of cells with a combination of BEZ235 and S3I-201 significantly induced the proapoptotic transcription factor, CHOP, and its targets, Bim and DR4. Knockdown of CHOP or Bim suppressed cell death stimulated by the combination treatment, implicating the involvement of these BEZ235/S3I-201-induced factors in pronounced cell death. Moreover, the BEZ235/S3I-201 combination enhanced TRAIL-induced cell death. Our results collectively suggest that blockage of Stat3 presents an effective strategy to overcome resistance to PI3K/Akt/mTOR inhibition.

The near full-length genome consisting of four segments of dsRNA from a chrysovirus infecting Korean Cryphonectria nitschkei BS122 strain (CnV1-BS122) was sequenced. The open reading frames of segments 1, 2, 3, and 4 were 2,889, 2,721, 2,475, and 2,232 nucleotides (nt) in length, respectively. Sequence analysis and homology searches of the amino acid sequences deduced from the ORFs of each segment revealed that segments 1, 2, 3, and 4 encoded RNA-dependent RNA polymerase, capsid protein, a putative cysteine protease, and replication-associated protein, respectively. The entire 5' ends of segments 1, 2, and 4 were 82, 242, and 698 nt in length, respectively; the sequence of the 5' end of segment 3 was not determined because of difficulty in amplification. The entire 3' end of segment 3 was 77 nt in length. Partial amplification of the 3' ends of segments 1, 2, and 4 yielded amplimers of 7, 17, and 30 nt, respectively.

Titanium dioxide (TiO2) nanofibers were fabricated by electrospinning a hybrid solution, which is a mixture of the TiO2 sol precursor, polymer, and solvent. The structure and gas sensing properties of TiO2 nanofibers were investigated. By calcining at 600 °C, the polymeric components were decomposed and a multi-layered random network structure of TiO2 nanofibers was obtained. Polycrystalline TiO2 nanofibers consist of tetragonal anatase and rutile TiO2 phases. The diameter ranged from 400 nm to 500 nm and the grain size was about 15 nm. The TiO2 nanofibers-based sensor exhibited response to CO concentration as low as 1 ppm at 200 °C.

Well ordered monolayer arrays of hollow SnO 2 shell were fabricated by combining colloidal templating and pulsed laser deposition methods. With increasing the thickness of SnO 2 hollow films, their half-spherical morphologies gradually develop to a bell-shaped one. While the surface of SnO 2 thin films is fairly flat, the surface of hollow SnO 2 shells consists of protruding crystals, indicating morphological dependency on the presence of substrate. Because of the low population of contiguous hollow SnO 2 shells, arrays of hollow SnO 2 showed higher dc electrical resistance than those of thin films.

Electrical modeling of the chemical gas sensors was successfully applied to TiO2 nanofiber gas sensors by developing an equivalent circuit model where the junction capacitance as well as the resistance can be separated from the comparable stray capacitance. The Schottky junction impedance exhibited a characteristic skewed arc described by a Cole–Davidson function, and the variation of the fit and derived parameters with temperature, bias, and NO2 gas concentration indicated definitely a physicochemical sensing mechanism based on the Pt|TiO2 Schottky junctions against the conventional supposition of the enhanced sensitivity in nanostructured gas sensors with high grain boundary/surface area. Analysis on a model Pt|TiO2|Pt structure also confirmed the characteristic impedance response of TiO2 nanofiber sensors.