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Epithelial-to-mesenchymal transition results in loss of specialized epithelial cell contacts and acquisition of mesenchymal invasive capacity. The transcription repressor zinc finger E-box-binding homeobox 1 (ZEB1) binds to E-boxes of gene promoter regions to suppress the expression of epithelial genes. ZEB1 has inconsistent molecular weights, which have been attributed to posttranslational modifications (PTM). We performed mass spectrometry and identified K811 acetylation as a novel PTM in ZEB1. To define the role of ZEB1 acetylation in regulating function, we generated ZEB1 acetyl-mimetic (K811Q) and acetyl-deficient (K811R) mutant-expressing non–small cell lung cancer cell lines (NSCLC). We demonstrate that the K811R ZEB1 (125 kDa) has a shorter protein half-life than wild-type (WT) ZEB1 and K811Q ZEB1 (∼225 kDa), suggesting that lack of ZEB1 acetylation in the lower molecular weight form affects protein stability. Further, the acetylated form of ZEB1 recruits the nucleosome remodeling and deacetylase (NuRD) complex to bind the promoter of its target genes mir200c-141 and SEMA3F. RNA-sequencing revealed that WT ZEB1 and K811Q ZEB1 downregulate the expression of epithelial genes to promote lung adenocarcinoma invasion and metastasis, whereas the K811R ZEB1 does not. Our findings establish that the K811 acetylation promotes ZEB1 protein stability, interaction with other protein complexes, and subsequent invasion/metastasis of lung adenocarcinoma via epithelial-to-mesenchymal transition. Implications: The molecular mechanisms by which ZEB1 is regulated by K811 acetylation to promote protein stability, NuRD complex and promoter interactions, and function are relevant to the development of treatment strategies to prevent and treat metastasis in patients with NSCLC.

One of the mechanisms by which cancer cells acquire hyperinvasive and migratory properties with progressive loss of epithelial markers is the epithelial-to-mesenchymal transition (EMT). We have previously reported that in different cancer types, including nonsmall cell lung cancer (NSCLC), the microRNA-183/96/182 cluster (m96cl) is highly repressed in cells that have undergone EMT. In the present study, we used a novel conditional m96cl mouse to establish that loss of m96cl accelerated the growth of Kras mutant autochthonous lung adenocarcinomas. In contrast, ectopic expression of the m96cl in NSCLC cells results in a robust suppression of migration and invasion in vitro, and tumor growth and metastasis in vivo. Detailed immune profiling of the tumors revealed a significant enrichment of activated CD8+ cytotoxic T lymphocytes (CD8+ CTLs) in m96cl-expressing tumors, and m96cl-mediated suppression of tumor growth and metastasis was CD8+ CTL-dependent. Using coculture assays with naïve immune cells, we show that m96cl expression drives paracrine stimulation of CD8+ CTL proliferation and function. Using tumor microenvironment-associated gene expression profiling, we identified that m96cl elevates the interleukin-2 (IL2) signaling pathway and results in increased IL2-mediated paracrine stimulation of CD8+ CTLs. Furthermore, we identified that the m96cl modulates the expression of IL2 in cancer cells by regulating the expression of transcriptional repressors Foxf2 and Zeb1, and thereby alters the levels of secreted IL2 in the tumor microenvironment. Last, we show that in vivo depletion of IL2 abrogates m96cl-mediated activation of CD8+ CTLs and results in loss of metastatic suppression. Therefore, we have identified a novel mechanistic role of the m96cl in the suppression of lung cancer growth and metastasis by inducing an IL2-mediated systemic CD8+ CTL immune response.

Potential ZEB1 acetylation-dependent target genes.

qPCR Primer Sequences

Antibody information

S1. K811 acetylation is integral to ZEB1 molecular weight. S2. &tilde225 kDa ZEB1 band regulates protein stability. S3. K811 acetylation facilitates ZEB1/NuRD interaction. S4. K811 acetylation directs the ZEB1/NuRD complex to bind at genomic promoters. S5. K811 acetylation decreased epithelial expression levels. S6. RNA-seq reveals K811 acetylation downregulates the expression of epithelial genes. S7. K811 acetylation in ZEB1 KO cells promotes lung adenocarcinoma migration and invasion. S8. K811 acetylation enhances metastatic potential of murine NSCLC cells.

Epithelial-to-mesenchymal transition results in loss of specialized epithelial cell contacts and acquisition of mesenchymal invasive capacity. The transcription repressor zinc finger E-box-binding homeobox 1 (ZEB1) binds to E-boxes of gene promoter regions to suppress the expression of epithelial genes. ZEB1 has inconsistent molecular weights, which have been attributed to posttranslational modifications (PTM). We performed mass spectrometry and identified K811 acetylation as a novel PTM in ZEB1. To define the role of ZEB1 acetylation in regulating function, we generated ZEB1 acetyl-mimetic (K811Q) and acetyl-deficient (K811R) mutant-expressing non–small cell lung cancer cell lines (NSCLC). We demonstrate that the K811R ZEB1 (125 kDa) has a shorter protein half-life than wild-type (WT) ZEB1 and K811Q ZEB1 (∼225 kDa), suggesting that lack of ZEB1 acetylation in the lower molecular weight form affects protein stability. Further, the acetylated form of ZEB1 recruits the nucleosome remodeling and deacetylase (NuRD) complex to bind the promoter of its target genes mir200c-141 and SEMA3F. RNA-sequencing revealed that WT ZEB1 and K811Q ZEB1 downregulate the expression of epithelial genes to promote lung adenocarcinoma invasion and metastasis, whereas the K811R ZEB1 does not. Our findings establish that the K811 acetylation promotes ZEB1 protein stability, interaction with other protein complexes, and subsequent invasion/metastasis of lung adenocarcinoma via epithelial-to-mesenchymal transition.Implications:The molecular mechanisms by which ZEB1 is regulated by K811 acetylation to promote protein stability, NuRD complex and promoter interactions, and function are relevant to the development of treatment strategies to prevent and treat metastasis in patients with NSCLC.

Protein expression values of the immunoprecipitated samples

ChIP Primer Sequences

Summary Non-small cell lung cancer (NSCLC) is the deadliest form of cancer worldwide, due in part to its proclivity to metastasize. Identifying novel drivers of invasion and metastasis holds therapeutic potential for the disease. We conducted a gain-of-function invasion screen, which identified two separate hits, IMPAD1 and KDELR2, as robust, independent drivers of lung cancer invasion and metastasis. Given that IMPAD1 and KDELR2 are known to be localized to the ER-Golgi pathway, we studied their common mechanism of driving in vitro invasion and in vivo metastasis and demonstrated that they enhance Golgi-mediated function and secretion. Therapeutically inhibiting matrix metalloproteases (MMPs) suppressed both IMPAD1- and KDELR2-mediated invasion. The hits from this unbiased screen and the mechanistic validation highlight Golgi function as one of the key cellular features altered during invasion and metastasis.

One of the mechanisms by which cancer cells acquire hyperinvasive and migratory properties with progressive loss of epithelial markers is the epithelial-to-mesenchymal transition (EMT). We have previously reported that in different cancer types, including nonsmall cell lung cancer (NSCLC), the microRNA-183/96/182 cluster (m96cl) is highly repressed in cells that have undergone EMT. In the present study, we used a novel conditional m96cl mouse to establish that loss of m96cl accelerated the growth of Kras mutant autochthonous lung adenocarcinomas. In contrast, ectopic expression of the m96cl in NSCLC cells results in a robust suppression of migration and invasion in vitro, and tumor growth and metastasis in vivo. Detailed immune profiling of the tumors revealed a significant enrichment of activated CD8

The strong diffusion of Fiber Reinforced Cementitious Matrix (FRCM) composites for the reinforcement of historic masonry constructions requires suitable experimental techniques for assessing possible defects in the adhesion between FRCM and masonry, and between FRCM layers. To this aim, in this paper, an innovative nonlinear ultrasonic approach based on the Side-band Peak Count (SPC) technique is proposed. This approach is discussed and validated through experimental tests made on tuff substrates reinforced with FRCM mortars embedding a basalt fibers grid and having known artificial defects at the adhesion between tuff and FRCM as well as in the fiber grid

Lung cancer is a highly aggressive and metastatic disease responsible for approximately 25% of all cancer-related deaths in the United States. Using high-throughput in vitro and in vivo screens, we have previously established Impad1 as a driver of lung cancer invasion and metastasis. Here we elucidate that Impad1 is a direct target of the epithelial microRNAs (miRNAs) miR-200 and miR∼96 and is de-repressed during epithelial-to-mesenchymal transition (EMT); thus, we establish a mode of regulation of the protein. Impad1 modulates Golgi apparatus morphology and vesicular trafficking through its interaction with a trafficking protein, Syt11. These changes in Golgi apparatus dynamics alter the extracellular matrix and the tumor microenvironment (TME) to promote invasion and metastasis. Inhibiting Impad1 or Syt11 disrupts the cancer cell secretome, regulates the TME, and reverses the invasive or metastatic phenotype. This work identifies Impad1 as a regulator of EMT and secretome-mediated changes during lung cancer progression.

Background: SARS-CoV-2 infection is the cause of the respiratory illness COVID-19, which presents most frequently with respiratory symptoms SARS-CoV-2 cell entry requires interactions with ACE2 and TMPRSS2 on the surface of the host cell Cancer patients and, specifically, those with thoracic malignancies seem to experience poorer clinical outcomes Methods: We utilized bulk and single-cell transcriptional data from a combination of normal and malignant tissues and cells from aerodigestive and respiratory tracts to explore mechanisms governing the expression of ACE2 and TMPRSS2 Additionally, we determined the effect of EMT induction, ZEB1 modulation, and SARS-CoV-2 infection on ACE2 expression Results: Our bulk data suggests that aerodigestive and lung cancer models express a broad range of ACE2 and TMRPSS2, particularly in epithelial cells, and would serve as good models for studying SARS-CoV-2 infection We assessed the relationship between ACE2 and epithelial differentiation in numerous datasets, and found consistent positive correlations with transcriptional and microRNA signifiers of epithelial differentiation The miR-200 family – zinc finger E-box-binding homeobox 1 (ZEB1) pathway, which is an established regulator of EMT, also directly regulates ACE2 expression, likely via putative ZEB1 repressor sites located in the ACE2 promoter Furthermore, SARS-CoV-2 infection reduces ACE2 expression and shifts cells to a more mesenchymal phenotype with loss of EPCAM and upregulation of ZEB1 and other EMT-associated genes Conclusions: ACE2-positive cells are almost exclusively epithelial and unexpectedly rare, considering the devastating impact of this infection Following viral entry, SARS-CoV-2 infection induces molecular changes within the cells that are reminiscent of EMT, including increased ZEB1 ZEB1, in turn, appears to directly repress the expression of ACE2 This SARS-CoV-2-induced ACE2 deficiency, compounded by the downregulation of genes, including claudins, which play a critical role in restricting epithelial and endothelial permeability, exposes respiratory cells to increased risk of edema and acute respiratory distress syndrome (ARDS) Legal entity responsible for the study: The authors Funding: NIH/NCI R01-CA207295 (L A B ), NIH/NCI U01-CA213273 (L A B , J V H ), CCSG P30-CA01667 (L A B ), University of Texas SPORE in Lung Cancer P5-CA070907 (L A B , D L G , J V H , C M G ), the Department of Defense (LC170171;L A B ), Khalifa Bin Zayed Al Nahyan Foundation (C M G ), RP170067 (EMP), through generous philanthropic contributions to The University of Texas MD Anderson Lung Cancer Moon Shot Program and Andrew Sabin Family Fellowship, and The Rexanna Foundation for Fighting Lung Cancer Disclosure: C Gay: Research grant/Funding (self): Astra Zeneca J V Heymach: Advisory/Consultancy: AstraZeneca;Advisory/Consultancy: Boehringer Ingelheim;Advisory/Consultancy: Exelixis;Advisory/Consultancy: Genentech;Advisory/Consultancy: GlaxoSmithKline;Advisory/Consultancy: Guardant Health;Advisory/Consultancy: Hengrui;Advisory/Consultancy: Spectrum L A Byers: Advisory/Consultancy, Research grant/Funding (self): AstraZeneca;Advisory/Consultancy, Research grant/Funding (self): AbbVie;Advisory/Consultancy, Research grant/Funding (self): GenMab;Advisory/Consultancy: BergenBio;Advisory/Consultancy: Pharma Mar SA;Advisory/Consultancy, Research grant/Funding (self): Sierra Oncology;Advisory/Consultancy: Merck;Advisory/Consultancy: Bristol Myers Squibb;Advisory/Consultancy: Genentech;Advisory/Consultancy: Pfizer;Research grant/Funding (self): Tolero Pharmaceuticals All other authors have declared no conflicts of interest

Genetic aberrations driving pro-oncogenic and pro-metastatic activity remain an elusive target in the quest of precision oncology. To identify such drivers, we use an animal model of KRAS-mutant lung adenocarcinoma to perform an in vivo functional screen of 217 genetic aberrations selected from lung cancer genomics datasets. We identify 28 genes whose expression promoted tumor metastasis to the lung in mice. We employ two tools for examining the KRAS-dependence of genes identified from our screen: 1) a human lung cell model containing a regulatable mutant KRAS allele and 2) a lentiviral system permitting co-expression of DNA-barcoded cDNAs with Cre recombinase to activate a mutant KRAS allele in the lungs of mice. Mechanistic evaluation of one gene, GATAD2B, illuminates its role as a dual activity gene, promoting both pro-tumorigenic and pro-metastatic activities in KRAS-mutant lung cancer through interaction with c-MYC and hyperactivation of the c-MYC pathway., KRAS-driven lung cancers represent an aggressive form of NSCLC. In this study the authors perform an in vivo functional screening and identify GATAD2B as a driver of tumor growth and metastasis in KRAS-driven lung cancer.

Metastasis is the cause for 90% of cancer-related mortalities. Identification of genetic drivers promoting dissemination of tumor cells may provide opportunities for novel therapeutic strategies. We previously reported an in vivo gain-of-function screen that identified ∼30 genes with a functional role in metastasis promotion and characterized detailed mechanistic functions of two hits. In the present study, we characterized the contribution of one of the identified genes, MBIP (MAP3K12 binding inhibitory protein), towards driving tumor invasion and metastasis. We demonstrate that expression of MBIP significantly enhances the cellular proliferation, migration and invasion of NSCLC cells in vitro and metastasis in vivo. We functionally characterized that MBIP mediates activation of the JNK pathway and induces expression of matrix metalloproteinases (MMPs), which are necessary for the invasive and metastatic phenotype. Our findings establish a novel mechanistic role of MBIP as a driver of NSCLC progression and metastasis.

Mitogen-activated protein kinase kinase (MEK) inhibitors have failed to show clinical benefit in Kirsten rat sarcoma (KRAS)-mutant lung cancer due to various resistance mechanisms. To identify differential therapeutic sensitivities between epithelial and mesenchymal lung tumors, we performed in vivo small hairpin RNA (shRNA) screens, proteomic profiling, and analysis of patient tumor datasets, which revealed an inverse correlation between mitogen-activated protein kinase (MAPK) signaling dependency and a zinc finger E-box-binding homeobox 1 (ZEB1)-regulated epithelial-to-mesenchymal transition (EMT). Mechanistic studies determined that MAPK signaling dependency in epithelial lung cancer cells is due to the scaffold protein interleukin 17 receptor D (IL17RD), which is directly repressed by ZEB1. Lung tumors in multiple Kras mutant murine models with increased ZEB1 displayed low IL17RD expression, accompanied by MAPK-independent tumor growth and therapeutic resistance to MEK inhibition. Suppression of ZEB1 function with miR-200 expression or the histone deacetylase (HDAC) inhibitor mocetinostat sensitized resistant cancer cells to MEK inhibition and markedly reduced in vivo tumor growth, showing a promising combinatorial treatment strategy for KRAS-mutant cancers. In human lung tumor samples, high ZEB1 and low IL17RD expression correlated with low MAPK signaling, presenting potential markers that predict patient response to MEK inhibitors.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, due in part to the propensity of lung cancer to metastasize. Aberrant epithelial-to-mesenchymal transition (EMT) is a proposed model for the initiation of metastasis. During EMT cell-cell adhesion is reduced allowing cells to dissociate and invade. Of the EMT-associated transcription factors, ZEB1 uniquely promotes NSCLC disease progression. Here we apply two independent screens, BioID and an Epigenome shRNA dropout screen, to define ZEB1 interactors that are critical to metastatic NSCLC. We identify the NuRD complex as a ZEB1 co-repressor and the Rab22 GTPase-activating protein TBC1D2b as a ZEB1/NuRD complex target. We find that TBC1D2b suppresses E-cadherin internalization, thus hindering cancer cell invasion and metastasis., Non-small cell lung cancer (NSCLC) is often associated with metastasis to the lungs. Here, the authors perform independent screens and identify NuRD as a co-repressor of ZEB1, and demonstrate TBC1D2b as a downstream target of ZEB1/NuRD complex regulating NSCLC metastasis.

// C. Allison Stewart 1 , Pan Tong 2 , Robert J. Cardnell 1 , Triparna Sen 1 , Lerong Li 2 , Carl M. Gay 1 , Fatemah Masrorpour 1 , You Fan 1 , Rasha O. Bara 1 , Ying Feng 3 , Yuanbin Ru 3 , Junya Fujimoto 4 , Samrat T. Kundu 1 , Leonard E. Post 3 , Karen Yu 3 , Yuqiao Shen 3 , Bonnie S. Glisson 1 , Ignacio Wistuba 4 , John V. Heymach 1 , Don L. Gibbons 1 , Jing Wang 2 and Lauren Averett Byers 1 1 Department of Thoracic Head & Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 2 Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 3 BioMarin Pharmaceutical, San Rafael, CA 94901, USA 4 Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA Correspondence to: Lauren Averett Byers, email: lbyers@mdanderson.org Keywords: SCLC, SLFN11, ATM, EMT, PARP inhibitor Received: December 10, 2016 Accepted: January 19, 2017 Published: February 15, 2017 ABSTRACT Small cell lung cancer (SCLC) is one of the most aggressive forms of cancer, with a 5-year survival

Metastatic lung cancer is the leading cause of cancer-associated mortality worldwide, therefore necessitating novel approaches to identify specific genetic drivers for lung cancer progression and metastasis. We recently performed an in vivo gain-of-function genetic screen to identify driver genes of lung cancer metastasis. In the study reported here, we identify TMEM106B as a primary robust driver of lung cancer metastasis. Ectopic expression of TMEM106B could significantly promote the synthesis of enlarged vesicular lysosomes that are laden with elevated levels of active cathepsins. In a TFEB-dependent manner, TMEM106B could modulate the expression of lysosomal genes of the coordinated lysosomal expression and regulation (CLEAR) pathway in lung cancer cells and patient samples. We also demonstrate that TMEM106B-induced lysosomes undergo calcium-dependent exocytosis, thereby releasing active lysosomal cathepsins necessary for TMEM106B-mediated cancer cell invasion and metastasis in vivo, which could be therapeutically prevented by pharmacological inhibition of cathepsins. Further, in TCGA LUAD data sets, 19% of patients show elevated expression of TMEM106B, which predicts for poor disease-free and overall-survival., One of the major causes of cancer-related mortality is represented by metastatic lung cancer. Here the authors characterize the role of TMEM106B in driving metastatic lung adenocarcinoma and suggest that TMEM106B-mediated secretion of cathespin impacts cell migration and invasion of lung cancer cells, increasing metastatic spreading.

Metastatic lung cancer is one of the most lethal forms of cancer and molecular pathways driving metastasis are still not clearly elucidated. Metastatic cancer cells undergo an epithelial-mesenchymal transition (EMT) where they lose their epithelial properties and acquire a migratory and invasive phenotype. Here we identify that expression of microRNAs from the miR-200 family and the miR-183~96~182 cluster are significantly co-repressed in non-small cell lung cancer (NSCLC) cell lines and primary tumors from multiple TCGA data sets with high EMT scores. Ectopic expression of the miR-183~96~182 cluster inhibited cancer cell migration and invasion, while its expression was tightly modulated by miR-200. We identified Foxf2 as a common, novel and direct target of both these microRNA families. Foxf2 expression tightly correlates with the transcription factor Zeb1 and is elevated in mesenchymal-like metastatic lung cancer cells. Foxf2 expression induced robust EMT, migration, invasion and metastasis in lung cancer cells, whereas Foxf2 inhibition significantly repressed these phenotypes. We also demonstrated that Foxf2 transcriptionally represses E-Cadherin and miR-200, independent of Zeb1, to form a double negative feedback loop. We therefore identified a novel mechanism whereby the miR-200 family and the miR-183~96~182 cluster inhibit lung cancer invasion and metastasis by targeting Foxf2.

Dilatometric shrinkage data was utilized to study the sintering kinetics of the in-house synthesized nano-crystalline 3-mol% yttria-samaria codoped tetragonal zirconia polycrystal (TZP) ceramics. The objective was to determine activation energy (Q) of sintering and the sintering mechanism (n) relevant to the initial stage of sintering. The product of activation energy and sintering parameter, i.e., “nQ” was calculated from the shrinkage data acquired from the constant rate of heating experiment. The apparent activation energy of sintering (Q) was calculated using modified-“Dorn” method. Modified Johnson’s equation was used to determine value of “n” using the activation energy obtained from the Dorn method. Stepwise isothermal dilatometry technique was utilized as an independent method to determine the “n” value. The activation energy of sintering was in the range of 400–525 kJ mol−1 and found to be dependent on the dopant concentration. The value of “n” was found to be ~0.33 for both 3 mol% yttria-doped (3Y-TZP) and yttria-samaria codoped (3(Y,S)-TZP) TZP, whereas for 3 mol% samaria-doped tetragonal zirconia (3S-TZP), the value of “n” was ~0.40. From the obtained “n” values, it may be concluded that grain boundary diffusion (GBD) was the dominant sintering mechanism in 3Y-TZP and 3(Y,S)-TZP, whereas an intermediate of GBD and volume diffusion influences the initial sintering stage in the 3S-TZP.

We have synthesized and characterized two different nanoassemblies of polydiacetylene (PDA), PDA nanovesicles and PDA nanocrystals, in order to study the influence of chain packing geometry of the two nanoassemblies on their third order nonlinear optical properties. The second molecular hyperpolarizability γ(-ω,ω,-ω,ω）of the two samples was investigated by antiresonant ring interferometric nonlinear spectroscopic (ARINS) technique using femtosecond modelocked Ti:sapphire laser in the spectral range of 720 nm - 820 nm. The observed spectral dispersion of γ has been explained in the framework of three-essential states model involving the ground state, a one-photon excited state and a two-photon excited state. The observed optical nonlinearity has been correlated with the electronic structure of the two samples. The energy of two-photon state, transition dipole moments and line width of the transitions have been estimated. Our studies show that the arrangement of polymer chains in polydiacetylene nanocrystals is more favorable for higher nonlinearity.

Production fluid typically leaves a well in multiple phases, including: oil, gas, water, and solids. It is necessary to separate these components in order to recover the oil and gas, treat the water, dispose of sand, etc. In the past decade there has been a trend to move processing equipment to the seabed. As offshore production continues to be developed in deeper water, it is imperative to develop separator technology that will be designed for the hydrostatic forces and large amounts of produced water that is expected to occur over time. This paper explores two novel subsea linear pipe designs that allow subsea processing to be accomplished more cost effectively than existing technologies. The process undertaken for designing the two novel subsea linear pipe separation technologies: (gas-liquid) and (gas-liquid-solids) is described. To validate the efficiency and effectiveness of these two systems, a Computational Fluid Dynamics (CFD) analysis and cost analysis have been performed for each separator to provide both a realistic view of the actual separation efficiency of the systems, as well as comparative cost efficiency to existing technology in use today. Separators utilizing the pipe classification can be made lighter and cheaper when compared to existing subsea separation modules. In addition, they can be fabricated out of standard pipe sizes, thus cutting costs due to high availability and ease of fabrication. These factors are made even more attractive when modularized for ease of installation and field expansion. Applications of the process and design of these special separators are discussed along with their potential advantages in offshore deepwater seabed based developments. With continued research and testing, the gas-liquid and gas-liquid-solid linear pipe separator designs have the potential to reshape the subsea separation industry.

We have investigated the spectral dispersion of real and imaginary parts of third-order nonlinear optical susceptibility of silver nanospherical particles in the aqueous solution using Z-scan and antiresonant ring interferometric nonlinear spectroscopic (ARINS) techniques by femtosecond pulses from the modelocked Ti:Sapphire laser over the spectral range of 720-820 nm. The observed characteristic of imaginary part shows a peak at similar to 766 nm which coincides with the peak of spectral dispersion of real part induced thermally. The observed peak in the dispersion of nonlinear absorption coefficient at similar to 766 nm is attributed to the two-photon absorption. This two-photon resonance peak is attributed to l =2 plasmon (quadrupolar mode) arising from the phase retardation effects in the spherical nanoparticles present in our polydispersed sample with their diameters (80 +/- 20 nm) beyond the quasi-static limit. (C) 2012 Elsevier B.V. All rights reserved.

Structural and optical properties of the Tb doped ZnO nanoparticles are systematically studied as a function of the Tb mole-fraction. Our study suggests that the Tb incorporates mostly on the surface and affects the optical properties of the ZnO nanoparticles by influencing the attachment of certain adsorbed groups, which are found to be responsible for the appearance of a broad green luminescence (GL) band in the photoluminescence spectra recorded for these nanoparticles. It has been found that the accumulation of Tb on the surface of the nanoparticles not only enhances the band edge to green luminescence intensity ratio under the vacuum condition but also increases the band gap energy by introducing a hydrostatic compressive strain in individual nanoparticles, which provides a unique opportunity to study the pressure dependence of the optical properties of nanoparticles without applying any external pressure. The hydrostatic compressive strain is explained in terms of the increase of the surface strain energy as a result of the Tb accumulation on the surface of the nanoparticles. The average value of the surface energy density for the particles has been estimated as a function of Tb mole-fraction. The pressure coefficient of the band gap which is obtained from the variation of the band gap energy with the hydrostatic strain has been found to decrease significantly with the particle size for the ZnO nanoparticles. (C) 2011 Elsevier Ltd. All rights reserved.

Keratin expression in stratified epithelia is tightly regulated during squamous cell differentiation. Keratins 5 and 14 are expressed in mitotically active basal layer cells, but their function is not well defined. Reported here is the possible role of K14 in regulation of cell proliferation/differentiation in stratified epithelial cells., Keratins are cytoplasmic intermediate filament proteins preferentially expressed by epithelial tissues in a site-specific and differentiation-dependent manner. The complex network of keratin filaments in stratified epithelia is tightly regulated during squamous cell differentiation. Keratin 14 (K14) is expressed in mitotically active basal layer cells, along with its partner keratin 5 (K5), and their expression is down-regulated as cells differentiate. Apart from the cytoprotective functions of K14, very little is known about K14 regulatory functions, since the K14 knockout mice show postnatal lethality. In this study, K14 expression was inhibited using RNA interference in cell lines derived from stratified epithelia to study the K14 functions in epithelial homeostasis. The K14 knockdown clones demonstrated substantial decreases in the levels of the K14 partner K5. These cells showed reduction in cell proliferation and delay in cell cycle progression, along with decreased phosphorylated Akt levels. K14 knockdown cells also exhibited enhanced levels of activated Notch1, involucrin, and K1. In addition, K14 knockdown AW13516 cells showed significant reduction in tumorigenicity. Our results suggest that K5 and K14 may have a role in maintenance of cell proliferation potential in the basal layer of stratified epithelia, modulating phosphatidylinositol 3-kinase/Akt–mediated cell proliferation and/or Notch1-dependent cell differentiation.

A decrease in the levels of the desmosomal plaque protein, plakophilin3 (PKP3), leads to a decrease in desmosome size and cell-cell adhesion. To test the hypothesis that PKP3 is required for desmosome formation, the recruitment of desmosomal components to the cell surface was studied in the PKP3 knockdown clones. The PKP3 knockdown clones showed decreased cell border staining for multiple desmosomal proteins, when compared to vector controls, and did not form desmosomes in a calcium switch assay. Further analysis demonstrated that PKP3, plakoglobin (PG) and E-cadherin are present at the cell border at low concentrations of calcium. Loss of either PG or E-cadherin led to a decrease in the levels of PKP3 and other desmosomal proteins at the cell border. The results reported here are consistent with the model that PG and E-cadherin recruit PKP3 to the cell border to initiate desmosome formation.

Mitotic progression requires the activity of the dual specificity phosphatase, cdc25C. Cdc25C function is inhibited by complex formation with two 14-3-3 isoforms, 14-3-3epsilon and 14-3-3gamma. To understand the molecular basis of specific complex formation between 14-3-3 proteins and their ligands, chimeric 14-3-3 proteins were tested for their ability to form a complex with cdc25C in vivo. Specific complex formation between cdc25C and 14-3-3epsilon in vivo requires a phenylalanine residue at position 135 (F135) in 14-3-3epsilon. Mutation of this residue to the corresponding residue present in other 14-3-3 isoforms (F135V) leads to reduced binding to cdc25C and a decrease in the ability to inhibit cdc25C function in vivo. Similarly, F135V failed to rescue the incomplete S phase and the G2 DNA damage checkpoint defects observed in cells lacking 14-3-3epsilon. A comparative analysis of the 14-3-3 structures present in the database suggested that the F135 in 14-3-3epsilon was required to maintain the integrity of a pocket that might be involved in secondary interactions with cdc25C. These results suggest that the specificity of the 14-3-3 ligand interaction may be dependent on structural motifs present in the individual 14-3-3 isoforms.

Stress-induced leakage current and time-dependent dielectric breakdown were investigated to examine the reliability of gate oxides grown on hydrogen- and deuterium-implanted silicon substrates. An order of magnitude improvement in charge-to-breakdown was observed for the deuterium-implanted devices as compared with the hydrogen-implanted ones. Such reliability improvement may be explained by the reduction of defects in the SiO2 and Si/SiO2 interface, such as Si dangling bonds, weak Si-Si bonds, and strained Si-O bonds due to the retention of implanted deuterium at the interface and in the bulk oxide as confirmed by secondary ion mass spectroscopy

Photoelectron transport in a photodetector in an ultra-high frame rate image sensor under the uniform illumination condition is investigated. The charge readout time in a multi-implant photodetector is estimated using a generalized model with both diffusion equation and continuity equations. The maximum effective diffusion length was assigned to each implanted region after taking into account the fringing field drift due to multiple implants. Assuming that the charge on each section is directly proportional to its area under uniform illumination, the total charge transport as a function of time is obtained by the superposition of charge contribution of all implanted regions. By increasing the number of implants in the photodetector it may be possible to collect more than 10 million frames s?1. The design effects are also investigated. The model showed excellent match with experimental results.

A dark red colored composite of C60 and (zinc, bismuth) borate glass was synthesized by solid-state reaction between C60 and frits of the glass at 650–700 °C in an argon atmosphere completely free from oxygen and moisture. An unusual change in color and the absorption spectrum of the glass upon composite formation with C60 indicated that the incorporated C60 underwent some sort of interaction with the glass. Such fullerene molecules bonded to relatively smaller units of bismuth-boron network, were extracted out by eluting a powdered sample with toluene. The composite itself and the toluene extract there of, were then characterized by ultraviolet–visible–near-infrared, infrared, and mass spectral studies. The results showed that the bismuth ions of the bismuth-boron network were bonded with the C60 cages through direct donation of their lone pair of electrons to the latter. The phenomenon of addition of boron to C60 via an “oxygen bridge,” which was observed in our earlier work, was also detected in this case. Studies on the nonlinear optical properties of the composite exhibited a moderate value of third order nonlinear susceptibility χ(3) (1.5 × 10−11 esu) and optical limiting properties of the composite. The result showed that the material had the prospect of being used in nonlinear optical devices.

Unconstraint depth photopolymerization has been used in recently developed 3D microfabrication process termed as Bulk lithography for the development of varying depth 3D microstructures. Prediction of the curing behaviour is the key for successful implementation of the process towards the development of the varying depth 3D microstructure. This paper presents the evolution of the cured voxel resulting from unconstraint depth photopolymerization under off-focus condition (on either side of the focal point). Variation in cured geometry is presented in the non-dimensional way under off-focus condition. The simulation carried out has predicted the significant variation of cured width under off-focus condition. This works points towards an alternative way of varying the geometry of the cured voxel for bulk lithography by positioning of the focal point relative to the resin surface.Copyright © 2014 by ASME

A new form of diffusion coefficient, termed the thermodynamic interdiffusion coefficient has been introduced in this investigation; it is related to the conventional chemical interdiffusion coefficient and thermodynamic properties of the system. Three main advantages in using the thermodynamic interdiffusion coefficient are described by applying this alternative approach of diffusion analysis, as an example, to the Al–Ni system. These include (1) a better interpretation of the relative diffusivities in the different phases of the same system, (2) estimation of appropriate values of the activation energy of the diffusion process and (3) determination of the thermodynamic intrinsic diffusion coefficient (also known as the tracer diffusion coefficient) in an intermediate phase with narrow homogeneity range, which otherwise is not possible without tracer diffusion experiments.

Two-photon optogalvanic transitions in Ar glow discharge with Nd: YAG laser pumped dye laser excitation in the frequency range 13520-16520 cm(-1) has been studied using linear and circular polarization. The intensities of two-photon optogalvanic transitions are very sensitive to changes in the incident laser power which is not the case with one-photon transitions. Intensity ratio for circular and linear polarized light for two photon transitions 6s'[1/2]degrees 1

Metastatic lung cancer is the leading cause of cancer related death in the world and novel approaches are necessary to elucidate specific genes that drive lung cancer progression and metastasis. Lung cancers commonly demonstrate genetic alteration of potent drivers like Kras, p53 or EGFR accompanied with hundreds of low frequency gene aberrations which are a mix of key driver events and nascent passenger mutations. We established a robust screening platform to selectively identify these functionally critical “driver” genes in lung cancers from a prioritized list of candidates selected by a multi-level cross-species comparison of our published high confidence transcriptome data from genetically-engineered mouse models and genomic data of human lung cancers from TCGA, focusing on elevated gene expression and/or gene amplification. We identified 225 putative candidate driver genes which were used to construct a lentiviral based cDNA expression library with unique molecular barcoding of individual cDNAs. Using a non-metastatic syngeneic mouse lung cancer model we generated individually transduced stable over-expressing lines for each gene. These candidate lines were used for a unique in vivo positive selection screen to identify functional metastasis drivers. We transplanted pools of 20 cDNA expressing lines into syngeneic mice and observed for primary tumor growth and occurrence of metastases in lungs and other organs. Metastatic drivers were identified by the relative enrichment of the unique barcode sequences in the genomic DNA from metastatic lesions over primary tumors. We have identified both known (e.g. MYC) and several novel (e.g. THRA, TMEM106B, GNAS) potential oncogenic and metastatic drivers. We validated our top hits for their individual in vivo metastatic potencies by gain-of-function/loss-of-function studies and identified TMEM106B as one of the primary drivers of metastasis. TMEM106B is a transmembrane protein localizing to the lysosomes and has been shown to drive expression of lysosomal genes. We hypothesize that elevated levels of TMEM106B is able to drive the expression and secretion of lysosomal enzymes thus making cancer cells hyper invasive and metastatic. We found strong correlation for expression of TMEM106B with several lysosomal genes in a panel of 1016 human lung cancers and are currently performing in depth studies to understand these mechanisms of TMEM106B driven metastasis. We are also analyzing the clinical relevance of TMEM106B based upon their expression pattern and prognostic utility across TCGA and other public datasets, along with in-house patient samples and tissue microarrays of resected and biopsy specimens. Identification of such novel players will significantly advance the field of cancer target discovery by identifying new drug targets and biomarkers, essential for effective treatment options for lung cancer patients. Citation Format: Samrat Kundu, Caitlin Grzeskowiak, Chad J. Creighton, Kenneth L. Scott, Don L. Gibbons. Identifying TMEM106B as a novel metastasis driver in non-small cell lung cancers through an in vivo gain-of-function screen. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 688.

s: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX Metastatic lung cancer is the leading cause of cancer related mortality in the world. Insights on specific genetic causes are limited and novel approaches are necessary to elucidate specific genes that drive lung cancer progression and metastasis. Extensive genomic and transcriptomic profiling of human lung cancers by consortia like ‘The Cancer Genome Atlas’ (TCGA) and the ‘International Cancer Genome Consortium’ (ICGC) has revealed a complex genetic landscape marked by abundant aberrations. In addition to acquiring mutations or amplification of well documented drivers like Kras, p53 or EGFR, lung cancers also demonstrate an enrichment of hundreds of low frequency gene aberrations which are a mix of key driver mutations and nascent passenger alterations caused by progressive genomic instability that could have definite role in cancer progression and metastasis. Our goal was to establish a robust screening platform to selectively identify these functionally critical “driver” genes in lung cancers. To identify a prioritized list of potential candidates we performed a multi-level cross-species comparison of our published high confidence transcriptome data from genetically-engineered mouse models of lung cancers and human lung cancer genomic data from TCGA, focusing on elevated gene expression and/or gene amplification. We identified an enriched list of 225 putative driver genes in lung cancers which were used to construct a lentiviral based cDNA expression library with unique molecular barcoding of individual cDNAs. Using a non-metastatic syngeneic mouse lung cancer model we generated individually transduced stable over-expressing lines for each of the 225 genes. These candidate lines were then entered into parallel in vitro and in vivo screens. In vitro testing includes cell invasion assays in a 96-well format, with positive hits defined as those scoring above 3x standard deviations relative to the control cells expressing mCherry. Positive hits were revalidated using standard 24-well invasion assays and also studied for growth phenotypes in 3D cultures. To selectively identify genes with functional role in metastasis, we performed a unique in vivo positive selection screen to test individual and combinatorial effects of lung cancer driver aberrations. For this we used pools of 20 cDNA expressing lines transplanted into syngeneic mice and observed for primary tumor growth and occurrence of metastases in lungs and other organs. Metastatic lesions and primary tumors were collected and genomic DNA from these was used to identify the unique individual barcodes by NGS barcode enrichment analysis. Metastatic drivers were identified by the relative enrichment of the unique barcode sequences for all the respective pool cDNAs in the metastatic tissue. We have identified both known (e.g. MYC) and several novel (e.g. THRA, TMEM106B, GNAS) potential oncogenic and metastatic drivers from the pooled screen which are currently being revalidated for their individual in vivo metastatic potencies. We are performing gain-of-function/loss-of-function studies for the identified driver genes to determine the pathways which they regulate. We are also analyzing the clinical relevance of these genes based upon their expression pattern and prognostic utility across TCGA and other public datasets, along with in-house patient samples and tissue microarrays of resected and biopsy specimens. Our unique high through-put approach will significantly advance the field of cancer target discovery by identifying new drug targets and biomarkers, essential for novel effective treatment options for lung cancer patients. Citation Format: Samrat T. Kundu, Caitlin Grzeskowiak, Chad J. Creighton, Kenneth L. Scott, Don L. Gibbons. A novel in vivo gain-of-function screen for metastasis drivers in non-small cell lung cancers. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B25.

The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis.

Structural and optical properties of the Tb doped ZnO nanoparticles are systematically studied as a function of the Tb mole-fraction. Our study suggests that the Tb incorporates mostly on the surface and affects the optical properties of the ZnO nanoparticles by influencing the attachment of certain adsorbed groups, which are found to be responsible for the appearance of a broad green luminescence (GL) band in the photoluminescence spectra recorded for these nanoparticles. It has been found that the accumulation of Tb on the surface of the nanoparticles not only enhances the band edge UV luminescence by suppressing the GL under the vacuum condition but also increases the band gap energy by introducing a hydrostatic compressive strain in individual nanoparticles, which provides a unique opportunity to study the pressure dependence of the optical properties of nanoparticles without applying any external pressure. The hydrostatic compressive strain is explained in terms of the increase of the surface strain energy as a result of the Tb accumulation on the surface of the nanoparticles. The average value of the surface energy density for the particles has been estimated as a function of Tb mole-fraction. The pressure coefficient of the band gap, which is obtained from the variation of the band gap energy with the hydrostatic strain, has been found to decrease significantly with the particle size for the ZnO nanoparticles.

The KRAS-variant is a germline single nucleotide polymorphism (SNP) within the 3'UTR of the KRAS gene predicted to disrupt a complementary binding site (LCS6) for the let-7 microRNA (miRNA). The KRAS-variant is associated with increased risk of various cancers, including lung cancer, ovarian cancer and triple-negative breast cancer, and is associated with altered tumor biology in head and neck cancer, colon cancer and melanoma. To better understand the molecular pathways that may be regulated or affected by the presence of the KRAS-variant allele in cancer cells, we examined its prevalence in the NCI-60 panel of cell lines and sought to identify common features of the cell lines that carry the variant allele. This study provides a step forward towards understanding the molecular and pathological significance of the KRAS-variant.