Catalytic Activity of Myoglobin Immobilized on Zirconium Phosphonates.

The adsorption and catalytic activity of myoglobin (Mb) on zirconium phosphonates (R-zirconium benzenephosphonate (R-ZrBP), R-zirconium carboxyethanephosphonate (R-ZrCEP), and a novel layered zirconium fluoride aminooctyl-N,N-bis(methylphosphonate) (ZrC8)) were investigated. The maximum adsorption was reached after 16 h of contact and was greater on hydrophobic supports such as R-ZrBP and ZrC8 compared to hydrophilic supports such as R-ZrCEP. The equilibrium adsorption isotherms fitted the Langmuir equation, suggesting the presence of a monolayer of protein molecules on the support surfaces. The catalytic activities of free Mb and of the obtained biocomposites were studied in terms of the oxidation of two aromatic substrates, o-phenylenediamine and 2-methoxyphenol (guaiacol), by hydrogen peroxide. The oxidation catalyzed by immobilized myoglobin followed the Michaelis-Menten kinetics, similar to oxidation by free Mb. The kinetic parameters, kcat and KM, were significantly affected by the adsorption process. Mb/R-ZrCEP was the most efficient biocatalyst obtained, probably because of the hydrophilic nature of the support. The effect of immobilization on the stability of Mb toward inactivation by hydrogen peroxide was also investigated, and an increased resistance was found. The biocomposites obtained can be stored at 4 °C for months without a significant loss of catalytic activity

MYC and human telomerase gene (TERC) copy number gain in early-stage non-small cell lung cancer

Objectives: We investigated the frequency of MYC and TERC increased gene copy number (GCN) in early-stage non-small cell lung cancer (NSCLC) and evaluated the correlation of these genomic imbalances with clinicopathologic parameters and outcome. Materials and Methods: Tumor tissues were obtained from 113 resected NSCLCs. MYC and TERC GCNs were tested by fluorescence in situ hybridization (FISH) according to the University of Colorado Cancer Center (UCCC) criteria and based on the receiver operating characteristic (ROC) classification. Results: When UCCC criteria were applied, 41 (36%) cases for MYC and 41 (36%) cases for TERC were considered FISH-positive. MYC and TERC concurrent FISH-positive was observed in 12 cases (11%): 2 (17%) cases with gene amplification and 10 (83%) with high polysomy. By using the ROC analysis, high MYC (mean ≥2.83 copies/cell) and TERC (mean ≥2.65 copies/cell) GCNs were observed in 60 (53.1%) cases and 58 (51.3%) cases, respectively. High TERC GCN was associated with squamous cell carcinoma (SCC) histology (P=0.001). In univariate analysis, increased MYC GCN was associated with shorter overall survival (P=0.032 [UCCC criteria] or P=0.02 [ROC classification]), whereas high TERC GCN showed no association. In multivariate analysis including stage and age, high MYC GCN remained significantly associated with worse overall survival using both the UCCC criteria (P=0.02) and the ROC classification (P=0.008). Conclusions: Our results confirm MYC as frequently amplified in early-stage NSCLC and increased MYC GCN as a strong predictor of worse survival. Increased TERC GCN does not have prognostic impact but has strong association with squamous histology

Prognostic impact of alternative splicing-derived hMENA isoforms in resected, node-negative, non-small-cell lung cancer

Risk assessment and treatment choice remain a challenge in early non-small-cell lung cancer (NSCLC). Alternative splicing is an emerging source for diagnostic, prognostic and therapeutic tools. Here, we investigated the prognostic value of the actin cytoskeleton regulator hMENA and its isoforms, hMENA(11a) and hMENA Delta v6, in early NSCLC. The epithelial hMENA(11a) isoform was expressed in NSCLC lines expressing E-CADHERIN and was alternatively expressed with hMENA Delta v6. Enforced expression of hMENA Delta v6 or hMENA(11a) increased or decreased the invasive ability of A549 cells, respectively. hMENA isoform expression was evaluated in 248 node-negative NSCLC. High pan-hMENA and low hMENA(11a) were the only independent predictors of shorter disease-free and cancer-specific survival, and low hMENA(11a) was an independent predictor of shorter overall survival, at multivariate analysis. Patients with low pan-hMENA/high hMENA(11a) expression fared significantly better (P <= 0.0015) than any other subgroup. Such hybrid variable was incorporated with T-size and number of resected lymph nodes into a 3-class-risk stratification model, which strikingly discriminated between different risks of relapse, cancer-related death, and death. The model was externally validated in an independent dataset of 133 patients. Relative expression of hMENA splice isoforms is a powerful prognostic factor in early NSCLC, complementing clinical parameters to accurately predict individual patient risk

Functional signaling pathway analysis of lung adenocarcinomas identifies novel therapeutic targets for KRAS mutant tumors

Little is known about the complex signaling architecture of KRAS and the interconnected RAS-driven protein-protein interactions, especially as it occurs in human clinical specimens. This study explored the activated and interconnected signaling network of KRAS mutant lung adenocarcinomas (AD) to identify novel therapeutic targets. Thirty-four KRAS mutant (MT) and twenty-four KRAS wild-type (WT) frozen biospecimens were obtained from surgically treated lung ADs. Samples were subjected to Laser Capture Microdissection and Reverse Phase Protein Microarray analysis to explore the expression/activation levels of 150 signaling proteins along with coactivation concordance mapping. An independent set of 90 non-small cell lung cancers (NSCLC) was used to validate selected findings by immunohistochemistry (IHC). Compared to KRAS WT tumors, the signaling architecture of KRAS MT ADs revealed significant interactions between KRAS downstream substrates, the AKT/mTOR pathway, and a number of Receptor Tyrosine Kinases (RTK). Approximately one-third of the KRAS MT tumors had ERK activation greater than the WT counterpart (p < 0.01). Notably 18% of the KRAS MT tumors had elevated activation of the Estrogen Receptor alpha (ER-α) (p=0.02).This finding was verified in an independent population by IHC (p=0.03). KRAS MT lung ADs appear to have a more intricate RAS linked signaling network than WT tumors with linkage to many RTKs and to the AKT-mTOR pathway. Combination therapy targeting different nodes of this network may be necessary to treat this group of patients. In addition, for patients with KRAS MT tumors and activation of the ER-α, anti-estrogen therapy may have important clinical implications

Viscoelastic material properties of the peripapillary sclera in normal and early-glaucoma monkey eyes

PURPOSE. To test the hypothesis that changes in the viscoelastic material properties of peripapillary sclera are present within monkey eyes at the onset of early experimental glaucoma detected by confocal scanning laser tomography (CSLT). METHODS. Short-term (3-9 weeks), moderate (Յ44 mm Hg) intraocular pressure (IOP) elevation was induced in one eye of each of eight male monkeys by lasering the trabecular meshwork. This procedure generated early experimental glaucoma, defined as the onset of CSLT-detected optic nerve head (ONH) surface change, in the treated eye. Scleral tensile specimens from the superior and inferior quadrants of the eight earlyglaucoma eyes were subjected to uniaxial stress relaxation and tensile tests to failure and the results compared with similar data obtained in a previous study of 12 normal (nonglaucomatous) eyes. Linear viscoelastic theory was used to characterize viscoelastic material property parameters for each specimen. Differences in each parameter due to quadrant and treatment were assessed by analysis of variance (ANOVA). RESULTS. Peripapillary sclera from the early-glaucoma eyes exhibited an equilibrium modulus (7.46 Ϯ 1.58 MPa) that was significantly greater than that measured in normal eyes (4.94 Ϯ 1.22 MPa; mean Ϯ 95% confidence interval, P Ͻ 0.01, ANOVA). Quadrant differences were not significant for the viscoelastic parameters within each treatment group. CONCLUSIONS. The long-term viscoelastic material properties of monkey peripapillary sclera are altered by exposure to moderate, short-term, chronic IOP elevations and these alterations are present at the onset of CSLT-detected glaucomatous damage to the ONH. Damage to and/or remodeling of the extracellular matrix of these tissues may underlie these changes in scleral material properties. (Invest Ophthalmol Vis Sci. 2005;46: 540 -546 1 The finite element method is a computer-based engineering technique that estimates the stresses (force/cross-sectional area) and strains (local deformation under those stresses) within a complex load-bearing structure. 2 The important aspects of a finite element model are the three-dimensional geometry and material properties of the load-bearing structure and the appropriate boundary and mechanical loading conditions. 2 Within such models of the monkey ONH, scleral material properties are needed to characterize accurately the important transition between the peripapillary sclera and the peripheral laminar beams. To characterize the material properties of peripapillary sclera, we constructed a controlled-environment testing apparatus capable of performing uniaxial biomechanical testing of soft tissues in tension. The overall response of a tissue to a load is the combination of instantaneous (elastic) and time-dependent (viscous) responses, which are governed by its viscoelastic material properties. All soft tissues are viscoelastic, and the characterization of the instantaneous and time-dependent aspects of these material properties is important in understanding a tissue's behavior as a load-bearing structure and its response to short-and long-term changes in the applied load. Accurate characterization of a tissue's material properties can lead to a broader understanding of the mechanisms that underlie tissue damage, as well as determine the resultant altered load-bearing behavior of a remodeled or healed tissue. In a previous report we performed quadrant-based uniaxial tensile testing of peripapillary sclera from normal rabbit and normal monkey eyes 3 and found no differences in the viscoelastic material properties of the peripapillary sclera by quadrant (superior, inferior, nasal, and temporal to the ONH) in either species. In addition, when normal rabbit and monkey sclera were compared with one another, peripapillary sclera from monkey eyes was stiffer and showed slower stress relaxation than sclera from rabbit eyes. The present study tests the hypothesis that changes in the viscoelastic material properties of monkey peripapillary sclera are present at the earliest detectable stage of glaucomatous damage to the ONH, defined as the onset of confocal scanning laser tomography (CSLT)-detected ONH surface change. MATERIALS AND METHODS Animals, Scleral Specimens, and Study Design All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. In the present study, a single scleral tensile specimen was generated from either the superior or inferior quadrant of eight eyes with experimental early glaucoma, yielding four early-glaucoma specimens for each quadrant. Each specimen underwent a multistage uniaxial tensile test. ViscoelasFrom th