Next-Generation Sequencing Workflow for NSCLC Critical Samples Using a Targeted Sequencing Approach by Ion Torrent PGM? Platform

Next-generation sequencing (NGS) is a cost-effective technology capable of screening several genes simultaneously; however, its application in a clinical context requires an established workflow to acquire reliable sequencing results. Here, we report an optimized NGS workflow analyzing 22 lung cancer-related genes to sequence critical samples such as DNA from formalin-fixed paraffin-embedded (FFPE) blocks and circulating free DNA (cfDNA). Snap frozen and matched FFPE gDNA from 12 non-small cell lung cancer (NSCLC) patients, whose gDNA fragmentation status was previously evaluated using a multiplex PCR-based quality control, were successfully sequenced with Ion Torrent PGM™. The robust bioinformatic pipeline allowed us to correctly call both Single Nucleotide Variants (SNVs) and indels with a detection limit of 5%, achieving 100% specificity and 96% sensitivity. This workflow was also validated in 13 FFPE NSCLC biopsies. Furthermore, a specific protocol for low input gDNA capable of producing good sequencing data with high coverage, high uniformity, and a low error rate was also optimized. In conclusion, we demonstrate the feasibility of obtaining gDNA from FFPE samples suitable for NGS by performing appropriate quality controls. The optimized workflow, capable of screening low input gDNA, highlights NGS as a potential tool in the detection, disease monitoring, and treatment of NSCLC.     

Investigating the Role of Conformational Effects on Laccase Stability and Hyperactivation under Stress Conditions

Fungal laccase from Steccherinum ochraceum 1833 displays remarkable stability under different harsh conditions: organic/buffer mixtures, thermal treatment, and microwave radiation. The behavior is particularly significant in the light of the sharp inactivation observed for two different fungal laccases. Laccase from S. ochraceum 1833 also displays hyperactivation under mild thermal treatment (60 °C). Molecular dynamics simulations at 80 °C explained how this laccase retains the geometry of the electron transfer pathway, thereby assuring electron transfer through the copper ions and thus maintaining its catalytic activity at high temperature. Spectroscopic studies revealed that the thermal activation corresponds to specific conformational changes in the protein. The results indicate that this laccase is potentially applicable under denaturing conditions that might be beneficial for the biotransformation of recalcitrant substrates.     

Effective wind actions on ideal and real structures

Gust buffeting of structures is analyzed by Double Modal Transformation, a method consisting in the joint transformation into principal components of structural displacements (modal analysis) and of wind loading (proper orthogonal decomposition). It commonly occurs that modal truncation applies to both the series of structural and loading modes; besides, many loading modes are almost orthogonal respect the dominant structural modes, so they do not contribute to structural response. This set of conditions allows the introduction of an effective action, defined as the loading process reconstructed retaining only those loading modes that excite the structure. This paper discusses this method and the related concepts by analyzing a set of simple ideal structures in closed form; this approach allows the formulation of general criteria aimed at defining the effective wind action as a function of non-dimensional structural and wind parameters. The numerical analysis of a real structure confirms the tendencies pointed out by the analytical solutions.     

Nutrition-based health: cell-based bioassays for food antioxidant activity evaluation

Food science has progressively evolved and now there are wide evidences that foods have biological activities that are beyond their classical nutritional value. In this field, the antioxidant activity of pure compounds, food, feed, and dietary supplements has been extensively studied and numerous analytical approaches and assay models have been developed, involving various systems from simple chemical assays to animal models and human studies. This article is an overview of different cell-based models that have been used for testing the antioxidant properties of food, feed, and dietary supplements. Advantages, drawbacks, and technical problems to develop and validate suitable, robust, and high-throughput cell-based bioassays for screening food antioxidant activity will be discussed.     

Towards biomimetic models of the reduced [FeFe]-hydrogenase that preserve the key structural features of the enzyme active site; a DFT investigation

[FeFe]-hydrogenases are the most efficient biological catalysts available for the H₂ evolution reaction. Their active site – the H-cluster – features a diiron subsite which has the peculiar characteristic of bearing cyanide groups hydrogen-bonded to the apoprotein as well as carbonyl ligands. Notably, one of the CO ligands is disposed in bridging position between the metal centers. This allows one of the Fe ions to retain a square pyramidal coordination – which determines the assumption of the so-called “rotated structure” – with a vacant coordination site in trans to the μ-CO group, ready to bind protons when the active site is in the Fe^IFe^I state. Many Fe^IFe^I biomimetic models have been synthesized and characterized so far, but most of them fail to reproduce the orientation of the diatomic ligands that is observed in the enzyme active site.     

Potential Mechanisms Linking Atherosclerosis and Increased Cardiovascular Risk in COPD: Focus On Sirtuins

The development of atherosclerosis is a multi-step process, at least in part controlled by the vascular endothelium function. Observations in humans and experimental models of atherosclerosis have identified monocyte recruitment as an early event in atherogenesis. Chronic inflammation is associated with ageing and its related diseases (e.g., atherosclerosis and chronic obstructive pulmonary disease). Recently it has been discovered that Sirtuins (NAD⁺-dependent deacetylases) represent a pivotal regulator of longevity and health. They appear to have a prominent role in vascular biology and regulate aspects of age-dependent atherosclerosis. Many studies demonstrate that SIRT1 exhibits anti-inflammatory properties in vitro (e.g., fatty acid-induced inflammation), in vivo (e.g., atherosclerosis, sustainment of normal immune function in knock-out mice) and in clinical studies (e.g., patients with chronic obstructive pulmonary disease). Because of a significant reduction of SIRT1 in rodent lungs exposed to cigarette smoke and in lungs of patients with chronic obstructive pulmonary disease (COPD), activation of SIRT1 may be a potential target for chronic obstructive pulmonary disease therapy. We review the inflammatory mechanisms involved in COPD-CVD coexistence and the potential role of SIRT1 in the regulation of these systems.     

Fat globule membranes in ewes' milk: The main enzyme activities during lactation

A trial was carried out on seven Massese ewes, from which individual milk samples were taken during lactation. All the enzyme activities investigated (oxidase activity of xanthine oxidoreductase, gamma-glutamyltranspeptidase, alkaline phosphatase and 5′-nucleotidase) were affected by the lactation phase, with the exception of the dehydrogenase activity of xanthine oxidoreductase (XOR). A higher oxidase activity (XO) of XOR was found when the diameter of milk fat globules was smaller. In addition, the absence of XO in colostrum led to the hypothesis that its antibacterial role is independent of its activity. The increase in alkaline phosphatase activity during lactation requires further research to better define the criteria for pasteurised sheep milk.     

An IR molding system for direct shaping of thermoplastics

A new molding technique is proposed to directly mold small parts from thermoplastic pellets. A small molding machine was designed and fabricated: the mold consisted of a transparent glass and a metallic frame; an infrared (IR) lamp provided the heat for the pellet melting. Molding tests were carried out by using three commercial thermoplastics and two metallic molds. Molding time and IR lamp power were changed to extract a moldability window for each combination of material and metallic mold. In the best process conditions, a high quality of the samples was obtained in terms of mold filling and surface aesthetics. POLYM. ENG. SCI., 2010. © 2010 Society of Plastics Engineers