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.     

Coupling hydrothermal liquefaction and aqueous phase reforming for integrated production of biocrude and renewable H2

Lignin-rich stream from lignocellulosic ethanol production was converted into biocrude by continuous hydrothermal liquefaction (HTL) while hydrogen was produced by aqueous phase reforming (APR) of the HTL aqueous by-product. The effects of Na₂CO₃ and NaOH were investigated both in terms of processability of the feedstock as well as yield and composition of the obtained products. A maximum biocrude yield of 27 wt% was reached in the NaOH-catalyzed runs. A relevant amount of dissolved phenolics were detected in the co-produced aqueous phase (AP), and removed by liquid–liquid extraction using butyl acetate or diethyl ether, preserving the APR catalyst stability and reaching an hydrogen yield up to 146 mmol H₂ L⁻¹ AP. Preliminary mass balances integrating HTL and APR showed that the hydrogen provided by APR may account for up to 46% of the hydrogen amount theoretically required for upgrading the HTL biocrude, thus significantly improving the process performance and sustainability.     

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.     

Influence of small rubber particles on the environmental stress cracking of high impact polystyrene

This article exploits the influence of rubber particle size (RPS) and rubber crosslinking on environmental stress cracking resistance (ESCR) of high impact polystyrene (HIPS), with special interest on the influence of small rubber particles fraction. Three commercial HIPS of high ESCR were selected and four batches of HIPS were prepared in‐house, including samples based on high cis and very high viscosity polybutadiene (PB). Their morphologies were analyzed by low angle laser light scattering, optical microscopy, and transmission electron microscopy, and the samples were submitted to flexural ESCR tests with fatty agents. The ESCR to sunflower oil was found to increase with the reduction of the rubber particles fraction smaller than 1–2 micron. Results have also confirmed that an increase in RPS is the key parameter to promote ESCR, although there is limit for RPS to be effective on ESCR improvement. The reduction of small rubber particles fraction in HIPS was achieved by using a high cis PB, that promotes low grafting efficiency of polystyrene onto PB backbone because of the low content of 1,2 vinyl isomer. Besides the ESCR improvements, HIPS with high cis PB showed higher elastic modulus and impact resistance than HIPS containing medium cis PB, which is desired for thickness reduction in food packaging and refrigeration cabinets. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Ownership, Investment Policies and Funding Choices of Italian Water Utilities: An Empirical Analysis

Nowadays, the efficient management of water has become the focus of vast debate, both in the academic literature and in the practical and regulatory field. Due to the growing importance and scarcity of water resources, it has become crucial to better understand how to improve the organizational efficiency of water utilities. By adopting an accounting perspective and using statistical methods, this paper analyzes whether and to what extent investment and financial strategies differ among clusters of water utilities with different ownership structures. The paper focuses on the Italian water industry, a context considered particularly appropriate due to the coexistence of utilities with different ownership structures. The main results of the paper show that ownership affects the level of investment as well as the financial structure and costs of water utilities. The evidence provided by this study should encourage national governments and regulatory authorities to select water utilities with the greatest investment potential measured in terms of financial efficiency and effectiveness.     

Interaction between Lipopolysaccharide and Gut Microbiota in Inflammatory Bowel Diseases

Lipopolysaccharides (LPSs) are bacterial surface glycolipids, produced by Gram-negative bacteria. LPS is known to determine acute inflammatory reactions, particularly in the context of sepsis. However, LPS can also trigger chronic inflammation. In this case, the source of LPS is not an external infection, but rather an increase in endogenous production, which is usually sustained by gut microbiota (GM), and LPS contained in food. The first site in which LPS can exert its inflammatory action is the gut: both GM and gut-associated lymphoid tissue (GALT) are influenced by LPS and shift towards an inflammatory pattern. The changes in GM and GALT induced by LPS are quite similar to the ones seen in IBD: GM loses diversity, while GALT T regulatory (Tregs) lymphocytes are reduced in number, with an increase in Th17 and Th1 lymphocytes. Additionally, the innate immune system is triggered, through the activation of toll-like receptor (TLR)-4, while the epithelium is directly damaged, further triggering inflammation. In this review, we will discuss the importance of the crosstalk between LPS, GM, and GALT, and discuss the possible implications.     

1923 Gleno Dam Break: Case Study and Numerical Modeling

On the morning of December 1, 1923, the Gleno Dam (located in the Central Italian Alps) suddenly collapsed a few days after the first complete reservoir filling. Nearly 4.5×106??m3 of water was released. The consequent inundation caused significant destruction along the downstream valley and a death toll of at least 356 lives. This failure is the only historical case of dam break caused by structural deficiencies that has occurred in Italy. As a result, it has deeply influenced the evolution of Italian regulations regarding dam design and hydraulic risk evaluation. However, in spite of its relevance, this event has never been characterized from a hydraulic standpoint. This paper reports the main information obtained from the analysis of a vast amount of historical documents regarding the Gleno Dam break to set up a case study useful for validating dam-break models in mountain settings. Moreover, it presents the main results of one-dimensional (1D) modeling of the dam break wave propagation accomplished with a first-order finite volume numerical scheme recently proposed in the literature for field applications. The overall effectiveness and reliability of the model are evaluated for this case characterized by very irregular topography. Finally, the practical relevance of several choices that the numerical reconstruction of this kind of event demands is tested.     

A monoclonal antibody to the ligand-binding domain of the neurokinin 1 receptor (NK1-R) for the neuropeptide substance P

For PCR-based genotyping using polymorphic microsatellite markers, DNA from decomposed postmortem human tissues was fractionated into six groups according to molecular size. The minimum required amounts of this degraded DNA, for detecting alleles at five microsatellite loci (ACTBP2, CMAG, HUMTH01, CYP19, and LPL) and one minisatellite locus (MCT118) were investigated respectively. The allele patterns were detected by electrophoresis of the PCR products on a 6%-denaturing polyacrylamide gel following silver staining. The detection of alleles for the loci with large allele size required more template DNA with higher molecular size than for that with small allele size. Amounts from 0.3 ng to 5 ng were needed for allele detection on genomic DNA from fresh blood. When the decomposed DNA mixture was used as the template, approximately ten times the amount of genomic DNA was required to detect alleles at the three loci of LPL, CYP19 and HUMTH01, while 24 to 67 times was required for the loci, CMAG, ACTBP2 and MCT118. It was demonstrated that a minimum molecular, size and amount of template DNA was needed for amplifying alleles of the six loci, and degraded DNA less than minimum size in the samples would prevent the detection of the loci which have large allele size.