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.     

Exploring New Potential Anticancer Activities of the G-Quadruplexes Formed by [(GTG2T(G3T)3] and Its Derivatives with an Abasic Site Replacing Single Thymidine

In this paper, we report our investigations on five {"type":"entrez-nucleotide","attrs":{"text":"T30175","term_id":"612273"}}T30175 analogues, prepared by replacing sequence thymidines with abasic sites (S) one at a time, in comparison to their natural counterpart in order to evaluate their antiproliferative potential and the involvement of the residues not belonging to the central core of stacked guanosines in biological activity. The collected NMR (Nuclear Magnetic Resonance), CD (Circular Dichroism), and PAGE (Polyacrylamide Gel Electrophoresis) data strongly suggest that all of them adopt G-quadruplex (G4) structures strictly similar to that of the parent aptamer with the ability to fold into a dimeric structure composed of two identical G-quadruplexes, each characterized by parallel strands, three all-anti-G-tetrads and four one-thymidine loops (one bulge and three propeller loops). Furthermore, their antiproliferative (MTT assay) and anti-motility (wound healing assay) properties against lung and colorectal cancer cells were tested. Although all of the oligodeoxynucleotides (ODNs) investigated here exhibited anti-proliferative activity, the unmodified {"type":"entrez-nucleotide","attrs":{"text":"T30175","term_id":"612273"}}T30175 aptamer showed the greatest effect on cell growth, suggesting that both its characteristic folding in dimeric form and its presence in the sequence of all thymidines are crucial elements for antiproliferative activity. This straightforward approach is suitable for understanding the critical requirements of the G-quadruplex structures that affect antiproliferative potential and suggests its application as a starting point to facilitate the reasonable development of G-quadruplexes with improved anticancer properties.     

The ANNS approach to DEM reconstruction

This research has 6 fundamental aims: (i) to present a modified version of Taylor's interpolation, one that is more effective and faster than the original; (ii) outline the capability of artificial neural networks (ANNs) to perform an optimal functional approximation of the digital elevation model reconstruction from a satellite map, using a small and independent sample of Global Positioning System observations; (iii) demonstrate experimentally how ANNs outperform the traditional and most used algorithm for the height interpolation (Taylor's interpolation); (iv) introduce a new ANN, the Conic Net, able to outperform the results of the classic and more known multilayer perceptron; (v) determine that Conic Nets, even when using Taylor's modified interpolation as input features, are able to optimally approximate the heights with one order of magnitude more than the original satellite map; and (vi) make evident the possibility to interpolate the DEM heights through an ANN, which learns a data set of known points.     

Extracellular Vesicles in Osteosarcoma: Antagonists or Therapeutic Agents?

Osteosarcoma (OS) is a skeletal tumor affecting mainly children and adolescents. The presence of distance metastasis is frequent and it is localized preferentially to the lung, representing the main reason for death among patients. The therapeutic approaches are based on surgery and chemotherapeutics. However, the drug resistance and the side effects associated with the chemotherapy require the identification of new therapeutic approaches. The understanding of the complex biological scenario of the osteosarcoma will open the way for the identification of new targets for its treatment. Recently, a great interest of scientific community is for extracellular vesicles (EVs), that are released in the tumor microenvironment and are important regulators of tumor proliferation and the metastatic process. At the same time, circulating extracellular vesicles can be exploited as diagnostic and prognostic biomarkers, and they can be loaded with drugs as a new therapeutic approach for osteosarcoma patients. Thus, the characterization of OS-related EVs could represent a way to convert these vesicles from antagonists for human health into therapeutic and/or diagnostic agents.     

Enhanced magnetic field sensitivity of spin-dependent transport in cluster-assembled metallic nanostructures

The emerging field of spintronics explores the many possibilities offered by the prospect of using the spin of the electrons for fast, nanosized electronic devices. The effect of magnetization acting on a current is the essence of giant or tunnel magnetoresistance. Although such spintronics effects already find technological applications, much of the underlying physics remains to be explored. The aim of this article is to demonstrate the importance of spin mixing in metallic nanostructures. Here we show that magnetic clusters embedded in a metallic matrix exhibit a giant magnetic response of more than 500% at low temperature, using a recently developed thermoelectric measurement. This method eliminates the dominating resistivity component of the magnetic response and thus reveals an intrinsic spin-dependent process: the conduction-electron spin precession about the exchange field as the electron crosses the clusters, giving rise to a spin-mixing mechanism with strong field dependence. This effect appears sensibly only in the smallest clusters, that is, at the level of less than 100 atoms per cluster.     

Saliva Proteomics as Fluid Signature of Inflammatory and Immune-Mediated Skin Diseases

Saliva is easy to access, non-invasive and a useful source of information useful for the diagnosis of serval inflammatory and immune-mediated diseases. Following the advent of genomic technologies and -omic research, studies based on saliva testing have rapidly increased and human salivary proteome has been partially characterized. As a proteomic protocol to analyze the whole saliva proteome is not currently available, the most common aim of the proteomic analysis is to discriminate between physiological and pathological conditions. The salivary proteome has been initially investigated in several diseases: oral squamous cell carcinoma and oral leukoplakia, chronic graft-versus-host disease, and Sjögren’s syndrome. Otherwise, salivary proteomics studies in the dermatological field are still in the initial phase, thus the aim of this review is to collect the best research evidence on the role of saliva proteomics analysis in immune-mediated skin diseases to understand the direction of research in this field. The results of PRISMA analysis reported herein suggest that human saliva analysis could provide significant data for the diagnosis and prognosis of several immune-mediated and inflammatory skin diseases in the next future.