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.     

Integration of InGaP/GaAs/Ge triple‐junction solar cells on deeply patterned silicon substrates

We report preliminary results on InGaP/InGaAs/Ge photovoltaic cells for concentrated terrestrial applications, monolithically integrated on engineered Si(001) substrates. Cells deposited on planar Ge/Si(001) epilayers, grown by plasma‐enhanced chemical vapor deposition, provide good efficiency and spectral response, despite the small thickness of the Ge epilayers and a threading dislocation density as large as 10⁷/cm². The presence of microcracks generated by the thermal misfit is compensated by a dense collection grid that avoids insulated areas. In order to avoid the excessive shadowing introduced by the use of a dense grid, the crack density needs to be lowered. Here, we show that deep patterning of the Si substrate in blocks can be an option, provided that a continuous Ge layer is formed at the top, and it is suitably planarized before the metalorganic chemical vapor deposition. The crack density is effectively decreased, despite that the efficiency is also lowered with respect to unpatterned devices. The reasons of this efficiency reduction are discussed, and a strategy for improvement is proposed and explored. Full morphological analysis of the coalesced Ge blocks is reported, and the final devices are tested under concentrated AM1.5D spectrum. Copyright © 2016 John Wiley & Sons, Ltd.     

A chemically enhanced biological process for lowering operative costs and solid residues of industrial recalcitrant wastewater treatment

An innovative process based on ozone-enhanced biological degradation, carried out in an aerobic granular biomass system (SBBGR - Sequencing Batch Biofilter Granular Reactor), was tested at pilot scale for tannery wastewater treatment chosen as representative of industrial recalcitrant wastewater. The results have shown that the process was able to meet the current discharge limits when the biologically treated wastewater was recirculated through an adjacent reactor where a specific ozone dose of 120 mg O₃/L_influent was used. The benefits produced by using ozone were appreciable even visually since the final effluent of the process looked like tap water. In comparison with the conventional treatment, the proposed process was able to reduce the sludge production by 25-30 times and to save 60% of operating costs.Molecular in situ detection methods were employed in combination with the traditional measurements (oxygen uptake rate, total protein content, extracellular polymeric substances and hydrophobicity) to evaluate microbial activity and composition, and the structure of the biomass. A stable presence of active bacterial populations was observed in the biomass with the simultaneous occurrence of distinctive functional microbial groups involved in carbon, nitrogen and sulphate removal under different reaction environments established within the large microbial aggregates. The structure and activity of the biomass were not affected by the use of ozone.     

Organochlorine pesticides in colostrums in case of normal and preterm labor (IASI, Romania)

Assessment of organochlorine pesticides (OCPs) in human body is important for human health because they have weak estrogenic or antiestrogenic effects and are considered endocrine disrupters. We used colostrum of women as indicator for levels of OCPs in human body for mothers with normal and preterm labor from eastern part of Romania. Sixty- three samples of colostrum were extracted by solid-phase extraction. Analyses were carried out using gas chromatography coupled to mass spectrometry (GC-MS). OCPs have been detected in all samples, with p,p’-dichlorodiphenyldichloroethylene (p,p’-DDE) and γ-hexachlorocyclohexane (γ-HCH) being at the highest concentrations. Of the organochlorines measured in clostrum samples from women in preterm labor, median levels of DDTs (470 ng/g) and HCHs (99 ng/g) were higher than for the same compounds from women in normal labor (median of DDTs = 268 ng/g and median of HCHs = 96 ng/g). Normal labor had higher median concentrations of HCB (19.5 ng/g) versus preterm labor (14 ng/g). Statistical data show high Spearman correlation coefficients between various OCPs. We found a good correlation between α-, γ-, β- and δ- HCH isomers (p < 0.001) for both normal and preterm labor. The most abundant target compound was p,p’-DDE (median value 96 ng/g, and 137 ng/g for mother with normal and preterm labor, respectively) in all colostrum samples. The estimated daily intakes of HCHs by infants exceeded corresponding Health Canada guidelines.     

Highly bright and photostable cyanine dye-doped silica nanoparticles for optical imaging: Photophysical characterization and cell tests

Spherical silica nanoparticles containing fluorescent trimethine indocyanine dyes (λ_abs = 547 nm, λ_em = 570 nm) were prepared using a water-in-oil microemulsion method. The nanoparticles were of 50 nm diameter and were almost monodispersed in aqueous solution at pH 5.5. Entrapment of dye molecules in the silica matrix stabilised photoemission over several hours of continuous irradiation. The photoemission intensity of the indocyanine was increased 13-fold over that recorded in solution. As each nanoparticle contained 110 dye molecules, the photoemission brightness of each particle was enhanced by three orders of magnitude. The fluorescent nanoparticles have been tested as imaging tools in in vitro tests. As an example of non-macrophagic cells, a highly differentiated neuronal cell line (GT1-7) was used and the results showed that the prepared nanoparticles can be incorporated into these cells with no apparent toxicity for up to three days.     

An effective multipurpose building block for 3D electropolymerisation: 2,2′-Bis(2,2′-bithiophene-5-yl)-3,3′-bithianaphthene

Novel thiophene-based oligomer, 2,2′-bis(2,2′-bithiophene-5-yl)-3,3′-bithianaphthene (TX), was designed and synthesized, and its electrochemical and spectral properties characterised. TX was readily polymerised electrochemically to form well organized conducting homopolymer films on various solid electrode substrates. Moreover, it was successfully used for deposition by electropolymerisation of electrochemically active thin films of co-polymers with three different monomers of functionalised bis(2,2′-bithienyl)methane derivatives. It appeared that TX was an effective crosslinker and 3D promoter in these electropolymerisations involving co-monomers intrinsically showing limited aptitude for the electropolymerisation or forming polymer films of low conductivity. This attractive TX ability stems from combination of its (i) high conjugation efficiency in each of the two planar moieties, (ii) intrinsic 3D structure on account of the presence of the central node, and (iii) intrinsic regioselectivity in electropolymerisation on account of the positions of the two available free α-thiophene sites.     

Electrospinning: A Novel Method to Incorporate POSS Into a Polymer Matrix

The effectiveness of electrospinning as a simple approach to disperse POSS into a polymer matrix at a nm‐level has been assessed. Electrospun and cast films were prepared by dissolving CA and epoxycyclohexylisobutyl POSS in the solvent mixture acetone/DMAc. The membranes were characterized by SEM, TEM and WAXD. Whereas films produced by casting showed µm‐sized POSS crystals, thus suggesting a small affinity between the polymer matrix and the POSS molecules, those prepared by electrospinning were characterized by a nanometric POSS distribution. This is explained by considering the peculiar solvent evaporation mechanism, occurring during the electrospinning process, which allows to produce nanofibers characterized by a silsesquioxane dispersion similar to that present in solution.