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.     

Homoserine Lactones,Methyl Oligohydroxybutyrates,and Other Extracellular Metabolites of Macroalgae‐Associated Bacteria of the Roseobacter Clade: Identification and Functions

Twenty‐four strains of marine Roseobacter clade bacteria were isolated from macroalgae and investigated for the production of quorum‐sensing autoinducers, N‐acylhomoserine lactones (AHLs). GC/MS analysis of the extracellular metabolites allowed us to evaluate the release of other small molecules as well. Nineteen strains produced AHLs, ranging from 3‐OH‐C10:0‐HSL (homoserine lactone) to (2E,11Z)‐C18:2‐HSL, but no specific phylogenetic or ecological pattern of individual AHL occurrence was observed when cluster analysis was performed. Other identified compounds included indole, tropone, methyl esters of oligomers of 3‐hydroxybutyric acid, and various amides, such as N‐9‐hexadecenoylalanine methyl ester (9‐C16:1‐NAME), a structural analogue of AHLs. Several compounds were tested for their antibacterial and antialgal activity on marine isolates likely to occur in the habitat of the macroalgae. Both AHLs and 9‐C16:1‐NAME showed high antialgal activity against Skeletonema costatum, whereas their antibacterial activity was low.     

Factors affecting acceptability of dry-cured ham throughout extended maturation under "bodega" conditions

The evolution of biochemical, instrumental colour and texture, sensory parameters and consumer acceptability of 12-month dry-cured hams maintained up to 26 months under “bodega” conditions (18 °C, 75% relative humidity) was assessed, in order to investigate the influence of extended ripening on their sensory characteristics and acceptability. Results demonstrated that ham acceptability showed no significant differences (p > 0.05) from 12 to 22 months, while it decreased significantly (p < 0.05) until 26 months. Principal component analysis of all data brought about a comprehensive explanation of the biochemical, instrumental and sensory parameters involved in the acceptability decrease. In fact, high pastiness and adhesiveness values, as measured by both sensory and instrumental methods, appeared to be most related to decreasing acceptability. Those attributes were the result of an excessive proteolysis, as revealed by biochemical maturation indices.     

Substrate constructed by an array of split ring resonators for a THz planar antenna

Metamaterials are artificial materials offering unique properties which render them useful for various applications. In the present paper, we examine whether it is possible to design a split ring resonators’ (SRRs) based metamaterial substrate for enhancing the performance of planar antennas operating at THz frequencies. Initially, the radiating characteristics of a simple rectangular patch antenna over two well-known SRR substrates are studied. The yielded results are then compared to those of two novel designs of metamaterial antenna substrate with SRRs of decreasing size which are proposed to improve the antenna’s performance.     

Use of High-Resolution X-Ray Micro-Computed Tomography for the Analysis of Internal Structural Changes in Maize Infected with <Emphasis Type="Italic">Fusarium verticillioides</Emphasis>

X-ray micro-computed tomography (X-ray micro-CT) is a non-destructive, three-dimensional (3D) imaging and analysis technique for the investigation of internal structure of a large variety of materials, including agricultural produce. As a relatively new method in the field of food science, X-ray micro-CT has been applied successfully to obtain micro-structural information of foods undergoing different physical and chemical changes. In this study, high-resolution X-ray micro-CT was used for non-destructive analysis of the internal structure of maize kernels infected with Fusarium verticillioides. The major anatomical features of the maize kernel were identified based on their differences in X-ray attenuation, i.e. the germ, scutellum, vitreous and floury endosperm. Fungal infection caused changes in the internal structure of the kernels over time, which included a decrease in total kernel volume and an increase in total volume of void space, with more voids observed in the germ and floury endosperm regions. No significant (P > 0.05) difference was observed between the control and the infected kernels; it was apparent that the changes observed in the infected kernels were not solely as a result of fungal growth. The grey level histograms of the control and infected kernels shifted to the lower grey value intensity range over time indicating an increase in void space within the kernels. In the 3D images, the increase in total volume of void space with fungal progression was clearer and the effect of fungal damage on the internal structure was evident.     

Weight loss and metabolic profiles in obese individuals using two different approaches

Intentional weight loss with a reduction in adipose tissue is associated with an improvement in medical complications linked to obesity. The aim of this study was to compare the effect of two different weight loss diets on obese individuals (BMI 30-40 kg m(-2)) for improvements in anthropometric measurements and blood biomarkers. Study 1 comprised a low energy diet (LED) of 5000-6000 kJ d(-1) for a slow but steady weight loss over 12 weeks. Study 2 comprised a very low energy diet (VLED) of 3000 kJ d(-1) using meal replacements for rapid weight loss over a shorter period of 4 weeks followed by 10 weeks of weight stabilisation to prevent rebound after rapid weight loss. Nutrition information sessions were given to both groups. Fasting blood samples, anthropometric measurements and 3-day food diaries were collected at baseline and again at completion of weight loss, at 12 weeks for LED group and 4 weeks for VLED group. Mean weight loss in the LED group (n = 18) was -3.17 kg (-3.7%) compared to a -6.54 kg (-7%) loss in the VLED group (n = 14) (p < 0.001). The VLED group experienced significantly greater reductions in fat mass, -13.9% compared to -8.9% for the LED group (p < 0.05). Significantly greater reductions in blood glucose (p < 0.05), cholesterol and LDL-C (p < 0.01 for both), and waist circumference (p≤ 0.05) were noted in VLED compared to those for LED diet. Short-term, rapid weight loss produced the desired ≥5% weight loss suggested to substantially reduce metabolic abnormalities associated with obesity, and to reduce health risks.     

Factors affecting bulk to total bacteria ratio in drinking water distribution systems

Bacteria in drinking water systems can grow in bulk water and as biofilms attached to pipe walls, both causing regrowth problems in the distribution system. While studies have focused on evaluating the factors influencing the bacteria in bulk water and in biofilms separately, there is a need for understanding biofilm characteristics relative to the bulk water phase. The current study evaluated the effects of chlorine and residence time on the presence of culturable bacteria in biofilms relative to that in bulk water. The results showed that when no chlorine residual was present in the system, the median ratio of bulk to total bacteria was 0.81, indicating that 81% of the bacteria were present in bulk water, whereas only 19% were present in the biofilm. As chlorine concentration increased to 0.2, 0.5, and 0.7 mg/L, the median percentage of bacteria present in bulk water decreased to 37, 28, and 31, respectively. On the other hand, as the residence times increased to 8.2, 12, 24, and 48h, the median percentage of bacteria present in bulk water increased to 7, 37, 58, and 88, respectively, in the presence of a 0.2mg/L chlorine residual. The common notion that biofilms dominate the distribution system is not true under all conditions. These findings suggest that bulk water bacteria may dominate in portions of a distribution system that have a low chlorine residual.     

Interplay between Humoral and CLA+ T Cell Response against Candida albicans in Psoriasis

Candida albicans (CA) infections have been associated with psoriasis onset or disease flares. However, the integrated immune response against this fungus is still poorly characterized in psoriasis. We studied specific immunoglobulins in plasma and the CA response in cocultures of circulating memory CD45RA⁻ cutaneous lymphocyte antigen (CLA)^+/− T cell with autologous epidermal cells from plaque and guttate psoriasis patients (cohort 1, n = 52), and also healthy individuals (n = 17). A complete proteomic profile was also evaluated in plaque psoriasis patients (cohort 2, n = 114) regarding their anti-CA IgA levels. Increased anti-CA IgA and IgG levels are present in the plasma from plaque but not guttate psoriasis compared to healthy controls. CA cellular response is confined to CLA⁺ T cells and is primarily Th17. The levels of anti-CA IgA are directly associated with CLA⁺ Th17 response in plaque psoriasis. Proteomic analysis revealed distinct profiles in psoriasis patients with high anti-CA IgA. C-C motif chemokine ligand 18, chitinase-3-like protein 1 and azurocidin were significantly elevated in the plasma from plaque psoriasis patients with high anti-CA levels and severe disease. Our results indicate a mechanism by which Candida albicans exposure can trigger a clinically relevant IL-17 response in psoriasis. Assessing anti-CA IgA levels may be useful in order to evaluate chronic psoriasis patients.     

Fe(PyTACN)‐Catalyzed cis‐Dihydroxylation of Olefins with Hydrogen Peroxide

A family of iron complexes with general formula [Fe(II)(^R,Y,XPyTACN)(CF₃SO₃)₂], where ^R,Y,XPyTACN=1‐[2′‐(4‐Y‐6‐X‐pyridyl)methyl]‐4,7‐dialkyl‐1,4,7‐triazacyclononane, X and Y refer to the groups at positions 4 and 6 of the pyridine, respectively, and R refers to the alkyl substitution at N‐4 and N‐7 of the triazacyclononane ring, are shown to be catalysts for efficient and selective alkene oxidation (epoxidation and cis‐dihydroxylation) employing hydrogen peroxide as oxidant. Complex [Fe(II)(^Me,Me,HPyTACN)(CF₃SO₃)₂] ( 7 ), was identified as the most efficient and selective cis‐dihydroxylation catalyst among the family. The high activity of 7 allows the oxidation of alkenes to proceed rapidly (30 min) at room temperature and under conditions where the olefin is not used in large amounts but instead is the limiting reagent. In the presence of 3 mol% of 7 , 2 equiv. of H₂O₂ as oxidant and 15 equiv. of water, in acetonitrile solution, alkenes are cis‐dihydroxylated reaching yields that might be interesting for synthetic purposes. Competition experiments show that 7 exhibits preferential selectivity towards the oxidation of cis olefins over the trans analogues, and also affords better yields and high [syn‐diol]/[epoxide] ratios when cis olefins are oxidized. For aliphatic substrates, reaction yields attained with the present system compare favourably with state of the art Fe‐catalyzed cis‐dihydroxylation systems, and it can be regarded as an attractive complement to the iron and manganese systems described recently and which show optimum activity against electron‐deficient and aromatic olefins.     

Singlet Oxygen Detection Using Red Wine Extracts as Photosensitizers

Moderate consumption of red wine provides beneficial effects to health. This is attributed to polyphenol compounds present in wine such as resveratrol, quercetin, gallic acid, rutin, and vanillic acid. The amount of these antioxidants is variable; nevertheless, the main beneficial effects of red wine are attributed to resveratrol. However, it has been found that resveratrol and quercetin are able to photosensitize singlet oxygen generation and conversely, gallic acid acts as quencher. Therefore, and since resveratrol and quercetin are some of the most important antioxidants reported in red wines, the aim of this research was to evaluate the photosensitizing ability of 12 red wine extracts through photo‐oxidation of ergosterol. The presence of ¹O₂ was detected by ergosterol conversion into peroxide of ergosterol through ¹H NMR analysis. Our results showed that 10 wine extracts were able to act as photosensitizers in the generation of singlet oxygen. The presence of ¹O₂ can damage other compounds of red wine and cause possible organoleptic alterations. Finally, although the reaction conditions employed in this research do not resemble the inherent conditions in wine making processing or storing, or even during its consumption, this knowledge could be useful to prevent possible pro‐oxidant effects and avoid detrimental effects in red wines.