Properties and Application of Cell-Free DNA as a Clinical Biomarker

Biomarkers are valuable tools in clinical practice. In 2001, the National Institutes of Health (NIH) standardized the definition of a biomarker as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. A biomarker has clinical relevance when it presents precision, standardization and reproducibility, suitability to the patient, straightforward interpretation by clinicians, and high sensitivity and/or specificity by the parameter it proposes to identify. Thus, serum biomarkers should have advantages related to the simplicity of the procedures and to the fact that venous blood collection is commonplace in clinical practice. We described the potentiality of cfDNA as a general clinical biomarker and focused on endothelial dysfunction. Circulating cell-free DNA (cfDNA) refers to extracellular DNA present in body fluid that may be derived from both normal and diseased cells. An increasing number of studies demonstrate the potential use of cfDNA as a noninvasive biomarker to determine physiologic and pathologic conditions. However, although still scarce, increasing evidence has been reported regarding using cfDNA in cardiovascular diseases. Here, we have reviewed the history of cfDNA, its source, molecular features, and release mechanism. We also show recent studies that have investigated cfDNA as a possible marker of endothelial damage in clinical settings. In the cardiovascular system, the studies are quite new, and although interesting, stronger evidence is still needed. However, some drawbacks in cfDNA methodologies should be overcome before its recommendation as a biomarker in the clinical setting.     

Characterisation of the vegetable rennets used for ‘Torta del Casar’ cheesemaking by a protein profile method

The purpose of this work was to develop a cheap, easy and fast procedure, based on protein analysis, that can be used in the characterisation of coagulants from thistle species, which can be used in Torta del Casar cheesemaking. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis showed high efficiency in the identification of Cynara cardunculus from other thistle species and in discriminating between the technological properties, milk‐clotting and casein degradation activities of the vegetable rennets used in this study. This technique allowed for the grouping of aqueous extracts from the flowers of C. cardunculus into five different groups, which exhibited variable ability to degrade milk caseins.     

Multivariate analysis of commercial and laboratory produced Sherry wine vinegars: influence of acetification and aging

One of the problems in the characterization of vinegars is the wide range of values obtained for the main physicochemical and sensorial parameters. The production method, raw material, and wood aging are factors of variation among wine vinegars. Twenty-seven vinegar samples corresponding to different wine substrates (Sherry wine, white wine) and different acetification methods (traditional method, acetification by submerged culture in acetators) were used to perform this study. Selected variables include those that play a major role in the final quality: aroma compounds and organic acids. Principal component analysis (PCA), cluster analysis (CA), linear discriminant analysis (LDA), and classification by artificial neural networks training by back-propagation (BPNN) were used to carry out the characterization. Sherry vinegars made by traditional methods and obtained either in the market or in the wineries were differentiated from Sherry vinegars obtained in the laboratory fermentor (aged or not in wood). Thus, the acetification method plays a major role in the characterization when these variables are considered.     

Inactivation effect of an 18-T pulsed magnetic field combined with other technologies on Escherichia coli

The inactivation effect of 18 T pulsed magnetic fields in combination with selected non-thermal technologies was studied on Escherichia coli ATCC 11775. The bacteria were subjected to a treatment of either ultrasound (20 kHz, 70 W, 242 μm), high hydrostatic pressure (207 MPa, 5 min), pulsed electric field (6.25 kV/cm, 5.6 ms), or anti-microbials (Nisin 77.5 mg/l, lysozyme 1 mg/ml) and 50 magnetic field pulses (18 T, 30 μs). No additional inactivation or cell damage due to exposure to the pulsed magnetic field at 42°C was observed.     

Cyclic nucleotide phosphodiesterase inhibition increases tyrosine phosphorylation and hyper motility in normal and pathological human spermatozoa.

Our objective was to determine the effect of phosphodiesterase (PDE) inhibition on: 1) tyrosine phosphorylation of human spermatozoa at the tail level; and 2) sperm motion parameters and hyperactivated motility. The study was conducted with normozoospermic and asthenozoospermic samples incubated under in vitro capacitating conditions. The main outcome measures were computer-assisted sperm motion analysis and fluorescent immunodetection of phosphotyrosine-containing proteins. Pentoxifylline (PTX) was used as PDE inhibitor because of its wide use in the clinic. PTX-treatment significantly increased sperm velocity, hyperactivated motility and tyrosine-phosphorylation, both in normo and asthenozoospermic samples. Tyrosine-phosphorylation of tail proteins was highly conspicuous in both types of samples, showing no differential pattern after PTX-treatment. Normozoospermic samples treated with pentoxifylline showed an increase in the number of spermatozoa displaying hyperactivated movement and tyrosine-phosphorylation at the tail level. Preliminary data on asthenozoospermic samples exhibiting altered motion characteristics and defective phosphorylation of sperm-tail proteins showed that both defects can be concomitantly overcome by pentoxifylline treatment. Tyrosine-phosphorylation of sperm-tail proteins is underlying the enhancement of hyperactivated motility resulting from PDE inhibition by pentoxifylline.     

Stabilization of supported platinum nanoparticles on γ-alumina catalysts by addition of tungsten

The thermal stabilization of Al₂O₃ using W⁶⁺ ions has been found useful to the synthesis of Pt/Al₂O₃ catalysts. The simultaneous and sequential methods were used to study the effect of W⁶⁺ upon Pt/γ-Al₂O₃ reducibility, Pt dispersion, and benzene hydrogenation. The W/Pt atomic ratios were from 0.49 to 12.4. In the first method we found that the W⁶⁺ ions delayed reduction of a fraction of Pt⁴⁺ atoms beyond 773 K. At the same time, W⁶⁺inhibited sintering of the metallic crystallites once they were formed on the surface. For the sequential sample with a W/Pt atomic ratio of 3.28 W⁶⁺ did not inhibit the H₂ reduction of Pt oxides even below of 773 K, the Pt oxides were reduced completely. After reduction at 1073 K, sequential samples impregnating Pt on WO_x–γ-Al₂O₃ were more active and stable during benzene hydrogenation. TOF of the reaction did not change when the W/Pt atomic ratio, preparation technique and reduction temperature changed and its value was of 1.1 s⁻¹. W⁶⁺ ions promoted high thermal stability of Pt crystallites when sequential catalysts were reduced at 1073 K and decreased their Lewis acidity_.     

Normal Skin Cells Increase Aggressiveness of Cutaneous Melanoma by Promoting Epithelial-to-Mesenchymal Transition via Nodal and Wnt Activity

Melanoma is a lethal form of skin cancer triggered by genetic and environmental factors. Excision of early-stage, poorly aggressive melanoma often leads to a successful outcome; however, left undiagnosed these lesions can progress to metastatic disease. This research investigates whether the exposure of poorly aggressive melanoma to certain normal skin cells can explain how non-metastatic melanoma becomes more aggressive while still confined to the skin. To this end, we used a serial co-culture approach to sequentially expose cells from two different, poorly aggressive human melanoma cell lines against normal cells of the skin beginning with normal melanocytes, then epidermal keratinocytes, and finally dermal fibroblasts. Protein extraction of melanoma cells occurred at each step of the co-culture sequence for western blot (WB) analysis. In addition, morphological and functional changes were assessed to detect differences between the serially co-cultured melanoma cells and non-co-cultured cells. Results show that the co-cultured melanoma cells assumed a more mesenchymal morphology and displayed a significant increase in proliferation and invasiveness compared to control or reference cells. WB analysis of protein from the co-cultured melanoma cells showed increased expression of Snail and decreased levels of E-cadherin suggesting that epithelial-to-mesenchymal transition (EMT) is occurring in these co-cultured cells. Additional WB analysis showed increased levels of Nodal protein and signaling and signs of increased Wnt activity in the co-cultured melanoma cells compared to reference cells. These data suggest that interaction between poorly aggressive melanoma cells with normal cells of the skin may regulate the transition from localized, poorly aggressive melanoma to invasive, metastatic disease via Nodal and/or Wnt induced EMT.     

Methodology for assessment of statistical planning effects on the S‐N curve determination using Monte Carlo simulations

One of the most important input data in fatigue analysis is the material fatigue properties. This research aims to present a methodology for assessment of statistical planning of fatigue experiments through a MatLab algorithm developed based on Monte Carlo simulations, which enables to simulate statistically the effects of main parameters used for defining the fatigue test setting and to verify their impact on the relative percentage difference (RPD) in fatigue properties estimation comparing to a reference material. The aspects treated here have not been clearly discussed in the standards. Therefore, the proposed recommendations combined with standards procedures is a tool for test engineers, permitting a fatigue test planning with more background and precision, which can help in decisions about which is the better setup, including the sample size, number of stress levels, stress value in each level, and replication. The methodology and good practices presented in this paper were demonstrated by means of actual data from the literature.     

Amino-functionalized carbon nanotubes for effectively improving the mechanical properties of pre-impregnated epoxy resin/carbon fiber

Pre-impregnated carbon fiber/epoxy resin (CF/epoxy prepreg) gained its popularity for significant stress applications, especially in the aerospace industry, owing to its excellent resistance and low specific mass. However, these CF/epoxy prepregs have a tendency to crack propagation. A solution for the prepregs fragility is the addition of carbon nanotubes (CNTs), especially those functionalized with amino groups, reinforcing the material due to its exceptional mechanical properties. In this work, the influence of the carbon chain length of two different amino-functionalized CNTs from diverse backgrounds (commercial and laboratory growth CNTs) is studied. The nanofillers were added in CF/epoxy prepregs by dry spraying without solvent aid. CNTs' samples were characterized by X-ray photoelectron spectroscopy, Raman spectroscopy, transmission electron microscopy, and thermogravimetric analysis (TGA), while the composites were analyzed by TGA, dynamic-mechanical analysis, and field emission scanning electron microscopy. The various surface treatment occurred at different levels according to the CNTs background, and all samples exhibited a distinct behavior. These differences were also observed in the composites' thermomechanical performance: CNTs functionalized with larger carbon chain amine presented the best results, with an increase of almost 100% in the storage moduli (E'), confirming the efficiency of amino-functionalized CNTs in the reinforcement of CF/epoxy prepregs.