•A novel laccase-modified screen printed electrode (SPE) has been developed.•Bionanocomposite sensor is optimized in terms of enzyme loading, pH and applied potential.•Linear range, sensitivity and ...LOD are assessed for BPA determination.•The biosensor has been challenged in tomato juice samples.
The relevance of Bisphenol A (BPA) in human health is well-known. For this reason we designed and developed a biosensor based on a bionanocomposite (laccase–thionine–carbon black)-modified screen-printed electrode. Thionine, a commercially available dye, was used as electrochemical mediator coupled with a nanostructured carbon black. By means of cyclic voltammetry, the interaction of thionine adsorbed on modified screen printed electrode with laccase/BPA reaction products has been studied. In addition, the immobilization of laccase by physical adsorption on the surface of thionine–carbon black modified screen printed electrodes was investigated. The response of the biosensor has been optimized in terms of enzyme loading, pH and applied potential reaching a linear concentration range of 0.5–50μM, a sensitivity of 5.0±0.1nA/μM and a limit-of-detection (LOD) of 0.2μM. The developed biosensor has been also challenged in tomato juice samples contained in metallic cans where release of BPA due to the epoxy resin coating can be assumed. A satisfactory recovery value comprised between 92% and 120% was obtained.
Three‐dimensional mixed‐mode crack propagation simulations were performed by means of the dual boundary element method code BEASY and 2 finite element method‐based crack propagation codes: ZENCRACK ...(ZC) and CRACKTRACER3D (CT3D). The stress intensity factors (SIFs) along the front of an initial semielliptical crack, initiated from the external surface of a shaft, were calculated for 4 different load cases: bending, press fit, shear, and torsion. The methods used for the SIF assessment along the crack front were the J‐integral for BEASY and ZC and the quarter point element stress method for CT3D. Subsequently, crack propagation simulations were performed, with the crack growth rate evaluated by using Paris' law, calibrated for the material at stake (American Society for Testing and Materials A469 steel). The kink angles were evaluated by using the minimum strain energy density and maximum tangential stress criteria for BEASY, the maximum energy release rate and maximum tangential stress for ZC, and the maximum principal asymptotic stress for CT3D. The results obtained in terms of SIFs and crack propagation life show very good agreement among the 3 codes. Also, the shape of the propagated crack, which is significantly out‐of‐plane for the shear and torsion loading, matched very well.
A thionine-modified carbon paste electrode for catechol and Bisphenol A (BPA) detection is presented. Graphite powder was modified by adsorbing thionine as electrochemical mediator. The ...electrochemical response of the modified carbon paste electrode (CPE) was determined before electrode modification with tyrosinase. Then, tyrosinase was added in order to assemble a biosensor. Once established the best operative conditions, an interelectrode reproducibility around 7% was obtained and the resulting biosensor showed improved sensitivities and (
S
=
139.6
±
1.1
nA/μM for catechol and
S
=
85.4
±
1.5
nA/μM for BPA) in comparison with the biosensor constructed without thionine (
S
=
104.4
±
0.5
nA/μM for catechol and
S
=
51.1
±
0.6
nA/μM for BPA) and low detection limits (0.15
μM for both the electrodes and analytes). Also the comparison with the results reported in the literature showed higher sensitivity and lower detection limit for our biosensor. Moreover the functioning of the thionine-tyrosinase CPE was validated following a biodegradation process of water polluted by BPA and comparing the time changes of BPA concentration inferred by the biosensor calibration curve and those determined by means of HPLC measurements.
This paper deals with the simulation of multiple crack propagation in friction stir welded butt joints and the aim is to assess the influence of process induced residual stresses on the fatigue ...behavior of the assembly. The distribution of the process induced residual stresses is mapped by means of the contour method; then, the computed residual stress field is superimposed, in the DBEM environment, to the stress field due to a remote fatigue traction load and the crack growth is simulated.
A two-parameters crack growth law is used for the crack propagation rate assessment. The Stress Intensity Factors are evaluated by the J-integral technique. Computational results have been compared with experimental data, provided by constant amplitude crack propagation tests on welded samples, showing the subdivision of the overall fatigue life in the two periods of crack initiation and crack propagation.
•Raman spectroscopy on nuclear and membrane regions of single human neuroblastoma cells.•Measurements are performed after irradiation by clinically relevant X-ray doses.•The acquired spectra are ...analyzed by PCA and interval-PCA methods.•The most relevant effects are evidenced in the spectra from the cell nucleus region.•Potential markers of an apoptotic process could be singled out for some X- ray doses.
Raman micro-spectroscopy was performed in vitro on nuclear and membrane regions of single SH-SY5Y human neuroblastoma cells after irradiation by graded X-ray doses (2, 4, 6, 8 Gy). The acquired spectra were analyzed by principal component analysis (PCA) and interval-PCA (i-PCA) methods. Biochemical changes occurring in the different regions of single cells as a consequence of the radiation exposure were observed in cells fixed immediately after the irradiation. The most relevant effects arose from the analysis of the spectra from the cell nucleus region. The observed changes were discussed in terms of the modifications in the cell cycle, resulting in an increase in the DNA-related signal, a protein rearrangement and changes in lipid and carbohydrates profiles within the nucleus. Potential markers of an apoptotic process in cell population irradiated with 6 and 8-Gy X-ray doses could have been singled out. No significant effects were found in spectra from cells fixed 24 h after the irradiation, thus suggesting the occurrence of repairing processes of the X-ray induced damage.
•Cellular acute heat shock response based on Fourier transform-IR spectroscopy.•Early cellular response to heat shock induces rearrangements of cellular components.•Cytoplasmic heat shock protein ...genes are essential to counteract acute heat shock.•Oxidative stress is not involved in early cellular response to heat shock.•Activation of early defence mechanisms may be decisive for cellular thermotolerance.
Heat stress is a significant challenge in dairy cattle herds, affecting milk production and quality, and generating important changes at the cellular level. Most in vitro research on heat shock (HS) effects on dairy cow mammary cells was focused on medium-long-term effects. In recent years, Fourier transform-infrared (FT-IR) micro-spectroscopy has been increasingly used to study the effects of several external stresses on different cell lines, down to the level of single cellular components, such as DNA/RNA, lipids, and proteins. In this study, the possible changes at the biochemical and molecular level induced by acute (30 min-2 h) HS in bovine mammary epithelial (BME-UV1) cells were investigated. The cells were exposed to different temperatures, thermoneutral (TN, 37 °C) and HS (42 °C), and FT-IR spectra were acquired to analyse the effects of HS on biochemical characteristics of BME-UV1 cellular components (proteins, lipids, and DNA/RNA). Moreover, cell viability assay, reactive oxygen species production, and mRNA expression of heat shock proteins (HSPA1A, HSP90AA1, GRP78, GRP94) and antioxidant genes (SOD1, SOD2) by RT-qPCR were also analysed. The FT-IR results showed a change already at 30 min of HS exposure, in the content of long-chain fatty acids, which probably acted as a response to a modification of membrane fluidity in HS cells compared with TN cells. After 2 h of HS exposure, modification of DNA/RNA activity and accumulation of aggregated proteins was highlighted in HS cells. The gene expression analyses showed the overexpression of HSPA1A and HSP90AA1 starting from 30 min up to 2 h in HS cells compared with TN cells. At 2 h of HS exposure, also the overexpression of GRP94 was observed in HS cells. Acute HS did not affect cell viability, reactive oxygen species level, and SOD1 and SOD2 gene expression of BME-UV1 cells. According to the results obtained, cells initiate early defence mechanisms in case of acute HS and probably this efficient response capacity may be decisive for tolerance to heat stress of dairy cattle.
•There is a strong experimental effort to validate the numerical procedure.•The proposed procedure predict residual fatigue life under mixed mode conditions.•A comparison is provided between ...different criteria for crack path assessment.
The subject for studies is a steel bar of circular cross-section with straight-fronted edge notch undergoing fatigue loads. Both the optical microscope measurements and the crack opening displacement (COD) method are used to monitor and investigate both crack depth and crack length during the tests. The variation of crack growth behavior is studied under cyclic axial and combined tension+torsion fatigue loading. Results show that cyclic Mode III loading superimposed on the cyclic Mode I leads to a fatigue life reduction. In parallel to the experimental activity, numerical calculations are performed based on three-dimensional DBEM analysis to determine the stress intensity factors along curvilinear surface crack front and fatigue life prediction. The experimental fatigue crack growth results obtained from round bar specimens have been compared with the numerical predictions. The computational DBEM results are found to be in satisfactory agreement with the experimental findings.
MicroRNAs are highly conserved, noncoding RNAs involved in post-transcriptional gene silencing. They have been shown to participate in a wide range of biological processes, including myogenesis and ...muscle regeneration. The goal of this study is to test the hypothesis that myo-miRs (myo = muscle + miR = miRNA) expression is altered in muscle from patients affected by myotonic dystrophy type 1 (DM1), the most frequently inherited neuromuscular disease in adults. In order to gain better insights about the role of miRNAs in the DM1 pathogenesis, we have also analyzed the muscular expression of miR-103 and miR-107, which have been identified in silico as attractive candidates for binding to the DMPK mRNA.
To this aim, we have profiled the expression of miR-133 (miR-133a, miR-133b), miR-1, miR-181 (miR-181a, miR-181b, miR-181c) and miR-206, that are specifically induced during myogenesis in cardiac and skeletal muscle tissues. miR-103 and miR-107, highly expressed in brain, heart and muscle have also been included in this study. QRT-PCR experiments have been performed on RNA from vastus lateralis biopsies of DM1 patients (n = 7) and control subjects (n = 4). Results of miRNAs expression have been confirmed by Northern blot, whereas in situ hybridization technique have been performed to localize misexpressed miRNAs on muscle sections from DM1 and control individuals.
Only miR-206 showed an over-expression in 5 of 7 DM1 patients (threshold = 2, fold change between 1.20 and 13.22, average = 5.37) compared to the control group. This result has been further confirmed by Northern blot analysis (3.37-fold overexpression, R2 = 0.89). In situ hybridization localized miR-206 to nuclear site both in normal and DM1 tissues. Cellular distribution in DM1 tissues includes also the nuclear regions of centralized nuclei, with a strong signal corresponding to nuclear clumps.
This work provides, for the first time, evidences about miRNAs misexpression in DM1 muscle tissues, adding a new element in the pathogenesis of this complex genetic disease.
The high frequency of
-promoting mutations and the increased expression of
mRNA in anaplastic thyroid cancer (ATC) make TERT a suitable molecular target for the treatment of this lethal neoplasm. In ...this study, we encapsulated an anti-
oligonucleotide in biocompatible nanoparticles and analyzed the effects of this novel pharmaceutical preparation in preclinical models of ATC. Biocompatible nanoparticles were obtained in an acidified aqueous solution containing chitosan, anti-
oligoRNAs, and poloxamer 188 as a stabilizer. The effects of these anti-
nanoparticles (Na-siTERT) were tested
on ATC cell lines (CAL-62 and 8505C) and
on xenograft tumors obtained by flank injection of CAL-62 cells into SCID mice. The Na-siTERT reduced the viability and migration of CAL-62 and 8505C cells after 48-hour incubation. Intravenous administration (every 48 hours for 13 days) of this encapsulated drug in mice hosting a xenograft thyroid cancer determined a great reduction in the growth of the neoplasm (about 50% vs. untreated animals or mice receiving empty nanoparticles), and decreased levels of Ki67 associated with lower hTERT expression. Moreover, the treatment resulted in minimal invasion of nearby tissues and reduced the vascularity of the xenograft tumor. No signs of toxicity appeared following this treatment. Telomere length was not modified by the Na-siTERT, indicating that the inhibitory effects of neoplasm growth were independent from the enzymatic telomerase function. These findings demonstrate the potential suitability of this anti-TERT nanoparticle formulation as a novel tool for ATC treatment.
.
Olive oil is the main fat source of the Mediterranean diet. This seasoning ingredient is highly appreciated for its unique taste, functional properties, and positive impact on human health. The ...determination of chemical composition is a demanding task in order to fully characterize this precious food product, ensure its quality, and prevent fraudulent practices. Among innovative techniques proposed for the oil analysis, surface-enhanced Raman spectroscopy (SERS) can be an extremely useful tool for olive oil characterization. In this frame, we have investigated five noncommercial olive oils produced in different parts of South Italy by using a commercial Raman microspectroscopy apparatus and home-made signal-enhancing SERS substrates. A wavelet-based data analysis has allowed us to efficiently remove the background and the noise from the acquired spectra. The analysis of these SERS spectra has enabled the quantification of the relative contents of carotene, oleic acid, and phenols. These relative contents differ in the examined samples. In addition, SERS response in the lipid region has indicated differences in the relative abundance of saturated fatty acids. The present results confirm the validity of the SERS technique as a rapid, nondestructive, and reliable analytical technique for identifying olive oil bioactive components.
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