Optimizing detection of RET and PPARg rearrangements in thyroid neoplastic cells using a home-brew tetracolor probe

BACKGROUND Fluorescence in situ hybridization (FISH) to identify specific DNA target sequences in the nuclei of nondividing cells of numerous solid neoplasms has contributed to the introduction of molecular cytogenetics as a useful adjunct to cytology, leading recently to the "marriage" of the 2 disciplines. Numerous cancer molecular markers can now be investigated using different technical approaches, at both the gene and expression levels, in biopsies of various suspected cancers, including differentiated thyroid carcinoma. The limited amount of bioptic material is often insufficient to carry out multiple tests, and optimizing handling of the biopsy is desirable. METHODS We have developed a home-brew tetracolor break-apart probe able to simultaneously identify the 2 most common genetic alterations in differentiated thyroid carcinoma: RET/PTC variants in papillary thyroid carcinoma and PAX8/PPARg fusion and variants in follicular thyroid carcinoma. RESULTS The probe had 100% specificity, 99.5% sensitivity, and >= 3% cutoff. The probe was tested on RET/PTC and PAX8/PPARg RT-PCR positive controls, and feasibility was assessed in 368 thyroid nodule fine-needle aspirations (FNA). In the latter analysis, 24 FNAs had split RET signal, and 9 had split PPARg signal. FISH analysis of available surgically removed nodules confirmed the sensitivity of FISH in detecting abnormal clones and oligoclones. CONCLUSIONS The home-brew tetracolor probe showed high feasibility, optimizing the use of the biological material in relation to the available molecular tests and maximizing the FISH experimental and slide-scoring times. This probe may be considered an alternative to RT-PCR when recovery and quality of RNA amplification from FNA are insufficient

Molecular weight of hydroxyethyl starch: is there an effect on blood coagulation and pharmacokinetics?

Background. The development of hydroxyethyl starches (HES) with low impact on blood coagulation but higher volume effect compared with the currently used HES solutions is of clinical interest. We hypothesized that high molecular weight, low-substituted HES might possess these properties. Methods. Thirty pigs were infused with three different HES solutions (20 ml kg−1) with the same degree of molar substitution (0.42) but different molecular weights (130, 500 and 900 kDa). Serial blood samples were taken over 24 h and blood coagulation was assessed by Thromboelastograph® analysis and analysis of plasma coagulation. In addition, plasma concentration and in vivo molecular weight were determined and pharmacokinetic data were computed based on a two-compartment model. Results. Thromboelastograph analysis and plasma coagulation tests did not reveal a more pronounced alteration of blood coagulation with HES 500 and HES 900 compared with HES 130. In contrast, HES 500 and HES 900 had a greater area under the plasma concentration-time curve [1542 (142) g min litre−1, P<0.001, 1701 (321) g min litre−1, P<0.001] than HES 130 [1156 (223) g min litre−1] and alpha half life (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} $\mathrm{t}_{{\alpha}}^{{\frac{1}{2}}}$ \end{document}) was longer for HES 500 [53.8 (8.6) min, P<0.01] and HES 900 [57.1 (12.3) min, P<0.01] than for HES 130 [39.9 (10.7) min]. Beta half life (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} $\mathrm{t}_{{\beta}}^{{\frac{1}{2}}}$ \end{document}), however, was similar for all three types of HES [from 332 (100) to 381 (63) min]. Conclusions. In low-substituted HES, molecular weight is not a key factor in compromising blood coagulation. The longer initial intravascular persistence of high molecular weight low-substituted HES might result in a longer lasting volume effec

Synthesis, Antitumor and Antiviral In Vitro Activities of New Benzotriazole-Dicarboxamide Derivatives

Cancer and viral infections continue to threaten humankind causing death worldwide. Hence, the discovery of new anticancer and antiviral agents still represents a major scientific goal. Heterocycles designed to mimic the chemical structure of natural pyrimidines and purines have been designed over the years, exerting their activity acting as false substrates on several different targets. We reported a series of bis-benzotriazole-dicarboxamide derivatives which inhibit viral helicase of poliovirus, and hence we planned structure modifications to obtain different series of new dicarboxamides. Here, the synthesis and characterization of 56 new compounds: 31 bis-benzotriazole dicarboxamides and 25 mono-substituted acidic derivatives are reported. The synthesized compounds were tested for their antiviral and antitumor activity. Mostly, compounds 4a, 4c and 4d showed antiviral activity against tested Picornaviruses, Coxsackievirus B5 and Poliovirus-1. Likewise, four derivatives (3b, 3d, 4d, 9b) showed notable antiproliferative activity inhibiting cell growth in two distinct antitumor screenings. Compound 3b was selected as the antitumor lead compound for the wide range of activity and the potency proved. The lead compound was proved to induce apoptosis in SK-MES1 tumor cells, in a dose-dependent manner

Effects of ivabradine on residual myocardial ischemia after PCI evaluated by stress echocardiography

Background. Residual angina after PCI is a frequently occurring disease. Ivabradine improves symptoms but its role in patients without left ventricular systolic dysfunction is still unclear. The aim was to quantify the effects of ivabradine in terms of MVO 2 indicators and diastolic function. Methods. Twenty-eight consecutive patients with residual angina after PCI were randomized to ivabradine 5 mg twice/day (IG) or standard therapy (CG). All patients performed a stress echocardiography at the enrollment and after 30 days. MVO 2 was estimated from double product (DP) and triple product (TP) integrating DP with ejection time (ET). Diastolic function was evaluated determining E and A waves, E′ measurements, and E/E′ ratio both at rest and at the peak of exercise. Results. The exercise time was longer in IG 9′49″ ± 48″ vs 8′09″ ± 59″ in CG (p=0.0001), reaching a greater workload (IG 139.3 ± 13.4 vs CG 118.7 ± 19.6 Watts; p=0.003). MVO 2 expressed with DP and TP was significantly higher in IG (DP: IG 24194 ± 2697 vs CG 20358 ± 4671.8, p=0.01; TP: IG 17239 ± 4710 vs CG 12206 ± 4413, p=0.007). At peak exercise, the ET was diminished in IG than CG. The analysis of diastolic function after the exercise revealed an increase of E and A waves, without difference in the E/A ratio. The E′ wave was higher in IG than CG, and in the same group, the differences between baseline and peak exercise were greater (ΔE′3.14 ± 0.7 vs 2.4 ± 1.13, p=0.047). The E/E′ ratio was reduced in patients treated with ivabradine (IG 10.2 ± 2.0 vs CG 7.9 ± 1.6, p=0.002). Conclusions. Ivabradine seems to produce a significant improvement of ischemic threshold, chronotropic reserve, and diastolic function

Breed of goat affects the prediction accuracy of milk coagulation properties using Fourier-transform infrared spectroscopy

The prediction of traditional goat milk coagulation properties (MCP) and curd firmness over time (CFt) parameters via Fourier-transform infrared (FTIR) spectroscopy can be of significant economic interest to the dairy industry and can contribute to the breeding objectives for the genetic improvement of dairy goat breeds. Therefore, the aims of this study were to (1) explore the variability of milk FTIR spectra from 4 goat breeds (Camosciata delle Alpi, Murciano-Granadina, Maltese, and Sarda), and to assess the possible discriminant power of milk FTIR spectra among breeds, (2) assess the viability to predict coagulation traits by using milk FTIR spectra, and (3) quantify the effect of the breed on the prediction accuracy of MCP and CFt parameters. In total, 611 individual goat milk samples were used. Analysis of variance of measured MCP and CFt parameters was carried out using a mixed model including the farm and pendulum as random factors, and breed, parity, and days in milk as fixed factors. Milk spectra for each goat were collected over the spectral range from wavenumber 5,011 to 925 × cm−1. Discriminant analysis of principal components was used to assess the ability of FTIR spectra to identify breed of origin. A Bayesian model was used to calibrate equations for each coagulation trait. The accuracy of the model and the prediction equation was assessed by cross-validation (CRV; 80% training and 20% testing set) and stratified CRV (SCV; 3 breeds in the training set, one breed in the testing set) procedures. Prediction accuracy was assessed by using coefficient of determination of validation (R2VAL), the root mean square error of validation (RMSEVAL), and the ratio performance deviation. Moreover, measured and FTIR predicted traits were compared in the SCV procedure by assessing their least squares means for the breed effect, Pearson correlations, and variance heteroscedasticity. Results showed the feasibility of using FTIR spectra and multivariate analyses to correctly assign milk samples to their breeds of origin. The R2VAL values obtained with the CRV procedure were moderate to high for the majority of coagulation traits, with RMSEVAL and ratio performance deviation values increasing as the coagulation process progresses from rennet addition. Prediction accuracy obtained with the SCV were strongly influenced by the breed, presenting general low values restricting a practical application. In addition, the low Pearson correlation coefficients of Sarda breed for all the traits analyzed, and the heteroscedastic variances of Camosciata delle Alpi, Murciano-Granadina, and Maltese breeds, further indicated that it is fundamental to consider the differences existing among breeds for the prediction of milk coagulation traits

The first case exploring the health management of dogs owned by a man suffering from Ebola (EVD) in Italy

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Characterization of milk composition, coagulation properties, and cheese-making ability of goats reared in extensive farms

The aims of this study were to explore the variability of milk composition, coagulation properties, and cheese-making traits of the Sarda goat breed, and to investigate the effects of animal and farm factors, and the geographic area (Central-East vs. South-West) of an insular region of Italy, Sardinia. A total of 570 Sarda goats reared in 21 farms were milk-sampled during morning milking. Individual milk samples were analyzed for composition, traditional milk coagulation properties (MCP), modeled curd-firming over time parameters (CFt), and cheese-making traits (cheese yield, %CY; recovery of nutrients, %REC; daily cheese yield, dCY). Farms were classified into 2 categories based on milk energy level (MEL; high or low), defined according to the average net energy of milk daily produced by the lactating goats. Milk yield and composition were analyzed using a mixed model including the fixed effects of MEL, geographic area, days in milk, and parity, and the random effect of farm within MEL and geographic area. Data about MCP, CFt, and the cheese-making process were analyzed using the same model, with the inclusion of the effects of animal and pendulum of the lactodynamograph instrument, allowing the measure of repeatability of these traits. Results showed that animal had greater influence on coagulation and cheese-making traits compared with farm effect. Days in milk influenced milk composition, whose changes partly reflected the modifications of %CY traits. Moreover, large differences were observed between primiparous and multiparous goats: primiparous goats produced less milk of better quality (higher fat, lower somatic cell and bacterial counts) and less cheese, but with higher recovery of fat and protein in the curd, compared with multiparous goats. The repeatability was very high, for both coagulation (84.0 to 98.8%) and cheese-making traits (89.7 to 99.9%). The effect of MEL was significant for daily productions of milk and cheese, coagulation time, and recovery of protein in the curd, which were better in high-MEL farms. As regards geographic area, milk composition and percentage cheese yield were superior in the Central-East area, whereas daily milk and cheese production and MCP were better in the South-West. This result was explainable by the phenomenon of crossbreeding Sarda goats with Maltese bucks, which occurred with greater intensity in the South-West than in the Central-East area of the island. The results provided by this study could be of great interest for the goat dairy sector. Indeed, the methods described in the present study could be applicable for other farming methods, goat breeds, and geographic areas. The collection of a wide range of phenotypes at individual animal level is fundamental for the characterization of local populations and can be used to guarantee breed conservation and the persistence of traditional farming systems, and to increase farmers' profit