Lundingsanders5468

High performance liquid chromatography with ultra-violet detection (HPLC-UV) and gas chromatography-mass spectrometry (GC-MS) methods were developed and validated for the determination of chlorambucil (CLB) and valproic acid (VPA) in plasma, as a part of experiments on their anticancer activity in chronic lymphocytic leukemia (CLL). CLB was extracted from 250 µL of plasma with methanol, using simple protein precipitation and filtration. Chromatography was carried out on a LiChrospher 100 RP-18 end-capped column using a mobile phase consisting of acetonitrile, water and formic acid, and detection at 258 nm. The lowest limit of detection LLOQ was found to be 0.075 μg/mL, showing sufficient sensitivity in relation to therapeutic concentrations of CLB in plasma. The accuracy was from 94.13% to 101.12%, while the intra- and inter-batch precision was ≤9.46%. For quantitation of VPA, a sensitive GC-MS method was developed involving simple pre-column esterification with methanol and extraction with hexane. Chromatography was achieved on an HP-5MSUI column and monitored by MS with an electron impact ionization and selective ion monitoring mode. Using 250 µL of plasma, the LLOQ was found to be 0.075 μg/mL. The accuracy was from 94.96% to 109.12%, while the intra- and inter-batch precision was ≤6.69%. Thus, both methods fulfilled the requirements of FDA guidelines for the determination of drugs in biological materials.This study aimed to describe glutathione peroxidase 4 (GPx4) in rat oocytes, preimplantation embryos, and female genital organs. After copulation, Sprague Dawley female rats were euthanized with anesthetic on the first (D1), third (D3), and fifth days of pregnancy (D5). Ovaries, oviducts, and uterine horns were removed, and oocytes and preimplantation embryos were obtained. Immunohistochemical, immunofluorescent, and Western blot methods were employed. Using immunofluorescence, we detected GPx4 in both the oocytes and preimplantation embryos. Whereas in the oocytes, GPx4 was homogeneously diffused, in the blastomeres, granules were formed, and in the blastocysts, even clusters were present mainly around the cell nuclei. Employing immunohistochemistry, we detected GPx4 inside the ovary in the corpus luteum, stroma, follicles, and blood vessels. In the oviduct, the enzyme was present in the epithelium, stroma, blood vessels, and smooth muscles. In the uterus, GPx4 was found in the endometrium, myometrium, blood vessels, and stroma. Moreover, we observed GPx4 positive granules in the uterine gland epithelium on D1 and D3 and cytoplasm of fibroblasts forming in the decidua on D5. read more Western blot showed the highest GPx4 levels in the uterus and the lowest levels in the ovary. Our results show that the GPx4 is necessary as early as in the preimplantation development of a new individual because we detected it in an unfertilized oocyte in a blastocyst and not only after implantation, as was previously thought.Multiplex PCR methods have been frequently used for authentication of meat product adulteration. Through screening of new species-specific primers designed based on the mitochondrial DNA sequences, a septuple PCR method is ultimately developed and optimized to simultaneously detect seven species including turkey (110 bp), goose (194 bp), pig (254 bp), sheep (329 bp), beef (473 bp), chicken (612 bp) and duck (718 bp) in one reaction. The proposed method has been validated to be specific, sensitive, robust and inexpensive. Taken together, the developed septuple PCR assay is reliable and efficient, not only to authenticate animal species in commercial meat products, but also easily feasible in a general laboratory without special infrastructures.In this work, a new family of poly(thiourethane) shape memory thermosetting actuators was developed and characterized. These materials can be easily prepared from mixtures of two different aliphatic diisocyanates and a trithiol in the presence of a latent catalyst, allowing an easy manipulation of the formulation. Rheological studies of the curing process confirm the latent character of the formulations. The glass transition temperatures and the mechanical properties can be modified by varying the proportion of diisocyanates (hexamethylene diisocyanate, HDI, and isophorone diisocyanate, IPDI) with stoichiometric amounts of trimethylolpropane tris(3-mercaptopropionate). The shape-memory behavior was deeply investigated under three different conditions unconstrained, partially constrained, and fully constrained. Tests were performed in single cantilever bending mode to simulate conditions closer to real complex mechanics of thermomechanical actuators under flexural performances. The complex recovery process in single cantilever bending mode was compared with that obtained using tensile mode. The results evidenced that the amount of recovery force in fully constrained conditions, or energy released during the recovery process in partially constrained, can be modulated by simply changing the proportion of both diisocyanates. A simple model based on Timoshenko beam theory was used for the prediction of the amount of work performed. The reported results are an important guideline to design shape-memory materials based on poly(thiourethane) networks, establishing criteria for the choice of the material depending on the expected application.The clinical relevance of circulating tumor cell clusters (CTC-clusters) in breast cancer (BC) has been mostly studied using the CellSearch®, a marker-dependent method detecting only epithelial-enriched clusters. However, due to epithelial-to-mesenchymal transition, resorting to marker-independent approaches can improve CTC-cluster detection. Blood samples collected from healthy donors and spiked-in with tumor mammospheres, or from BC patients, were processed for CTC-cluster detection with 3 technologies CellSearch®, CellSieve™ filters, and ScreenCell® filters. In spiked-in samples, the 3 technologies showed similar recovery capability, whereas, in 19 clinical samples processed in parallel with CellSearch® and CellSieve™ filters, filtration allowed us to detect more CTC-clusters than CellSearch® (median number = 7 versus 1, p = 0.0038). Next, samples from 37 early BC (EBC) and 23 metastatic BC (MBC) patients were processed using ScreenCell® filters for attaining both unbiased enrichment and marker-independent identification (based on cytomorphological criteria).