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Preemptive antifungal therapy: Do diagnostics help?
Published in Mahmoud A. Ghannoum, John R. Perfect, Antifungal Therapy, 2019
Vidya Jagadeesan, Margaret Powers-Fletcher, Kimberly E. Hanson
The FDA cleared protocol for Fungitell defines a single positive value of ≥80 pg/mL as positive, and levels from 60 to 79 pg/mL as “equivocal” results. The ≥80 pg/mL was established in a multicenter validation study [85]. It is important to note that optimal thresholds may be different for children and that these have not been extensively defined [86]. Additionally, the Fungitell cut-off is higher than the values established for the Fungitec-G and Wako assays. A 20 pg/mL threshold was set for Fungitec-G test based on the observation that plasma BDG levels in 60 healthy adult controls were all <10 pg/mL and 37 of 41 patients with autopsy-verified or microbiologically documented fungal infections had concentrations above this level (sensitivity 90% [95% CI; 77%–97%]) [87]. At a concentration >20 pg/mL, the Fungitec-G test also showed 100% specificity when analyzing 59 samples from subjects with non-fungal infection or fever of non-infectious origin [87]. The dissimilarity of cut-off values among chromogenic test kits may be related to differences in the affinity/reactivity of reagents used in each assay. Reagents utilized in the test Fungitell are extracted from Limulus polyphemus, a different genera of horseshoe crab than is used in Fungitec-GT (Tachypleus tridentatus) [84].
Application of anti-Xa assay in monitoring unfractionated heparin therapy in contemporary antithrombotic management
Published in Expert Review of Hematology, 2023
Michael Safani, Steve Appleby, Ryan Chiu, Emmanuel J Favaloro, Emanuel T. Ferro, Jimmy Johannes, Milan Sheth
5.1. The anti-Xa assay represents a functional assay that measures antithrombin-catalyzed inhibition of factor Xa only and does not account for anti-IIa activity of UFH. The UFH in the plasma sample inhibits the enzymatic conversion of a Xa-specific chromogenic substrate to a colored product by factor Xa. The chromogen generated is proportional to the amount of residual factor Xa remaining after neutralization by the UFH-anti-thrombin complex and is inversely proportional to the UFH concentration [42]. There is a significant positive correlation between UFH dose and anti-Xa activity and this linear relationship was demonstrated in a neonatal and pediatric ECMO trial [43,44]. This association remained significant even after adjusting for anti-thrombin levels [44]. Calibration curves are created using multiple concentrations of UFH to calculate the UFH concentration in the patient plasma. Notably, in some assays, antithrombin is added to the test while in others it is not. Therefore, in assays with supplemented antithrombin, the results of the in vitro test may not reflect the patient’s in vivo heparin activity [35].
The inhibitory effect of boric acid on hypoxia-regulated tumour-associated carbonic anhydrase IX
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2022
Zainab Saad Yusuf, Tugba Kevser Uysal, Ender Simsek, Alessio Nocentini, Sameh Mohamed Osman, Claudiu T. Supuran, Özen Özensoy Güler
For determination of protein levels, cell lysates of the six-well plates were seeded one day before the experiment and treated with the determined IC50 concentration of BA or AZA for 24 h under normoxic and hypoxic conditions. Cells were washed with PBS and then were treated in a lysis buffer (1% NP-40, 50 mM Tris, and 150 mM NaCl) with protease inhibitors on ice. The lysates were collected by centrifugation at 13,000 × g for 15 min at 4 °C. These supernatants were collected, and the aliquots were stored at −20 °C. These supernatants were used to detect the levels of CA IX and HIF-1α levels using commercially available enzyme-linked immunosorbent assay (ELISA) kits. These protein levels were determined by using CA IX ELISA kit and HIF1α ELISA kit according to the manufacturer’s instructions. Of 40 µL of the cell lysates was seeded into the microplates and with 10 µL of protein conjugate and 50 µL of streptavidin-horseradish to reach a volume of 100 µL. The microplates were incubated at 37 °C for 1 h and then washed five times with buffer. Of 50 µL of chromogen A and 50 µL of chromogen B were added to the plates and incubated at 37 °C for 10 min. Of 50 µL of the stop solution was then added and the microplates were read at 450 nm using the Epoch micro-plate reader (BioTek, Cambridge, MA).
Quercetin and Luteolin Improve the Anticancer Effects of 5-Fluorouracil in Human Colorectal Adenocarcinoma In Vitro Model: A Mechanistic Insight
Published in Nutrition and Cancer, 2022
Mehmet Kadir Erdoğan, Can Ali Ağca, Hakan Aşkın
The determination of vascular endothelial growth factor (VEGF), which is an important angiogenesis regulator in cancer formation, was determined by the human VEGF ELISA method (61). 5 × 104 cells were treated for 24 h with IC50 doses of quercetin, luteolin, 5-FU and combinations. Medium was removed, cells were washed with cold PBS and collected with a scraper, incubated in a mixture of incubation buffer and standard diluent buffer for 2 h at room temperature and washed with wash buffer. Then, respectively, HU-VEGF biotin conjugate and streptavidin-HRP reagents were added and incubated for 30 min. After aspiration and washing, stabilized chromogen was added and kept in the dark at room temperature for 30 min. Stop solution was then added and after 2 h the absorbances were measured at 450 nm wavelength with ELISA reader. The standard curve was plotted for each sample, the results were compared to the standard curve, and the amount of VEGF for each treatment group was calculated as pg/ml.