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Liposome/Viral Hybrid Gene Delivery Systems
Published in Kenneth L. Brigham, Gene Therapy for Diseases of the Lung, 2020
To eliminate the problem of low transfection efficiency and to determine whether plasmid replication, as opposed to retention, was responsible for the prolongation in transgene expression, cells were transfected and then grown in media containing hygromycin (38), which is toxic to cells. Since all four plasmids contained a hygromycin-resistance gene, only transfected cells would survive hygromycin. If plasmids replicated when cells divided, subsequent daughter cells would survive in hygromycin. With only retention of plasmids (as opposed to replication), plasmids would be diluted in subsequent generations, and daughter cells would begin to die. Finally, a hygromycin-resistance gene in a non-replicating plasmid could induce integration of the plasmid into the host genome and production of a population of dividing, hygromycin-resistant (and luciferase expressing) cells. As a control for the number of cells which might spontaneously develop resistance to hygromycin, cells were also transfected with the RSVluc plasmid, a plasmid expressing the luciferase gene under the control of the RSV promoter but not expressing the hygromycin-resistance gene.
Immunological Approaches
Published in Adorjan Aszalos, Modern Analysis of Antibiotics, 2020
Deborah E. Dixon, Susan J. Steiner, Stanley E. Katz
Foglesong and LeFeber [24] developed a RIA for determination of hygromycin B in feeds. Recoveries of 97–103% of hygromycin B were made from various feed mixtures. There was no significant cross-reactivity with the other antibiotics usually added in combination with hygromycin B. Monensin, tylosin, bacitracin, streptomycin, and chlortetracycline all showed less than 0.1% cross-reactivity with the hygromycin B antibody.
High-throughput discovery of novel small-molecule inhibitors of acid Ceramidase
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2023
Mazen Aseeri, José Luis Abad, Antonio Delgado, Gemma Fabriàs, Gemma Triola, Josefina Casas
The A375 cell line stably overexpressing ASAH1 under the control of a tetracycline/doxycycline-responsive promoter was kindly provided by Dr. Carmen Bedia and Prof. Thierry Levade17. The antibiotic selection of this cell line was performed with blasticidin (3 µg/mL) and hygromycin B (250 µg/mL). Ectopic expression of AC was induced with doxycycline at 1 µg/mL for 24 h before use. Cells were suspended in the appropriate volume of a 0.25 M saccharose solution with the proteases inhibitors aprotinin (1 mg/mL), leupeptin (1 mg/mmL) and PMSF (100 mM). The suspension was submitted to three cycles of a 5 s sonication (probe) at 10 watts/5 s resting on ice. The cell lysate was centrifuged at 600 g for 5 min. The supernatant was collected and protein concentration was determined with BSA as a standard using the bicinchoninic acid (BCA) protein determination kit (Thermo Scientific) according to the manufacturer’s instructions.
Identification of a new structural family of SGK1 inhibitors as potential neuroprotective agents
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2023
Ines Maestro, Enrique Madruga, Patricia Boya, Ana Martínez
U2OS-iMLS (± PRKN) FlpIn TRex cells. Human osteosarcoma U2OS cell line with stable inducible expression of the internal mitochondrial localisation signal (iMLS) (NIPSNAP11-53)-EGFP-mCherry33. They were generated with the FlpIn system using the pcDNA5-MLS-EGFP-mCherry plasmid. As those cells do not express PRKN, U2OS-iMLS were transduced with a lentiviral particle expressing PRKN. Both cell lines were culture with DMEM with glutamine supplemented with 10% FBS and 1% Pen-Strep at 37 °C and 5% CO2. Cells were selected with 100 µg/mL hygromycin and 5 µg/mL blasticidin. Additional 2 µg/mL puromycin were used for U2OS-iMLS-PRKN cells. To induce the expression of the mitophagy reporter, 500 µg/mL doxycycline was added for the last 24 h. These cells were obtained from Prof. Anne Simonsen Lab (Faculty of Medicine, Institute of Basic Medical Sciences and Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, University of Oslo, Norway).
Inhibitory effect of sixteen pharmaceutical excipients on six major organic cation and anion uptake transporters
Published in Xenobiotica, 2021
Ruicong Ma, Gentao Li, Xue Wang, Yajuan Bi, Youcai Zhang
Probenecid (PROB, 4-(dipropylsulfamoyl) benzoic acid), quinine (QUI), rifampicin (RIF), and poly-D-lysine were purchased from Sigma-Aldrich Chem Co. (St. Louis, MO, USA). 6-Carboxyfluorescein (6-CF) was obtained from Aladdin (Shanghai, China). 4-(4-(Dimethylamino) styryl)-N-methylpyridinium (ASP+) was purchased from Molecular Probes (Eugene, OR, USA). Dibromofluorescein (DBF) was bought from Tokyo Chemical Industry Co. (Tokyo, Japan). Fluorescein-methotrexate (FMTX) was bought from Biotium (San Francisco, CA, USA). Hygromycin B was purchased from Solarbio (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and trypsin were bought from Biological Industries (Beit Haemek, Galilee, Israel). The detailed information of 16 excipients investigated in the present study is shown in Table 1. Other chemicals were commercial products with the highest purity grade available.