ABSTRACT
A pulsating flow of medium was used to alleviate diffusion and transport limitations in a hollow fiber bioreactor containing a human hepatoblastoma cell line. The strategy is easy to implement but effective. The pulsating flow is introduced by a solenoid pinch valve at the outlet of the bioreactor and regulated by a timing circuit. In a permeability test, the system with pulsating flow had much less membrane fouling as compared to the control, a conventional hollow fiber unit. In hepatocyte culture test runs, the pulsating-flow bioreactor demonstrated the ability to maintain a higher cell viability. Histological sections indicated significantly smaller necrotic regions in the pulsating-flow bioreactor as compared to the conventional unit.
Subject(s)
Bioartificial Organs , Liver, Artificial , Pulsatile Flow , Cell Line, Tumor , Cell Survival , Hepatoblastoma , Hepatocytes/physiology , Humans , Liver NeoplasmsABSTRACT
Fluorescent in situ hybridization was combined with flow cytometry to detect the expression of the double-stranded-RNA-induced protein kinase (PKR) in single cells. Labeled anti-sense oligonucleotide was used to target the specific mRNA while the protein was targeted with an antibody. It was demonstrated that the PKR-mRNA signal could be protected through a lengthy immunostaining procedure. The expression pattern of the PKR-mRNA with respect to DNA content was shown to be comparable to that of 18S ribosomal RNA.
Subject(s)
Protein Kinases/genetics , RNA, Messenger/metabolism , Cell Line, Tumor , Flow Cytometry/methods , Fluorescent Dyes , Humans , In Situ Hybridization, Fluorescence/methods , Protein Kinases/metabolism , RNA, Double-Stranded/metabolism , RNA, Messenger/genetics , Reproducibility of ResultsABSTRACT
Previous studies of antibody binding domains have established many crucial features that include important structural positions, canonical formations, and the geometric correlations with the binding site nature and topography. In this work, position-specific frequency and hierarchical clustering analysis are used to explore the statistical pattern of the residues in the complementarity determining regions of human antibodies. In addition, Shannon's information entropy is computed for the entire heavy and light chains and compared with germline patterns to seek variability due to antibody clonal selection. Results are compared with reported analyses based on structural data and ligand-protein contact point computations based on Protein Data Bank records. Observations derived from the present sequence analysis are consistent with previous structural based methods. In the absence of structural data, methods used in this work can be effective and efficient computational tools used for identifying residues that are important for antigen targeting and predicting the probable amino acid distribution expected at these positions. The results in turn can be applied to help design or plan mutagenesis experiments to improve the binding properties of antibodies.
