Search

Abstract: A dual reporter cell assay (DRCA) that allows real-time detection of herpes simplex virus (HSV) infection was developed. This was achieved by stable transfection of cells with an expression cassette that contains the dual reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP), under the control of an HSV early gene promoter. Baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cell lines were used as parental cell lines because the former is permissive for both HSV serotypes, HSV-1 and HSV-2, whereas the latter is susceptible to infection only by HSV-2. The DRCA permitted differential detection of HSV-1 and HSV-2 by observation of EGFP-positive cells, as substantiated by screening a total of 35 samples. The BHK-based cell line is sensitive to a viral titer as low as a single plaque-forming unit with a robust assay window as measured by a chemiluminescent assay. Evaluations of the DRCA with representative acyclovir-sensitive and acyclovir-resistant HSV strains demonstrated that their drug susceptibilities were accurately determined by a 48-h format. In summary, this novel DRCA is a useful means for serotyping of HSV in real time as well as a rapid screening method for determining anti-HSV susceptibilities. [Copyright &y& Elsevier]

The biochemical measure of success in assisted cartilage repair is normally judged by repair tissue cell density, mRNA and protein expression, and accumulation of extracellular matrix molecules. Existing methods to solubilize cartilage matrix proteoglycans and cellular DNA for quantification, such as papain digestion, often destroy one or more species of the above-named parameters, in order to render others measurable. We have therefore developed a methodology to measure specific levels of mRNA, protein, DNA, glycosaminoglycan, and collagen content on single pulverized 10-mg samples of cartilage, or tissue-engineered cartilage, using successive extractions in concentrated guanidine hydrochloride (GuCl) and guanidine thiocyanate (GITC) solutions. Conditions were developed to solubilize most cellular proteins, DNA, proteoglycans, and some matrix proteins with an initial GuCl extraction step. A subsequent extraction with GITC was essential to solubilize the majority of the cellular RNA. Guanidine-insoluble material was rendered soluble by papain digestion, to enable quantification of collagen, residual glycosaminoglycan, and residual unextracted DNA in individual samples. In general, total collagen, GAG, and DNA content measured in multivalent-extracted samples was similar to that obtained with samples digested directly with papain. Moreover, we were able to reliably detect, in these same multivalent extracts, expressed mRNA as well as specific cellular and extracellular matrix proteins. This multivalent assay could be applied to a variety of cells cultured in biopolymers and to tissues from which biochemical components may be otherwise difficult to extract. [Copyright &y& Elsevier]

Abstract: Mass spectrometric (MS) isotopomer analysis has become a standard tool for investigating biological systems using stable isotopes. In particular, metabolic flux analysis uses mass isotopomers of metabolic products typically formed from 13C-labeled substrates to quantitate intracellular pathway fluxes. In the current work, we describe a model-driven method of numerical bias estimation regarding MS isotopomer analysis. Correct bias estimation is crucial for measuring statistical qualities of measurements and obtaining reliable fluxes. The model we developed for bias estimation corrects a priori unknown systematic errors unique for each individual mass isotopomer peak. For validation, we carried out both computational simulations and experimental measurements. From stochastic simulations, it was observed that carbon mass isotopomer distributions and measurement noise can be determined much more precisely only if signals are corrected for possible systematic errors. By removing the estimated background signals, the residuals resulting from experimental measurement and model expectation became consistent with normality, experimental variability was reduced, and data consistency was improved. The method is useful for obtaining systematic error-free data from 13C tracer experiments and can also be extended to other stable isotopes. As a result, the reliability of metabolic fluxes that are typically computed from mass isotopomer measurements is increased. [Copyright &y& Elsevier]