Understanding quantitative polymerase chain reaction bioanalysis issues before validation planning: Japan Bioanalysis Forum discussion group.

Investigating the biodistribution of cell and gene therapy products may play an important role in evaluating their safety and pharmacology. As quantitative polymerase chain reaction (qPCR) is often used for these analyses, it is essential to improve the reliability of bioanalysis performed using qPCR. In this report, the authors discuss the use of qPCR in nonclinical studies, as it can be used to detect target DNA/RNA and it is quantitative and applicable for long-term analysis.

Simultaneous measurement of mouse and human albumin in chimeric mice with humanized livers.

The degree of human hepatocyte replacement in chimeric mice with humanized liver has previously been shown to correlate with human plasma albumin measurements. However, there are no reports that directly compare the remaining endogenous mouse albumin with the newly expressed human albumin following engraftment. To better understand the disposition of serum albumin in PXB-mice, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to simultaneously quantitate both human and mouse albumin from plasma.

Data on long-term survival of the NOD/Shi-scid IL-2Rγ (NOG) mouse in two facilities.

The objective of this study was to investigate an appropriate observation period for an evaluation of tumorigenicity in NOD/Shi-scid IL-2 Rγ (NOG) mice. At SNBL, 19 male and 19 female NOG mice were observed the general condition from 7 weeks old up to 68 weeks old and at FBRI, 7 male and 16 female NOG mice were observed the general condition throughout the lifespan from 7 weeks old. The survival rate started to decline rapidly around 54 to 56 weeks of age in both facilities without a facility difference.

Biodistribution studies for cell therapy products: Current status and issues.

Information on the biodistribution (BD) of cell therapy products (CTPs) is essential for prediction and assessment of their efficacy and toxicity profiles in non-clinical and clinical studies. To conduct BD studies, it is necessary to understand regulatory requirements, implementation status, and analytical methods. This review aimed at surveying international and Japanese trends concerning the BD study for CTPs and the following subjects were investigated, which were considered particularly important: 1) comparison of guidelines to understand the regulatory status of BD studies in a global setting; 2) case studies of the BD study using databases to understand its current status in cell therapy; 3) case studies on quantitative polymerase chain reaction (qPCR) used primarily in non-clinical BD studies for CTPs; and 4) survey of imaging methods used for non-clinical and clinical BD studies.

Evaluation of domain of unknown function 1220 (DUF1220) for detection of human genome by quantitative polymerase chain reaction: Potential use in assessing the biodistribution of transplanted therapeutic human cells.

The biodistribution profile of cell-based therapy products in animal models is important for evaluation of their safety and efficacy. Because of its quantitative nature and sensitivity, the quantitative polymerase chain reaction (qPCR) is a useful method for detecting and quantifying xenogeneic cell-derived DNA in animal models, thereby allowing a biodistribution profile to be established. Although the restriction endonuclease family from Arthrobacter luteus (Alu) of repetitive elements in human genome sequences has been used to assess the biodistribution of human cells, high background signals are detected.

The Arabidopsis synaptotagmin SYTA regulates the cell-to-cell movement of diverse plant viruses.

Synaptotagmins are a large gene family in animals that have been extensively characterized due to their role as calcium sensors to regulate synaptic vesicle exocytosis and endocytosis in neurons, and dense core vesicle exocytosis for hormone secretion from neuroendocrine cells. Thought to be exclusive to animals, synaptotagmins have recently been characterized in Arabidopsis thaliana, in which they comprise a five gene family. Using infectivity and leaf-based functional assays, we have shown that Arabidopsis SYTA regulates endocytosis and marks an endosomal vesicle recycling pathway to regulate movement protein-mediated trafficking of the Begomovirus Cabbage leaf curl virus (CaLCuV) and the Tobamovirus Tobacco mosaic virus (TMV) through plasmodesmata (Lewis and Lazarowitz, 2010).

N-terminal deletions of the phiX174 external scaffolding protein affect the timing and fidelity of assembly.

The first alpha-helices of Microviridae external scaffolding proteins function as coat protein substrate specificity domains. Mutations in this helix can lengthen the lag phase before progeny production. 5' deletion genes, encoding N-terminal deletion proteins, were constructed on plasmids and in the øX174 genome.

Scaffolding proteins altered in the ability to perform a conformational switch confer dominant lethal assembly defects.

In the phiX174 procapsid crystal structure, 240 external scaffolding protein D subunits form 60 pairs of asymmetric dimers, D(1)D(2) and D(3)D(4), in a non-quasi-equivalent structure. To achieve this arrangement, alpha-helix 3 assumes two different conformations: (i) kinked 30 degrees at glycine residue 61 in subunits D(1) and D(3) and (ii) straight in subunits D(2) and D(4). Substitutions for G61 may inhibit viral assembly by preventing the protein from achieving its fully kinked conformation while still allowing it to interact with other scaffolding and structural proteins.

Eliminating the requirement of an essential gene product in an already very small virus: scaffolding protein B-free øX174, B-free.

Unlike most viral assembly systems, two scaffolding proteins, B and D, mediate bacteriophage øX174 morphogenesis. The external scaffolding protein D is highly ordered in the atomic structure and proper function is very sensitive to mutation. In contrast, the internal scaffolding protein B is relatively unordered and extensive alterations do not eliminate function.

Characterization and function of putative substrate specificity domain in microvirus external scaffolding proteins.

Microviruses (canonical members are bacteriophages phiX174, G4, and alpha3) are T=1 icosahedral virions with an assembly pathway mediated by two scaffolding proteins. The external scaffolding protein D plays a major role during morphogenesis, particularly in icosahedral shell formation. The results of previous studies, conducted with a cloned chimeric external scaffolding gene, suggest that the first alpha-helix acts as a substrate specificity domain, perhaps mediating the initial coat-external scaffolding protein interaction.

Identification of an interacting coat-external scaffolding protein domain required for both the initiation of phiX174 procapsid morphogenesis and the completion of DNA packaging.

The phiX174 external scaffolding protein D mediates the assembly of coat protein pentamers into procapsids. There are four external scaffolding subunits per coat protein. Organized as pairs of asymmetric dimers, the arrangement is unrelated to quasi-equivalence.