Short tandem repeat profiling: part of an overall strategy for reducing the frequency of cell misidentification

The role of cell authentication in biomedical science has received considerable attention, especially within the past decade. This quality control attribute is now beginning to be given the emphasis it deserves by granting agencies and by scientific journals. Short tandem repeat (STR) profiling, one of a few DNA profiling technologies now available, is being proposed for routine identification (authentication) of human cell lines, stem cells, and tissues. The advantage of this technique over methods such as isoenzyme analysis, karyotyping, human leukocyte antigen typing, etc., is that STR profiling can establish identity to the individual level, provided that the appropriate number and types of loci are evaluated. To best employ this technology, a standardized protocol and a data-driven, quality-controlled, and publically searchable database will be necessary. This public STR database (currently under development) will enable investigators to rapidly authenticate human-based cultures to the individual from whom the cells were sourced. Use of similar approaches for non-human animal cells will require developing other suitable loci sets. While implementing STR analysis on a more routine basis should significantly reduce the frequency of cell misidentification, additional technologies may be needed as part of an overall authentication paradigm. For instance, isoenzyme analysis, PCR-based DNA amplification, and sequence-based barcoding methods enable rapid confirmation of a cell line’s species of origin while screening against cross-contaminations, especially when the cells present are not recognized by the species-specific STR method. Karyotyping may also be needed as a supporting tool during establishment of an STR database. Finally, good cell culture practices must always remain a major component of any effort to reduce the frequency of cell misidentification

Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues

Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues

Calculation of the relative metastabilities of proteins in subcellular compartments of Saccharomyces cerevisiae

[abridged] Background: The distribution of chemical species in an open system at metastable equilibrium can be expressed as a function of environmental variables which can include temperature, oxidation-reduction potential and others. Calculations of metastable equilibrium for various model systems were used to characterize chemical transformations among proteins and groups of proteins found in different compartments of yeast cells. Results: With increasing oxygen fugacity, the relative metastability fields of model proteins for major subcellular compartments go as mitochondrion, endoplasmic reticulum, cytoplasm, nucleus. In a metastable equilibrium setting at relatively high oxygen fugacity, proteins making up actin are predominant, but those constituting the microtubule occur with a low chemical activity. A reaction sequence involving the microtubule and spindle pole proteins was predicted by combining the known intercompartmental interactions with a hypothetical program of oxygen fugacity changes in the local environment. In further calculations, the most-abundant proteins within compartments generally occur in relative abundances that only weakly correspond to a metastable equilibrium distribution. However, physiological populations of proteins that form complexes often show an overall positive or negative correlation with the relative abundances of proteins in metastable assemblages. Conclusions: This study explored the outlines of a thermodynamic description of chemical transformations among interacting proteins in yeast cells. The results suggest that these methods can be used to measure the degree of departure of a natural biochemical process or population from a local minimum in Gibbs energy.Comment: 32 pages, 7 figures; supporting information is available at http://www.chnosz.net/yeas

Original scientific paper Isolation and partial characterization of protease from

Abstract: Enzymatic characteristics of a protease from a medically important, referent strain of Pseudomonas aeruginosa ATCC 27853 were determined. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS--PAGE, and gel filtration, it was estimated that the molecular mass of the purified enzyme was about 15 kDa. Other enzymatic properties were found to be: pH optimum 7.1, pH stability between 6.5 and 10; temperature optimum around 60 °C while the enzyme was stable at 60 °C for 30 min. Inhibition of the enzyme was observed with metal chelators, such as EDTA and 1,10-phenanthroline, suggesting that the protease is a metalloenzyme. Furthermore, the enzyme contains one mole of zinc ion per mole of enzyme. The protease was stable in the presence of different organic solvents, which enables its potential use for the synthesis of peptides