Development of a Large-Scale Pathogen Screening Test for the Biosafety Evaluation of Canine Mesenchymal Stem Cells.

BACKGROUND: The action of mesenchymal stem cells (MSCs) is the subject of intense research in the field of regenerative medicine, including their potential use in companion animals, such as dogs. To ensure the safety of canine MSC batches for their application in regenerative medicine, a quality control test must be conducted in accordance with Good Manufacturing Practices (GMP). Based on guidance provided by the European Medicines Agency, this study aimed to develop and validate a highly sensitive and robust, nucleic acid-based test panel for the detection of various canine pathogens. Analytical sensitivity, specificity, amplification efficiency, and linearity were evaluated to ensure robust assessment. Additionally, viable spike-in controls were used to control for optimal nucleic acid extraction. The conventional PCR-based and real-time PCR-based pathogen assays were evaluated in a real-life setting, by direct testing MSC batches. RESULTS: The established nucleic acid-based assays displayed remarkable sensitivity, detecting 100-1 copies/reaction of template DNA. They also exhibited high specificity and efficiency. Moreover, highly effective nucleic acid isolation was confirmed by the sensitive detection of spike-in controls. The detection capacity of our optimized and validated methods was determined by direct pathogen testing of nine MSC batches that displayed unusual phenotypes, such as reduced cell division or other deviating characteristics. Among these MCS batches of uncertain purity, only one tested negative for all pathogens. The direct testing of these samples yielded positive results for important canine pathogens, including tick-borne disease-associated species and viral members of the canine infectious respiratory disease complex (CIRDC). Notably, samples positive for the etiological agents responsible for enteritis (CPV), leptospirosis (Leptospira interrogans), and neosporosis (Neospora caninum) were also identified. Furthermore, we conducted biosafety evaluation of 12 MSC batches intended for therapeutic application. Eleven MSC batches were found to be free of extraneous agents, and only one tested positive for a specific pathogen, namely, canine parvovirus. CONCLUSION: In this study, we established and validated reliable, highly sensitive, and accurate nucleic acid-based testing methods for a broad spectrum of canine pathogens.

TATA binding protein associated factor 3 (TAF3) interacts with p53 and inhibits its function.

BACKGROUND: The tumour suppressor protein p53 is a sequence specific DNA-binding transcription regulator, which exerts its versatile roles in genome protection and apoptosis by affecting the expression of a large number of genes. In an attempt to obtain a better understanding of the mechanisms by which p53 transcription function is regulated, we studied p53 interactions. RESULTS: We identified BIP2 (Bric-à-brac interacting protein 2), the fly homolog of TAF3, a histone fold and a plant homeodomain containing subunit of TFIID, as an interacting partner of Drosophila melanogaster p53 (Dmp53). We detected physical interaction between the C terminus of Dmp53 and the central region of TAF3 both in yeast two hybrid assays and in vitro. Interestingly, DmTAF3 can also interact with human p53, and mammalian TAF3 can bind to both Dmp53 and human p53. This evolutionarily conserved interaction is functionally significant, since elevated TAF3 expression severely and selectively inhibits transcription activation by p53 in human cell lines, and it decreases the level of the p53 protein as well. CONCLUSION: We identified TAF3 as an evolutionarily conserved negative regulator of p53 transcription activation function.

Daxx-like protein of Drosophila interacts with Dmp53 and affects longevity and Ark mRNA level.

Daxx-like protein (DLP), the Drosophila homolog of Daxx, binds Drosophila melanogaster p53 (Dmp53) through its C-terminal region. We generated DLP mutants and found that although DLP expression is developmentally regulated, it is not essential for the execution of the developmental program. The effects DLP mutations show in the loss of heterozygosity assay and on phenotypes resulting from Dmp53 overexpression indicate a genetic interaction between DLP and Dmp53. In contrast to Dmp53 mutants, however, loss of DLP does not result in radiosensitivity indicating that it does not play an essential role in the activation of Dmp53-dependent response after ionizing radiation, and DLP is also not required for the irradiation-induced activation of reaper. In contrast, DLP is involved in the transcriptional regulation of Ark, because Ark mRNA level is decreased in DLP mutants and increased upon ectopic overexpression of DLP. Interestingly, DLP mutants have reduced longevity and reduced female fertility. Altogether, our data suggest complex functions for DLP, which include an anti-apoptotic effect exerted through repression of some Dmp53 functions, and activation of some proapoptotic genes.