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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.
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Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability.
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ADN Helicasas/metabolismo , Reparación del ADN/genética , Recombinación Homóloga , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Daño del ADN/genética , Daño del ADN/efectos de la radiación , ADN Helicasas/genética , ADN Polimerasa II/genética , ADN Polimerasa II/metabolismo , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Reparación del ADN/efectos de la radiación , Replicación del ADN/genética , Replicación del ADN/efectos de la radiación , Inestabilidad Genómica , Mutación/genética , Antígeno Nuclear de Célula en Proliferación/genética , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Proteína SUMO-1/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Sumoilación , Rayos Ultravioleta/efectos adversosRESUMEN
DNA double-strand breaks (DSBs) can be generated not only by reactive agents but also as a result of replication fork collapse at unrepaired DNA lesions. Whereas ubiquitylation of proliferating cell nuclear antigen (PCNA) facilitates damage bypass, modification of yeast PCNA by small ubiquitin-like modifier (SUMO) controls recombination by providing access for the Srs2 helicase to disrupt Rad51 nucleoprotein filaments. However, in human cells, the roles of PCNA SUMOylation have not been explored. Here, we characterize the modification of human PCNA by SUMO in vivo as well as in vitro. We establish that human PCNA can be SUMOylated at multiple sites including its highly conserved K164 residue and that SUMO modification is facilitated by replication factor C (RFC). We also show that expression of SUMOylation site PCNA mutants leads to increased DSB formation in the Rad18(-/-) cell line where the effect of Rad18-dependent K164 PCNA ubiquitylation can be ruled out. Moreover, expression of PCNA-SUMO1 fusion prevents DSB formation as well as inhibits recombination if replication stalls at DNA lesions. These findings suggest the importance of SUMO modification of human PCNA in preventing replication fork collapse to DSB and providing genome stability.
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Roturas del ADN de Doble Cadena , Antígeno Nuclear de Célula en Proliferación/metabolismo , Sumoilación , Replicación del ADN , Histonas/metabolismo , Recombinación Homóloga , Humanos , Mutación , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/genética , Proteína SUMO-1/metabolismoRESUMEN
Human Ape2 protein has 3' phosphodiesterase activity for processing 3'-damaged DNA termini, 3'-5' exonuclease activity that supports removal of mismatched nucleotides from the 3'-end of DNA, and a somewhat weak AP-endonuclease activity. However, very little is known about the role of Ape2 in DNA repair processes. Here, we examine the effect of interaction of Ape2 with proliferating cell nuclear antigen (PCNA) on its enzymatic activities and on targeting Ape2 to oxidative DNA lesions. We show that PCNA strongly stimulates the 3'-5' exonuclease and 3' phosphodiesterase activities of Ape2, but has no effect on its AP-endonuclease activity. Moreover, we find that upon hydrogen-peroxide treatment Ape2 redistributes to nuclear foci where it colocalizes with PCNA. In concert with these results, we provide biochemical evidence that Ape2 can reduce the mutagenic consequences of attack by reactive oxygen species not only by repairing 3'-damaged termini but also by removing 3'-end adenine opposite from 8-oxoG. Based on these findings we suggest the involvement of Ape2 in repair of oxidative DNA damage and PCNA-dependent repair synthesis.
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Daño del ADN , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Exodesoxirribonucleasas/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Adenina/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , Endonucleasas , Humanos , Peróxido de Hidrógeno/farmacología , Enzimas Multifuncionales , Oxidación-Reducción , Antígeno Nuclear de Célula en Proliferación/análisisRESUMEN
Mesenchymal stem cells (MSC) are emerging as an effective therapeutic tool in treating canine osteoarthritis (OA). In this report, we focused on the questions of whether MSC transplantation has long-term beneficial effects for the improvement in motion and also evaluated the safety of MSC injection. Visceral adipose tissue, a surgical waste obtained during routine ovariectomy served as a source of allogeneic MSCs and used to treat OA. Altogether, fifty-eight dogs were transplanted in the study suffering from OA in the elbow (42 animals), hip (5), knee (8), ankle (2), and hock (1). The effect of MSC transplantation was evaluated by the degree of lameness at a 4-5-years follow-up period based on the owners' subjective observations. The results showed that 83% of the OA patients improved or retained improvement in lameness. Clinical safety of the treatment was assessed by evaluating the coincidence of tumors or other diseases and other adverse reactions (such as local inflammation) after MSC cell therapy. Two incidences of local inflammation for <1 week at the site of injection were reported. No other adverse reactions were detected post-treatment. Sixteen dogs died during the study, 4 due to cancer and 12 due to other diseases, diagnosed by veterinarians. Overall, our survey suggests that MSC transplantation has long-term beneficial effects in reducing lameness. Moreover, no enrichment in a specific cause of death was observed in the transplanted animals, compared to reported literature. Our data suggest that MSC treatment could be an effective and safe long-term therapy for canine OA.
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DNA damage, such as abasic sites and DNA strand breaks with 3'-phosphate and 3'-phosphoglycolate termini present cytotoxic and mutagenic threats to the cell. Class II AP endonucleases play a major role in the repair of abasic sites as well as of 3'-modified termini. Human cells contain two class II AP endonucleases, the Ape1 and Ape2 proteins. Ape1 possesses a strong AP-endonuclease activity and weak 3'-phosphodiesterase and 3'-5' exonuclease activities, and it is considered to be the major AP endonuclease in human cells. Much less is known about Ape2, but its importance is emphasized by the growth retardation and dyshematopoiesis accompanied by G2/M arrest phenotype of the APE2-null mice. Here, we describe the biochemical characteristics of human Ape2. We find that Ape2 exhibits strong 3'-5' exonuclease and 3'-phosphodiesterase activities and has only a very weak AP-endonuclease activity. Mutation of the active-site residue Asp 277 to Ala in Ape2 inactivates all these activities. We also demonstrate that Ape2 preferentially acts at mismatched deoxyribonucleotides at the recessed 3'-termini of a partial DNA duplex. Based on these results we suggest a novel role for human Ape2 as a 3'-5' exonuclease.
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Disparidad de Par Base , Exodesoxirribonucleasas/metabolismo , Sitios de Unión , ADN/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Desoxirribonucleótidos/química , Desoxirribonucleótidos/metabolismo , Endonucleasas , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/aislamiento & purificación , Humanos , Enzimas Multifuncionales , Mutación , Hidrolasas Diéster Fosfóricas/metabolismo , Especificidad por SustratoRESUMEN
Lysophospholipids, particularly lysophosphatidylcholine (lyso-PC), have been implicated in modulating T cell functions at the sites of inflammation and atherosclerosis. Although the chemotactic and immunomodulatory effects are well documented, the exact signaling pathway of lyso-PC action is poorly defined. In this work, we studied the earliest biochemical events in T cells triggered by lyso-PC. A marked and immediate tyrosine phosphorylation was induced in the leukemic T cell line, Jurkat. Phosphorylation of cellular substrates included src family kinase, p56(lck) and syk family kinase, ZAP70. The lyso-PC induced tyrosine phosphorylation was largely dependent on the presence of functional p56(lck). Tyrosine phosphorylation was followed by the elevation of intracellular Ca(2+) concentration. The magnitude of the mobilization of the intracellular Ca(2+) was similar in the absence of the p56(lck) activity in JCaM1.6 cells as in Jurkat cells, however, it was slightly but reproducibly delayed compared to that in the wild type cells. Inhibition of the Ser/Thr kinases and tyrosine kinases with staurosporine and genistein, respectively, decreased the rise in the intracellular Ca(2+) content. Moreover, pertussis toxin completely blocked the Ca(2+) signal supporting the role of the G-protein coupled LPC receptor in this event.