The Förster Resonance Energy Transfer (FRET) method has wide application in modern science for studying protein–protein interactions and conformational changes. FRET allows to assess molecular interactions by measuring energy transfer between acceptor and donor fluorophores coupled to the molecule(s) of interest. The method demands high precision in experimental design, experimental settings and correct data interpretation. Therefore, we tested several parameters to estimate FRET measurement accuracy in our Nikon wide-field fluorescence FRET system. The experiments were performed in a HEK-293 cell line transfected with DNA constructs expressing Calcium Release-Activated Channel (CRAC) subunits STIM1 and ORAI1 coupled to donor fluorophore Cyan Fluorescent Protein (CFP) and acceptor fluorophore Yellow Fluorescent Protein (YFP), respectively. Exposure time and approach of data analysis varied throughout experiments in order to optimise FRET data quality. Dependence of FRETeff values on measurement quality and donor/acceptor fluorophore ratio in the cells was estimated. We demonstrated that, using the wide-field fluorescence FRET system, minimising the exposure of fluorophores before measurement using neutral density (ND) filters considerably minimises undesirable photo-bleaching of the fluorophores. There was a strong correlation between the CFP/YFP ratio in the cells and the observed FRET level, suggesting that only cells with certain donor/acceptor ratio might be comparable. We also showed impact of FRET measurement quality, defined as accordance of FRET pixels to Gaussian distribution, on FRET artefacts. Knowledge obtained during our experiments may be important for approbating similar wide-field fluorescence FRET systems to study two separate molecule interactions and for understanding the correct setup of the experiments and data interpretation.
The study characterises canine adipose-derived stem cells (cASCs) in comparison to human ASCs (hASCs) and tests their safety in a canine model after intravenous administration. cASCs from two dogs were cultured under hypoxic conditions in a medium supplemented with autologous serum. They were plastic adherent, spindle-shaped cells that expressed CD73, CD90, and CD44 but lacked CD45, CD14, HLA-DR, and CD34. cASCs differentiated toward adipogenic, osteogenic, and chondrogenic lineages, although adipogenic differentiation capacity was low. Blast transformation reaction demonstrated that these cells significantly suppress T-cell proliferation, and this ability is dose-dependent. Intravenous administration of a cell freezing medium, therapeutic dose of cASCs (2 × 106 live cells/kg), and five times higher dose of cASCs showed no significant side effects in two dogs. Microscopic tissue lesions were limited to only mild, non-specific changes. There were no signs of malignancy. The results of the study indicate that cASCs are similar to hASCs and are safe for therapeutic applications in a canine model. The proposed methodology for ASC preparation on a non-routine basis, which includes individually optimised cell culture conditions and offers risk-adapted treatment, could be used for future personalised off-the-shelf therapies, for example, in myocardial infarction or stroke.
Human bocavirus 1 (HBoV1) is a parvovirus recently found to be a possible aetiologic agent of acute respiratory disease in children. We conducted the first clinical and molecular study on this virus in Latvia (LV) and Lithuania (LT). The aim of the study was to determine the occurrence of HBoV1 in respiratory tract samples taken from hospitalised children with acute respiratory tract infections in LV and LT. In total 186 children with age one to 50 months, and who fulfilled criteria of acute respiratory tract infection, including lower respiratory tract infections, with or without fever, were included in this study. A nasopharyngeal aspirate was obtained from each patient on admission. DNA was isolated and polimerase chain reaction (PCR) performed targeting the HBoV1 NS1sequence. HBoV1 positive samples were sequenced and phylogenetic analysis was performed. HBoV1 sequence was detected in 42 (32%) of 130 LV and in 8 (14%) of 56 LT samples. In LV the majority of patients with HBoV1 infection were observed in February while in LT in October. The phylogenetic tree for HBoV1 indicated that isolates of HBoV1 cluster closely and include almost all of the isolates in this study. HBoV1 is common in Latvia and Lithuania and might be a significant pathogen that contributes to acute respiratory tract infections in children.