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Introduction: Canine adipose-derived mesenchymal stem cells (cAD-MSCs) hold therapeutic promise due to their regenerative potential, particularly within their secretome. However, concerns arise regarding the impact of in vitro cultivation necessitated for storing therapeutic doses, prompting this study to comprehensively explore the impact of in vitro aging on gene expression and secretome composition. Methods: The study involved collecting abdominal adipose tissue samples from nine healthy female dogs, from which cAD-MSCs were extracted and cultured. Stem cells were validated through trilineage differentiation assays and flow cytometry immunophenotyping. Gene expression profiling using RT-qPCR array, and cAD-MSCs secretome LC-MS/MS analysis, were conducted at passages 3 and 6 to reveal gene expression and protein composition alterations during in vitro culture. Results and Discussion: The results demonstrate that the gene expression and secretome composition of cAD-MSCs were impacted by in vitro aging. Among many alterations in gene expression between two passages, two significant downregulations were noted in the MSC-associated PTPRC and IL10 genes. While the majority of proteins and their functional characteristics were shared between passages, the influence of cell aging on secretome composition is highlighted by 10% of proteins being distinctively expressed in each passage, along with 21 significant up- and downregulations. The functional attributes of proteins detected in passage 3 demonstrated a greater inclination towards supporting the regenerative capacity of cAD-MSCs. Moreover, proteins in passage 6 exhibited a noteworthy correlation with the blood coagulation pathway, suggesting an elevated likelihood of coagulation events. To the best of our knowledge, this study presents the first original perspective on the changes in secretome composition that occur when cAD-MSCs age in vitro. Furthermore, it contributes to broadening the currently restricted knowledge base concerning the secretome of cAD-MSCs. In conclusion, our findings show that the regenerative potential of cAD-MSCs, as well as their secretome, may be compromised by in vitro aging. Therefore, our study suggests a preference for earlier passages when considering these cells for therapeutic applications.
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Research background: Baranjski kulen is one of the most popular fermented meat sausages originating from Croatia. It has protected geographical indication, and is traditionally produced in the Baranja region of Croatia. Kulenova seka is a fermented sausage very similar to baranjski kulen, but it has a different calibre and consequently, a shorter time of production. In recent decades, due to the high demand and popularity of these products, industrially produced baranjski kulen and kulenova seka have become available on the market. This work aims to identify specific characteristics of traditional and industrial sausages baranjski kulen and kulenova seka on proteome, peptidome and metabolome level, which could potentially lead to better optimization of the industrial production process in order to obtain an equivalent to the traditional product. Experimental approach: Protein profiles of baranjski kulen and kulenova seka (traditional and industrial) were analysed using two-dimensional gel electrophoresis followed by differential display analysis and protein identification by mass spectrometry. Peptidomic profile was analysed via liquid chromatography-tandem mass spectrometry. Furthermore, aroma profiles were investigated via headspace solid phase microextraction and gas chromatography-mass spectrometry. Results and conclusions: The major identified characteristics of each product were: industrial baranjski kulen: specific degradation of myosin-1 and titin, overabundance of stress-related proteins and increased phenylalanine degradation; traditional baranjski kulen: decreased concentration of phenylalanine and overabundance of fructose-bisphosphate aldolase A and carbonic anhydrase 3; industrial kulenova seka - specific myosin-4 and haemoglobin subunit alpha degradation process; traditional kulenova seka - overabundance of dihydropyrimidine dehydrogenase [NADP(+)] and myosin light chain 1/3, skeletal muscle isoform, degradation of albumin and myoglobin, decreased concentrations of almost all free amino acids and increased amounts of smoke-derived volatile compounds. Presented results showed that potential product type-specific quality markers for each sausage could be found. Novelty and scientific contribution: In this preliminary communication, the first insights into protein degradation processes and generation of peptides, free amino acids and aroma compounds of industrial and traditional baranjski kulen and kulenova seka are presented. Although further research is needed to draw general conclusions, the specific profile of proteins, peptides, amino acids and volatile compounds represents the first step in the industrial production of sausages that meet the characteristics of traditional flavour.
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Bacterium Acinetobacter baumannii is a leading cause of nosocomial infections. The occurrence of antibiotic-resistant A. baumannii isolates outside hospitals suggests that monitoring of this pathogen in environmental samples is needed. Survival of pandrug-resistant A. baumannii was followed on selective plates with and without carbapenems in water and soil. After a few days of starvation, A. baumannii lost the ability to be cultivated at 44°C on plates supplemented with carbapenems. Once cultivated on plates without carbapenems and/or at 36°C, A. baumannii could grow again at 44°C in the presence of carbapenems. Comparative proteomic analysis revealed that impaired membrane integrity and reduced function of efflux pumps due to elevated temperature combined with antibiotic exposure were the main reasons for this phenomenon. Loss of thermotolerance in the presence of antibiotics points to the need for temperature adjustment in long-term monitoring of A. baumannii in environmental samples, to avoid the underestimation of viable bacteria.
