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To improve the prognosis of bladder and prostate cancer, highly specific and sensitive biomarkers are needed for early detection, prognosis prediction, and therapeutic stratification. Extracellular vesicles (EV) from plasma could fill this gap due to their potential to serve as cancer biomarkers. However, the enrichment of EV is a major challenge, because the highly abundant plasma proteins are interfering with analytical downstream applications like mass spectrometry (MS). Therefore, the purity requirements of the EV samples must be carefully considered when selecting or developing a suitable EV enrichment method. The aim of this study was to compare a self-designed EV enrichment method based on density cushion centrifugation (DCC) combined with size exclusion chromatography (SEC) and concentration (method 1) with the exoRNeasy midi kit from Qiagen (method 2) and with unprocessed plasma. Furthermore, the single steps of method 1 were evaluated for their effectiveness to enrich EV from plasma. The results showed that the EV samples enriched with method 1 contained the highest levels of EV and exosome markers with simultaneously low levels of highly abundant plasma proteins. In summary, the combination of DCC, SEC and concentration proved to be a promising approach to discover EV-based biomarkers from plasma of cancer patients.

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Genotypes exhibiting an increased mutation rate, called hypermutators, can propagate in microbial populations because they can have an advantage due to the higher supply of beneficial mutations needed for adaptation. Although this is a frequently observed phenomenon in natural and laboratory populations, little is known about the influence of parameters such as the degree of maladaptation, stress intensity, and the genetic architecture for adaptation on the emergence of hypermutators. To address this knowledge gap, we measured the emergence of hypermutators over ~1,000 generations in experimental Escherichia coli populations exposed to different levels of osmotic or antibiotic stress. Our stress types were chosen based on the assumption that the genetic architecture for adaptation differs between them. Indeed, we show that the size of the genetic basis for adaptation is larger for osmotic stress compared to antibiotic stress. During our experiment, we observed an increased emergence of hypermutators in populations exposed to osmotic stress but not in those exposed to antibiotic stress, indicating that hypermutator emergence rates are stress type dependent. These results support our hypothesis that hypermutator emergence is linked to the size of the genetic basis for adaptation. In addition, we identified other parameters that covaried with stress type (stress level and IS transposition rates) that might have contributed to an increased hypermutator provision and selection. Our results provide a first comparison of hypermutator emergence rates under varying stress conditions and point towards complex interactions of multiple stress-related factors on the evolution of mutation rates.

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OBJECTIVES: Esmolol-based cardioplegic arrest offers better cardioprotection than crystalloid cardioplegia but has been compared experimentally with blood cardioplegia only once. We investigated the influence of esmolol crystalloid cardioplegia (ECCP), esmolol blood cardioplegia (EBCP) and Calafiore blood cardioplegia (Cala) on cardiac function, metabolism and infarct size in non-infarcted and infarcted isolated rat hearts. METHODS: Two studies were performed: (i) the hearts were subjected to a 90-min cardioplegic arrest with ECCP, EBCP or Cala and (ii) a regional myocardial infarction was created 30 min before a 90-min cardioplegic arrest. Left ventricular peak developed pressure (LVpdP), velocity of contractility (dLVP/dtmax), velocity of relaxation over time (dLVP/dtmin), heart rate and coronary flow were recorded. In addition, the metabolic parameters were analysed. The infarct size was determined by planimetry, and the myocardial damage was determined by electron microscopy. RESULTS: In non-infarcted hearts, cardiac function was better preserved with ECCP than with EBCP or Cala relative to baseline values (LVpdP: 100 ± 28% vs 86 ± 11% vs 57 ± 7%; P = 0.002). Infarcted hearts showed similar haemodynamic recovery for ECCP, EBCP and Cala (LVpdP: 85 ± 46% vs 89 ± 55% vs 56 ± 26%; P = 0.30). The lactate production with EBCP was lower than with ECCP (0.6 ± 0.7 vs 1.4 ± 0.5 µmol/min; P = 0.017). The myocardial infarct size and (ECCP vs EBCP vs Cala: 16 ± 7% vs 15 ± 9% vs 24 ± 13%; P = 0.21) the ultrastructural preservation was similar in all groups. CONCLUSIONS: In non-infarcted rat hearts, esmolol-based cardioplegia, particularly ECCP, offers better myocardial protection than Calafiore. After an acute myocardial infarction, cardioprotection with esmolol-based cardioplegia is similar to that with Calafiore.

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OBJECTIVES: Blood cardioplegia (BCP) can be used in different ways to protect the heart from ischaemia-reperfusion injury during cardiac surgery. Because there could be differences between warm and cold intermittent cardioplegia with or without warm reperfusion, we investigated the influence of 2 blood cardioplegia solutions on cardiac function, metabolism and infarct size in stable and infarcted rat hearts. METHODS: The hearts of 32 male Wistar rats were excised and inserted into a blood-perfused isolated heart apparatus. In 16 hearts, an acute myocardial infarction was induced by ligation of the left anterior descending coronary artery at least 30 min before aortic clamping. After aortic clamping, either Calafiore or Buckberg BCP was administered. During reperfusion, coronary blood flow, left ventricular developed pressure and dp/dt max were recorded, and oxygen consumption and lactate production were determined. The infarct size after 90 min of reperfusion was measured by triphenyl tetrazolium chloride staining. The hearts of rats without infarction were investigated using transmission electron microscopy. RESULTS: In hearts without infarction, haemodynamic recovery was similar for Calafiore and Buckberg solutions: left ventricular developed pressure [Cala 62% of baseline (BL), Buck 58% BL] and dp/dt max (Cala 83% BL, Buck 89% BL). Coronary flow, which was slightly less in infarcted hearts, also recovered similarly after the administration of the 2 BCP solutions (Cala 65% BL, Buck 68% BL). During reperfusion, lactate production was similar (Cala 0.85 ml/min, Buck 1.0 ml/min), and the cellular oedema index and mitochondrial swelling were comparable between the 2 groups. In hearts with infarction, left ventricular developed pressure (Cala 58% BL, Buck 56% BL) and dp/dt max (Cala 79% BL, Buck 72% BL) showed similar recovery for reperfusion with Calafiore or Buckberg BCP. In addition, coronary flow recovered similarly (Cala 54% BL, Buck 57% BL). During reperfusion, myocardial oxygen consumption was lower in the Cala (67% BL) than in the Buck (82% BL) group, but lactate production was similar between the Cala (1.1 ml/min) and the Buck (1.1 ml/min) groups. Myocardial infarct size was also similar in the Cala group (24%) and in the Buck group (26%). CONCLUSIONS: In stable perfused rat hearts and in an in vitro model of acute myocardial infarction, the 2 BCP solutions offer equally good myocardial protection.

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BACKGROUND: In patients undergoing surgical myocardial revascularization for acute myocardial infarction, excellent myocardial protection can be achieved by blood cardioplegia. We investigated the influence of age on cardiac function, metabolism, and infarct size using Buckberg's blood cardioplegia (BCP). METHODS: The hearts of male Wistar rats ("adult", age 3 months, n = 8; "senile", age 24 months, n = 8) were excised and mounted on a blood-perfused isolated heart apparatus. An acute myocardial infarction was induced by coronary artery ligation for 30 min before aortic clamping and infusion of Buckberg's BCP. Throughout the experiment, functional parameters were recorded: coronary blood flow (normalized by heart weight), left ventricular peak developed pressure (LVpdP), and positive and negative derived left ventricular pressure over time (dLVPdtmax and dLVPdtmin). Oxygen consumption (MVO2) and lactate production of the hearts were calculated. The infarct size after 90 min of reperfusion (in % of the area at risk) was measured with triphenyl tetrazolium chloride staining of the myocardium. RESULTS: The baseline coronary flow normalized by heart weight was significantly lower in the senile hearts (1.6 ±â€¯0.4 ml/(min ∗ g)) compared with the adult hearts (2.0 ±â€¯0.3 ml/(min ∗ g); p = 0.04). After 90 min of aortic clamping, hemodynamic function of senile hearts recovered better than that of adult hearts: LVpdP (adult 57% of baseline [BL]; senile 88% BL; p = 0.044) and dLVPdtmax (adult 74% BL, senile 102% BL; p = 0.12). In contrast, myocardial infarct size was similar between the adult (26%) and senile (21%; p = 0.45) hearts, and coronary flow recovered to a similar extent (55% BL and 58% BL, respectively). During reperfusion, MVO2 (80% BL and 81% BL) and lactate production (1.2 and 1.3 µmol/min) were similar in the two groups. CONCLUSION: After acute myocardial infarction in a rat model, hearts recovered function after reperfusion with Buckberg's BCP solution. Hearts from aged animals recovered better than those from younger animals.

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