Biology subjects

Duska, F.

Publications and source records attributed to Duska, F..

3 recordsLinked to original sources

bioRxiv2024

Evaluating Bacterial Viability in Faecal Microbiota Transplantation: A Comparative Analysis of In Vitro Cultivation and Membrane Integrity Methods

BackgroundFaecal microbiota transplantation (FMT) is a developing therapy for disorders related to gut dysbiosis. Despite its growing application, standardized protocols for FMT filtrate preparation and quality assessment remain undeveloped. The viability of bacteria in the filtrate is crucial for FMTs efficacy and for validating protocol execution. We compared two methods--in vitro cultivation and membrane integrity assessment--for their accuracy, reproducibility, and clinical applicability in measuring bacterial viability in frozen FMT stool filtrate. MethodsBacterial viability in stool filtrate was evaluated using (i) membrane integrity through fluorescent DNA staining with SYTO9 and propidium iodide, followed by flow cytometry; and (ii) culturable bacteria counts (colony-forming units, CFU) under aerobic or anaerobic conditions. ResultsWe refined the bacterial DNA staining protocol integrated with flow cytometry for stool samples. Both the membrane integrity-based and cultivation-based methods exhibited significant variability in bacterial viability across different FMT filtrates, without correlation. The cultivation-based method showed a mean coefficient of variance of 17%, ranging from 5.3% to 52.9%. Conversely, the membrane integrity approach yielded highly reproducible results, with a median coefficient of variance for viable cells of 0.9%, ranging from 8.5% to 0.04%. ConclusionBacterial viability assessment using cultivation-dependent methods produces inconsistent outcomes. In contrast, the membrane integrity method offers robust and precise data, making it a viable option for routine faecal material evaluation in FMT.

microbiology↗

bioRxiv2019

Kinetic characteristics of propofol-induced inhibition of electron-transfer chain and fatty acid oxidation in human and rodent skeletal and cardiac muscles

IntroductionPropofol causes a profound inhibition of fatty acid oxidation (FAO) and reduces spare electron transfer chain (ETC) capacity in a range of human and rodent cells and tissues - a feature that might be related to the pathogenesis of Propofol Infusion Syndrome. We aimed to explore the mechanism of propofol-induced alteration of bioenergetic pathways by describing its kinetic characteristics.\n\nMethodsWe obtained samples of skeletal and cardiac muscle from Wistar rat (n=3) and human subjects: vastus lateralis from hip surgery patients (n=11) and myocardium from brain-dead organ donors (n=10). We assessed mitochondrial functional indices using standard SUIT protocol and high resolution respirometry in fresh tissue homogenates with or without short-term exposure to a range of propofol concentration (2.5-100 g/ml). After finding concentrations of propofol causing partial inhibition of a particular pathways, we used that concentration to construct kinetic curves by plotting oxygen flux against substrate concentration during its stepwise titration in the presence or absence of propofol. By spectrophotometry we also measured the influence of the same propofol concentrations on the activity of isolated respiratory complexes.\n\nResultsWe found that human muscle and cardiac tissues are more sensitive to propofol-mediated inhibition of bioenergetic pathways than rats tissue. In human homogenates, palmitoyl carnitine-driven respiration was inhibited at much lower concentrations of propofol than that required for a reduction of ETC capacity, suggesting FAO inhibition mechanism different from downstream limitation or carnitine-palmitoyl transferase-1 inhibition. Inhibition of Complex I was characterised by more marked reduction of Vmax, in keeping with non-competitive nature of the inhibition and the pattern was similar to the inhibition of Complex II or ETC capacity. There was no inhibition of Complex IV nor increased leak through inner mitochondrial membrane with up to 100 g/ml of propofol. If measured in isolation by spectrophotometry, propofol 10 g/ml did not affect the activity of any respiratory complexes.\n\nConclusionIn human skeletal and heart muscle homogenates, propofol in concentrations that are achieved in propofol-anaesthetized patients, causes a direct inhibition of fatty acid oxidation, in addition to inhibiting flux of electrons through inner mitochondrial membrane. The inhibition is more marked in human as compared to rodent tissues.

pharmacology and toxicology↗

bioRxiv2018

Lactate production without hypoxia in skeletal muscle during electrical cycling: Crossover study of femoral venous-arterial differences in healthy volunteers

BackgroundAim of the study was to compare metabolic response of leg skeletal muscle during functional electrical stimulation-driven unloaded cycling (FES) to that seen during volitional supine cycling.\n\nMethodsFourteen healthy volunteers were exposed in random order to supine cycling, either volitional (10-25-50 W, 10 min) or FES assisted (unloaded, 10 min) in a crossover design. Whole body and leg muscle metabolism were assessed by indirect calorimetry with concomitant repeated measurements of femoral venous-arterial differences of blood gases, glucose, lactate and amino acids.\n\nResultsUnloaded FES cycling, but not volitional exercise, led to a significant increase in across-leg lactate production (from -1.1{+/-}2.1 to 5.5{+/-}7.4 mmol/min, p<0.001) and mild elevation of arterial lactate (from 1.8{+/-}0.7 to 2.5{+/-}0.8 mM). This occurred without widening of across-leg VA O2 and CO2 gaps. Femoral SvO2 difference was directly proportional to VA difference of lactate (R2= 0.60, p=0.002). Across-leg glucose uptake did not change with either type of exercise. Systemic oxygen consumption increased with FES cycling to similarly to 25W volitional exercise (138{+/-}29% resp. 124{+/-}23% of baseline). There was a net uptake of branched-chain amino acids and net release of Alanine from skeletal muscle, which were unaltered by either type of exercise.\n\nConclusionsUnloaded FES cycling, but not volitional exercise causes significant lactate production without hypoxia in skeletal muscle. This phenomenon can be significant in vulnerable patients groups.

physiology↗