Measurement Of Oxygen Consumption Rate Research Articles

Too big to study? The biologging approach to understanding the behavioural energetics of ocean giants.

Wild animals are under selective pressure to optimise energy budgets; therefore, quantifying energy expenditure, intake and allocation to specific activities is important if we are to understand how animals survive in their environment. One approach toward estimating energy budgets has involved measuring oxygen consumption rates under controlled conditions and constructing allometric relationships across species. However, studying 'giant' marine vertebrates (e.g. pelagic sharks, whales) in this way is logistically difficult or impossible. An alternative approach involves the use of increasingly sophisticated electronic tags that have allowed recordings of behaviour, internal states and the surrounding environment of marine animals. This Review outlines how we could study the energy expenditure and intake of free-living ocean giants using this 'biologging' technology. There are kinematic, physiological and theoretical approaches for estimating energy expenditure, each of which has merits and limitations. Importantly, tag-derived energy proxies can hardly be validated against oxygen consumption rates for giant species. The proxies are thus qualitative, rather than quantitative, estimates of energy expenditure, and have more limited utilities. Despite this limitation, these proxies allow us to study the energetics of ocean giants in their behavioural context, providing insight into how these animals optimise their energy budgets under natural conditions. We also outline how information on energy intake and foraging behaviour can be gained from tag data. These methods are becoming increasingly important owing to the natural and anthropogenic environmental changes faced by ocean giants that can alter their energy budgets, fitness and, ultimately, population sizes.

Clinical Benefit and Improvement of Mitochondrial Function in Low Risk Myelodysplastic Syndrome Treated By Combination Ultra Coenzyme Q10 and L-Carnitine

Background: Structural mitochondrial abnormalities as well as alterations in gene expression of key mitochondrial proteins are well recognized in myelodysplastic syndrome (MDS), suggesting that mitochondrial dysfunction is important in the pathogenesis of this disease. Both Coenzyme Q10 and L- carnitine play an essential role in efficient cellular energy production. Clinical and cytogenetic response to high dose of Coenzyme Q10 in patients with MDS was demonstrated previously. The combination of L- carnitine and Coenzyme Q10 has shown a beneficial effect in other mitochondrial disorders. Aim: To assess the effect of combination of food supplements on the clinical course and mitochondrial function of peripheral blood cells obtained from patients with in low/intermediate- 1 risk MDS.Materials and Methods: Ultra-coenzyme Q10 (180 mg/day), L-carnitine (2000 mg/day) and a vitamin mineral complex were administered for 6 months; patients were allowed to continue treatment for an additional 6 months, if it was beneficial. Inclusion criteria were Hb<10.5g/dL or PLT<100x109/L or ANC<0.8X109/L. As of May 2017, 33 pts. have been enrolled in the study. Peripheral blood cells were assessed by the Seahorse XF analyzer which is a state- of- the- art tool for assessment of real time cellular respiration, by continuous measurement of Oxygen Consumption Rate (OCR) under different conditions in-vitro . Mitochondrial function assessment was performed at baseline and at six-month point. Results: The median age of the entire cohort was 75 years (range: 56-93), 20(61%) of pts. were male. Pts. were classified per IPSS-R: very low risk 7 (21%), low risk 21(64%),int. 5 (15%). Median Hb was 9.5 g/dL (6.4-12.2); 17 (52%) were transfusions dependent and 16(48%) failed treatment with erythropoietin stimulating agents (ESA). 28 patients completed treatment protocol. Six pts. (21.4%) showed a hematological Improvement according to IWG 2006 criteria (out of which 4 were transfusions dependent). Median time to the initial response was 9 weeks (5-24). At 3 months' time point 14 of 18 patients had an increased in their integral Quality of life (QOL) score per FACT questionnaire; mean improvement was 14.3 points per patient (p=0.011). The pre-treatment mitochondrial basal respiration and especially spare respiratory capacity tested on cells of 11 MDS cases were uniformly lower in comparison with healthy controls (p=0.04 and p=0.007,respectively). There was significant improvement of basal, as well as spare respiration in peripheral blood cells obtained from MDS patients after six months food supplements (p=0.0027 and p=0.02, respectively). Conclusions: We demonstrated for the first time that mitochondrial energy production in peripheral blood is impaired in patients with MDS. Furthermore, we showed that this is at least partially reversible with food supplementations known to augment mitochondrial function. Combination of Ultra coenzyme Q10 with L-carnitine and vitamin mineral complex leads to moderate clinical response in low/intermediate-1 risk MDS patients and improve QOL DisclosuresNo relevant conflicts of interest to declare.

Metabolic cost in healthy fit older adults and young adults during overground and treadmill walking

PurposeThe purpose of this study was to determine whether net metabolic cost of walking is affected by age per se.MethodsWe selected 10 healthy, active older adults (mean age 75 years) and 10 young adults (mean age 26 years), and determined their preferred overground walking speed. On the same day, in a morning and afternoon session, we had them walk at that speed overground and on a treadmill while we measured oxygen consumption rate. From the latter we subtracted the rate in sitting and calculated net metabolic cost.ResultsAnthropometrics were not different between the groups nor was preferred walking speed (1.27 m s−1 both groups). There was no difference in net metabolic cost of overground walking between older and young adults (e.g., in the morning 2.64 and 2.56 J kg−1 m−1, respectively, p > 0.05). In the morning session, net metabolic cost of walking was higher on the treadmill than overground in our older adults by 0.6 J kg−1 m−1 (p < 0.05), but not in young adults.ConclusionFirst, there is no effect of age per se on metabolic cost of overground walking. Second, older adults tend to have higher metabolic cost of walking on a treadmill than walking overground at preferred speed, and adaptation may take a long time. The commonly reported age-related elevation of metabolic cost of walking may be due to confounding factors causing preferred walking speed to be lower in older adults, and/or due to older adults reacting differently to treadmill walking than young adults.

High-resolution spatiotemporal pHe and pO2 imaging in head and neck and oesophageal carcinoma cells

BackgroundpO2 and pH are physiological parameters relevant for different processes in health and disease, including wound healing and cancer progression. Head and neck squamous cell carcinomas (HNSCC) and oesophageal squamous cell carcinomas (ESCC) have a high rate of local recurrence that is partly related to treatment-resistant residual tumour cells. Hence, novel diagnostic tools are required to visualise potential residual tumour cells and thereby improve treatment outcome for HNSCC and ESCC patients. We developed a device to spatiotemporally measure oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) to distinguish HNSCC and ESCC cells from healthy cells in vitro, exploiting general metabolic differences between cancer cells and healthy cells.MethodsOCR and ECAR were measured via a newly developed device named STO2p-Q (SpatioTemporal O2 and pH Quantification) using the VisiSens technology based on ratiometric fluorescence imaging, facilitating spatiotemporal resolution. Results were confirmed using extracellular flux analyses (Seahorse technology).ResultsSTO2p-Q is described and used to measure OCR and ECAR in HNSCC and ESCC cell lines and normal fibroblast and epithelial cells as components of the tumour microenvironment. OCR measurements showed differences amongst HNSCC and ESCC cell lines and between HNSCC/ESCC and normal cells, which on average had lower OCR than HNSCC/ESCC cells. Both OCR and ECAR measurements were independently verified using the Seahorse technology. Additionally, using STO2p-Q, HNSCC/ESCC, and normal cells could be spatially resolved with a resolution in the low millimetre range.ConclusionsWe developed a method to spatiotemporally measure OCR and ECAR of cells, which has many potential in vitro applications and lays the foundation for the development of novel diagnostic tools for the detection of cancerous tissue in HNSCC and ESCC patients in vivo.

BRAF-Like and RAS-Like Thyroid Cancer is Characterized by Distinct Metabolic Phenotypes

Abstract Background: Next generation sequencing has led to the categorization of thyroid cancer (TC) based on the molecular and transcriptomic profile into the BRAF-like and RAS-like tumors. The BRAF-RAS score (BRS) has been developed to quantify the extent to which the gene expression profile resembles either the BRAFV600E- or RAS-mutant profiles and is utilized as a continuous measure from -1 to +1, respectively. Oncogene-driven signaling pathways have an impact on the intracellular metabolism– glycolysis and oxidative phosphorylation (OXPHOS). There are limited data on the genotype-metabolic phenotype correlation in TC cells. Therefore, the goal of this study was to perform a comprehensive analysis of the association between the BRS and TC cell metabolism. Methods: We analyzed the mRNA expression of key enzymes involved in glycolysis (lactate dehydrogenase LDHA) and OXPHOS (ATP synthase) in 496 BRAF-like and RAS-like human TC tissue samples based on The Cancer Genome Atlas. We performed an in vitro study using 8 TC cell lines– 4 BRAF-like with BRS between -1 and 0, and 4 RAS-like with BRS between 0 and +1. OXPHOS was determined by measuring oxygen consumption rate (OCR) of each cell line using Seahorse XF Cell Mito Stress Test Kit, while Seahorse XF Glycolysis Stress Test Kit was used to measure the extracellular acidification rate (ECAR) due to anaerobic glycolysis The association between the OCR, ECAR and BRS was tested using the Pearson correlation coefficient (r). Student T-test was used to compare the continuous variables between the groups with a p-value of ≤ 0.05 as statistically significant. Results:RAS-like tumors were associated with higher mRNA expression of the OXPHOS marker ATP synthase than BRAF-like lesions, as evidenced by a moderate positive correlation (r = 0.5) between ATP synthase expression and BRS. BRAF-like tumors were characterized by a relatively higher mRNA expression of a glycolytic enzyme LDHA, as documented by a moderate negative correlation (r = -0.6) between LDHA expression and BRS. Consistently, functional in vitro studies revealed that RAS-like cell lines had a higher OXPHOS compared with BRAF-like cell lines (maximum OCR: 2538.4±1601.3 vs 548.5±276.5, p=0.049, respectively). The glycolysis rate was comparable between RAS-like and BRAF-like cell lines (maximum ECAR: 1290.4±520.5 vs 2399.9±1413.1, p=0.19, respectively). There was a strong positive correlation between the BRS and OCR (r=0.78) and a low to negligible negative correlation between BRS and ECAR (r=-0.26) in examined TC cell lines. Conclusions:BRAF-like and RAS-like tumors are characterized by distinct metabolic phenotypes with RAS-like TC more likely to utilize OXPHOS to meet metabolic demands. Therapeutic strategies targeting oncogene-driven signaling pathways and cancer metabolism based on distinct metabolic phenotypes may provide an individualized approach to TC therapy.

Oxygen consumption rates in deep-sea hydrothermal vent scale worms: Effect of life-style, oxygen concentration, and temperature sensitivity

Deep-sea hydrothermal vents are a challenging environment inhabited by very specialized species. To reap the benefits of the local primary production, species need to cope with a number of constraints among which low oxygen is probably the most basic. This hypoxia is further complicated by the highly variable temperature these species experience. We studied the response of deep-sea hydrothermal species of scale worms (Annelida, Polynoidae) to varying levels of oxygen and showed that they were capable of compensating a decrease of environmental oxygen concentration (= oxyregulators), down to values of about 30 μmol l−1. This contrasts with shallow-water temperate species, for which oxygen consumption is directly proportional to its concentration (= oxyconformers). We measured oxygen consumption rates in 11 species from hydrothermal vents, as well as 2 species from the general deep-sea, and compared them to three shallow-water species. Life-style (free-living vs. commensal) and habitat of origin (shallow-water, deep-sea, and hydrothermal vent) did not affect oxygen consumption rates. In agreement with thermodynamic expectations, as temperature increases, oxygen consumption increases as well for all species. The sensitivity of oxygen consumption to temperature variation in the shallow-water species is however smaller than that from the deep-sea hydrothermal vent species. This unexpected result could correspond to a pronounced increase of activity (avoidance behaviour) in the vent species, which was not observed for the shallow-water species.

ILF3-AS1 promotes the aerobic glycolysis and proliferation of melanoma cells by regulating miR-493-5p/PDK1 pathway.

The aim of this study was to investigate the role of ILF3-AS1 in regulating the survival of melanoma and its molecular mechanism. The relative expression level of ILF3-AS1 in melanoma was assessed by qPCR. The effect of ILF3-AS1 and PDK1 on the cell viability was tested by MTT assay. Glucose uptake colorimetric assay, lactate assay, the measurements of extracellular acidification rate (ECAR) and Oxygen consumption rate (OCR) were performed to test the effect of ILF3-AS1 and PDK1 on the cellular glycolysis. Luciferase assay was conducted to detect the interactions of ILF3-AS1, miR-493-5p and PDK1. RNA immunoprecipitation chip (RIP) assay was used to detect the enrichments of ILF3-AS1 and miR-493-5p in the complex. Protein level of PDK1 was detected by western blot analysis. qPCR revealed that ILF3-AS1 was upregulated in human melanoma cell lines. MTT assay showed that ILF3-AS1 knockdown blunted cell proliferation, which was rescued by the overexpression of PDK1. Glucose uptake colorimetric assay, lactate assay, the measurements of ECAR and OCR indicated that ILF3-AS1 promoted glycolysis through PDK1. Western blotting results showed that ILF3-AS1 overexpression promoted PDK1 expression, which was prevented by miR-493-5p overexpression in SK-MEL-1 cells. ILF3-AS1 promotes the aerobic glycolysis and survival of melanoma cells involving miR-493-5p/PDK1 pathway.

Aberrant energy metabolism causes chondrocyte dysfunction in osteoarthritis

Purpose: Metabolic pathways are a series of chemical reactions by which cells break-down nutrients to acquire the energy and processes building blocks that they need to maintain critical cellular processes. The details of chondrocyte metabolism and how it rewires in osteoarthritis (OA) disease progression is not well characterised. Central carbon metabolism (CCM) is an interconnected metabolic system that transforms carbon through glycolysis, gluconeogenesis, the pentose phosphate pathway, and the tricarboxylic acid (TCA) cycle pathway into energy that is essential for the physiology of chondrocytes. The aim of this research is to identify how changes in the energy metabolic state, in particular the central carbon metabolism, contributes to OA. Methods: OA and non-OA cartilage specimens were harvested from patients who required total knee arthroplasty (n=8 patient samples). In order to investigate the metabolism differences an integrated isotype assisted U-13C metabolic flux labelling approach was utilized on chondrocytes growing in limited glucose conditions. Isotopologue profiles of key metabolites were obtained by liquid chromatography/mass spectrometry (LC/MS). Expression profiles of genes regulating energy metabolism of OA cartilage of human and OA rat models were determined using RNA-Seq analysis. We further used extracellular flux assays, which allow for direct evaluation of cellular bioenergetic profiles ex vivo by measuring oxygen consumption rate (OCR, measure of oxidative phosphorylation) and extracellular acidification rate (ECAR, measure of aerobic glycolysis), to test the bio-energetic profile of OA chondrocytes. Results: Firstly, U-13C-labeling identified significant metabolic differences between OA chondrocytes vs. non-OA chondrocytes. In particular, OA chondrocytes displayed remarkably diverse rate of glucose utilization. Higher levels of glucose 6-phosphate (G6P), fructose 1,6-disphosphate (F16DP), glyceraldehyde 3-phosphate (GA3P) and lactate (LAC) were detected in OA chondrocytes. Furthermore, there was also a marked increase in 13C labelled enrichment of oxaloacetic acid (OAA) in TCA cycle of OA chondrocytes, while decrease in the rest substrates such as alpha-Ketoglutarate (aKG) and succinate (SUC). Together, these results demonstrated altered metabolism in OA chondrocytes (Figure 1). Secondly, analysis of the metabolic pathway differentially expressed genes revealed that genes involved in central carbon metabolism were altered in OA chondrocytes compared to non-OA cells. Thirdly, mitochondria and glycolysis stress tests utilizing seahorse technology revealed that basal glycolysis was increased in OA chondrocytes (Non-OA: 46.72±11.03; OA: 81.45±24.91 mpH/min, p<0.05) while basal mitochondrial oxidative phosphorylation was decreased (Non-OA: 24.26±10.35; OA: 6.94±3.06 pmol/min, p<0.05). Stress tests also showed that maximum glycolysis capacity was higher in OA chondrocytes (Non-OA: 47.86±15.01; OA: 91.67±36.31 mpH/min, p<0.05) while maximum oxidative phosphorylation capacity increased in non-OA chondrocytes (Non-OA: 121.22±54.70; OA: 40.25±9.83 pmol/min, p<0.01). These results demonstrated that OA chondrocytes switch to conditions of higher glycolysis and lower mitochondrial oxidative during OA progression. Conclusions: Altogether, this study, which is the first comprehensive comparative analysis of metabolism in chondrocytes, provides a foundation for targeting metabolism for therapeutic benefit in OA.

Hyperglycemia-induced severe mitochondrial bioenergetic deficit of lacrimal gland contributes to the early onset of dry eye in diabetic mice

Dry eye and diabetic keratopathy represent the major diabetic complications in ocular surface. Here we found that diabetic mice exhibited the early onset of reduced tear secretion and lacrimal gland weight compared to the symptoms of diabetic keratopathy. Considering to the high bioenergetic needs in lacrimal gland and cornea, we hypothesized that hyperglycemia may cause different severity of mitochondrial bioenergetic deficit between them. Through the measurement of oxygen consumption rate (OCR) and basal extracellular acidification rate (ECAR), we found the apparent alterations of mitochondrial bioenergetic profiles in diabetic lacrimal gland and cornea, accompanied with the mtDNA damage and copy number reduction, as well as the reduced glutathione content. Comparative analysis revealed that mouse lacrimal gland cells exhibited 2–3 folds higher of basal, ATP production, maximal OCR and basal ECAR than corneal epithelial cells in normoglycemia. However, the differences were slightly significant or even not detected in hyperglycemia. Accordingly, the mitochondrial bioenergetic metabolism of lacrimal gland was more compromised than that of corneal epithelium in diabetic mice. Through the administration of mitochondrial-targeted antioxidant SkQ1, the severity of dry eye and diabetic keratopathy was significantly attenuated with the improved mitochondrial function. These results indicate that the susceptibility of mitochondrial bioenergetic deficit in diabetic lacrimal gland may contribute to the early onset of dry eye, while mitochondria-targeted antioxidant possesses therapeutic potential for diabetic dry eye and keratopathy.

Real-Time Assessment of Mitochondrial Toxicity in HepG2 Cells Using the Seahorse Extracellular Flux Analyzer.

The liver is the primary organ responsible for drug detoxification. Drug-induced liver injury (DILI) is a leading cause of attrition during drug development and is one of the main reasons that drugs are withdrawn from the market. Hence, the prevention of DILI plays a central role in the overall drug-discovery process. Most of the liver's energy supply comes in the form of adenosine triphosphate (ATP), which is largely generated by mitochondria. This article describes the evaluation of drug-induced mitochondrial dysfunction using the Seahorse Extracellular Flux Analyzer (Agilent). The described protocols detail the accurate measurement of ATP production rate in HepG2 cells after exposure to a panel of potentially toxic compounds. This assay measures changes in extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as indicators of glycolysis and mitochondrial respiration-the two major energy-generating pathways in a cell. This assay provides a useful model to predict mitochondrial dysfunction-mediated DILI. © 2021 Wiley Periodicals LLC. Basic Protocol: Measurement of cellular ECAR, OCR, and ATP production in live HepG2 cells Support Protocol 1: Culturing and maintaining of HepG2 cells Support Protocol 2: Determining optimal cell density per well.

Relative comparison of strobilurin fungicides at environmental levels: Focus on mitochondrial function and larval activity in early staged zebrafish (Danio rerio)

Strobilurin fungicides are used globally and have been detected in microgram per liter concentrations in aquatic environments. Here, we determined the potential toxicity of four commonly used strobilurins (azoxystrobin, kresoxim-methyl, pyraclostrobin, trifloxystrobin) on mitochondrial function and locomotor activity of larval zebrafish at an environmentally relevant level. As the mode of action of strobilurins in fungi is binding to cytochrome bc1 in mitochondrial complex III, we evaluated exposure effects on mitochondrial oxidative phosphorylation of zebrafish, by measuring oxygen consumption rates, mitochondria-related enzyme activities, and transcripts levels for genes associated with the electron transfer chain and citric acid cycle. We found that 50 nM pyraclostrobin and trifloxystrobin lowered basal respiration, oligomycin-induced ATP respiration, and maximal respiration of embryos. Dysfunction in mitochondrial bioenergetics was associated with changes in mitochondrial complex III activity and transcripts of oxidative respiration and stress-related genes. Lower activity of complex III, and reduced cytb mRNA levels were hypothesized to contribute to reduced electron supply to complex IV and V. Both coxI and atp6 were up-regulated, suggesting a compensatory response to impaired oxidative respiration. Cluster analysis indicated that strobilurin-induced oxidative stress and cytb transcript were related to impaired oxidative phosphorylation. We also assessed larval behavior responses, as reduced ATP can affect activity. We observed that pyraclostrobin and trifloxystrobin induced hypoactive responses in zebrafish. At 50 nM, azoxystrobin and kresoxim-methyl exerted no effects on mitochondrial function nor locomotion of zebrafish. Studies such as this are important for determining sublethal toxicity to these fungicides, as widespread detection of strobilurins in aquatic environments suggests there is a potential for adverse effects in aquatic organisms.

Oxygen Consumption Evaluation: An Important Indicator of Metabolic State, Cellular Function, and Cell Fate Along Neural Deregulation.

Depletion of oxygen (O2) levels and reduction in the ATP synthesis (or even its complete blockage) are important characteristics of mitochondrial dysfunction; features that are often correlated with neurodegeneration. The measurement of oxygen consumption rate (OCR) is thus essential to evaluate cellular metabolism, survival, and neuroprotective strategies. In the present chapter, we describe the oxygen consumption assay using a Clark-type oxygen electrode in different types of samples named cells suspension (from primary and established cell culture), brain slices (ex vivo), and fresh brain tissues. In addition, we demonstrate herein how the program Oxygraph can be used in order to analyze the data and different approaches to normalize it.

Time-Resolved Fluorescence Assay for Measuring Oxygen Consumption Rates in Staphylococcus aureus.

Oxygen consumption is a fundamental characteristic of staphylococcal physiology reflecting the energy and metabolic state of the bacterial cell. During aerobic growth, oxygen consumption rates (OCR) depend on nutrient availability and vary at different growth stages. The measurement of oxygen consumption rates provides a versatile tool to characterize the impact of various mutations, environmental cues, and antibiotics on bacterial growth and fitness. In this chapter, we describe a MitoXpress® Xtra-based oxygen consumption assay for fast and reliable determination of respiration rates in Staphylococcus aureus. This highly reproducible and simple method requires a minimal set of reagents and allows rapid screening of multiple samples through real-time determination of the OCR with an oxygen-sensing probe and fluorescence plate reader.

The small molecule SERCA activator CDN1163 increases energy metabolism in human skeletal muscle cells

Background and objectiveA number of studies have highlighted muscle-specific mechanisms of thermogenesis involving futile cycling of Ca2+ driven by sarco (endo)plasmic reticulum Ca2+-ATPase (SERCA) and generating heat from ATP hydrolysis to be a promising strategy to counteract obesity and metabolic dysfunction. However, to the best of our knowledge, no experimental studies concerning the metabolic effects of pharmacologically targeting SERCA in human skeletal muscle cells have been reported. Thus, in the present study, we aimed to explore the effects of SERCA-activating compound, CDN1163, on energy metabolism in differentiated human skeletal muscle cells (myotubes). MethodsIn this study, we used primary myotube cultures derived from muscle biopsies of the musculus vastus lateralis and musculi interspinales from lean, healthy male donors. Energy metabolism in myotubes was studied using radioactive substrates. Oxygen consumption rate was assessed with the Seahorse XF24 bioanalyzer, whereas metabolic genes and protein expressions were determined by qPCR and immunoblotting, respectively. ResultsBoth acute (4 ​h) and chronic (5 days) treatment of myotubes with CDN1163 showed increased uptake and oxidation of glucose, as well as complete fatty acid oxidation in the presence of carbonyl cyanide 4-(trifluromethoxy)phenylhydrazone (FCCP). These effects were supported by measurement of oxygen consumption rate, in which the oxidative spare capacity and maximal respiration were enhanced after CDN1163-treatment. In addition, chronic treatment with CDN1163 improved cellular uptake of oleic acid (OA) and fatty acid β-oxidation. The increased OA metabolism was accompanied by enhanced mRNA-expression of carnitine palmitoyl transferase (CPT) 1B, pyruvate dehydrogenase kinase (PDK) 4, as well as increased AMP-activated protein kinase (AMPK)Thr172 phosphorylation. Moreover, following chronic CDN1163 treatment, the expression levels of stearoyl-CoA desaturase (SCD) 1 was decreased together with de novo lipogenesis from acetic acid and formation of diacylglycerol (DAG) from OA. ConclusionAltogether, these results suggest that SERCA activation by CDN1163 enhances energy metabolism in human myotubes, which might be favourable in relation to disorders that are related to metabolic dysfunction such as obesity and type 2 diabetes mellitus.

Ex vivo Assessment of Mitochondrial Function in Human Peripheral Blood Mononuclear Cells Using XF Analyzer.

Cellular health and function, as we know today, depend on a large extent on mitochondrial function. The essential function of mitochondria is the energy production, more precisely ATP production, via oxidative phosphorylation. Mitochondrial energy production parameters therefore represent important biomarkers. Studies on human cells have mainly been performed on in vitro cell cultures. However, peripheral blood mononuclear cells (PBMCs) are particularly suitable for such examinations. That's why this protocol describes a method to measure key parameters of mitochondrial function in freshly isolated PBMCs with the latest technology, the XF Analyzer. For this ex vivo approach PBMCs are first isolated out of human anticoagulated blood. Next, they are attached to the surface of special microplates pre-coated with Poly-D-Lysine. During the subsequent measurement of oxygen consumption rate (OCR) as well as extracellular acidification rate (ECAR) the stress reagents oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone and antimycin A are injected. Several mitochondrial parameters can be calculated from the results obtained. The application of this protocol allows the analysis of various influences, such as pharmaceuticals or environmental factors, on human cells.

Cell Energy Budget Platform for Multiparametric Assessment of Cell and Tissue Metabolism.

Specific bioenergetic signature reports on the current metabolic state of the cell, which may be affected by metabolic rearrangement, dysfunction or dysregulation of relevant signaling pathways, altered physiological condition or energy stress. A combined analysis of respiration , glycolytic flux, Krebs cycle activity, ATP levels, and total biomass allows informative initial assessment. Such simple, high-throughput, multiparametric methodology, called cell energy budget (CEB ) platform, is presented here and demonstrated with particular cell and tissue models. The CEB uses a commercial fluorescent lanthanide probe pH-Xtra™ to measure extracellular acidification (ECA) associated with lactate (L-ECA) and combined lactate/CO2 (T-ECA), a phosphorescent probe MitoXpress®-Xtra to measure oxygen consumption rate (OCR), a bioluminescent ATP kit, and an absorbance-based total protein assay. All the assays are performed on a standard multi-label reader. Using the same readouts, the CEB approach can be extended to more detailed mechanistic studies, by targeting specific pathways in cell bioenergetics and measuring other cellular parameters, such as NAD(P)H, Ca2+, mitochondrial pH, membrane potential, redox state, with conventional fluorescent or luminescent probes.

Antimicrobial susceptibility testing by measuring bacterial oxygen consumption on an integrated platform.

Cellular respiration is a fundamental feature of metabolic activity and oxygen consumption can be considered as a reliable indicator of bacterial aerobic respiration, including for facultative anaerobic bacteria like E. coli. Addressing the emerging global health challenge of antimicrobial resistance, we performed antimicrobial susceptibility testing using the bacterial oxygen consumption rate (OCR) as a phenotypic indicator. We demonstrated that microbial exposure to antibiotics showed systematic OCR variations, which enabled determining minimum inhibitory concentrations for three clinically relevant antibiotics, ampicillin, ciprofloxacin, and gentamicin, within a few hours. Our study was performed by using photoluminescence-based oxygen sensing in a microchamber format, which enabled reducing the sample volume to a few hundred microliters. OCR modeling based on exponential bacterial growth allowed estimating the bacterial doubling time for various culture conditions (different types of media, different culture temperature and antibiotic concentrations). Furthermore, correlating metabolic heat production data, as obtained by nanocalorimetry in the same type of microchamber, and OCR measurements provided further insight on the actual metabolic state and activity of a microbial sample. This approach represents a new path towards more comprehensive microbiological studies performed on integrated miniaturized systems.

Dynamic oxygen-17 MRI with adaptive temporal resolution using golden-means-based 3D radial sampling.

The aim of this study was to develop a high-resolution 3D oxygen-17 (17 O) MRI method to delineate the kinetics of 17 O-enriched water (H217 O) across the entire mouse brain after a bolus injection via the tail vein. The dynamic 17 O signal was acquired with a golden-means-based 3D radial sampling scheme. To achieve adequate temporal resolution with preserved spatial resolution, a k-space-weighted view sharing strategy was used in image reconstruction with an adaptive window size tailored to the kinetics of the 17 O signal. Simulation studies were performed to determine the adequate image reconstruction parameters. The established method was applied to delineating the kinetics of intravenously injected H217 O in vivo in the post-stroke mouse brain. The proposed dynamic 17 O-MRI method achieved an isotropic resolution of 1.21 mm (0.77 mm nominal) in mouse brain at 9.4T, with the temporal resolution increased gradually from 3 s at the initial phase of rapid signal increase to 15 s at the steady-state. The high spatial resolution enabled the delineation of the heterogeneous H217 O uptake and washout kinetics in stroke-affected mouse brain. The current study demonstrated a 3D 17 O-MRI method for dynamic monitoring of 17 O signal changes with high spatial and temporal resolution. The method can be utilized to quantify physiological parameters such as cerebral blood flow and blood-brain barrier permeability by tracking injected H217 O. It can also be used to measure oxygen consumption rate in 17 O-oxygen inhalation studies.

Yeast cell wall mannan rich fraction modulates bacterial cellular respiration potentiating antibiotic efficacy

Now more than ever there is a demand to understand the mechanisms surrounding antibiotic resistance and look for alternative ways to impact phenotypic antibiotic outcome. Cellular energetics can be impacted by many bacteriostatic and bactericidal antibiotics, which affect metabolism and energy output, resulting in a reduction of cell growth or induction of cell death respectively. In this study, we provide evidence that a mannan rich fraction (MRF) from the cell wall of Saccharomyces cerevisiae modulates growth of antibiotic susceptible and resistant Escherichia coli and potentiates bactericidal antibiotic efficiency through modulation of bacterial cellular respiration. The role of MRF in modulating bactericidal impact and cellular metabolic state were assessed in E. coli by monitoring microbial growth and by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XFe96 Analyser, respectively. This work further illustrates the link between bacterial susceptibility to antibiotics (phenotypic resistance) and resistance through modulation of bacterial metabolism. This is the first example of yeast MRF enabling collateral sensitivity to antibiotics in vitro and supports the search for alternative strategies to promote animal health without contributing to the growing issue of antimicrobial resistance.

271 Beta-adrenergic agonists increase maximal output of oxidative phosphorylation in bovine satellite cells

Abstract Beta-adrenergic agonists (βAA) are widely used supplements in beef production to improve feed efficiency and increase lean muscle mass, yet little is known about the molecular mechanism by which βAA achieve this outcome. Our objective was to identify the influence of Ractopamine HCl (RH) and Zilpaterol HCl (ZH) on mitochondrial function in muscle stem cells. Satellite cells were isolated from skeletal muscle of cattle (n = 4) and yak (n = 1) at harvest. Cells were exposed to ZH, RH, or no βAA in replicates of four. Real-time measurements of oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were recorded using intracellular flux analysis. Incubation of bovine (n = 4) cells with either ZH or RH increased maximal respiration (P = 0.004) and spare respiratory capacity (P = 0.013). When yak cells were included, the response to treatment was similar (P = 0.006 and P = 0.006), but an animal effect was also observed (P &amp;lt; 0.02), presumably due to differences in genetic background. These findings indicate that βAA-treated cells have the capacity to respond more efficiently to increased energy demands than non-supplemented cells. Beta-adrenergic agonists improve the efficiency of muscle stem cells in part by modifying mitochondrial function.