Measurement Of Oxygen Consumption Research Articles

Conservation energetics of beluga whales: using resting and swimming metabolism to understand threats to an endangered population.

The balance between energetic costs and acquisition in free-ranging species is essential for survival, and provides important insights regarding the physiological impact of anthropogenic disturbances on wild animals. For marine mammals such as beluga whales (Delphinapterus leucas), the first step in modeling this bioenergetic balance requires an examination of resting and active metabolic demands. Here, we used open-flow respirometry to measure oxygen consumption during surface rest and submerged swimming by trained beluga whales, and compared these measurements with those of a commonly studied odontocete, the Atlantic bottlenose dolphin (Tursiops truncatus). Both resting metabolic rate (3012±126.0 kJ h-1) and total cost of transport (1.4±0.1 J kg-1m-1) of beluga whales were consistent with predicted values for moderately sized marine mammals in temperate to cold-water environments, including dolphins measured in the present study. By coupling the rate of oxygen consumption during submerged swimming with locomotor metrics from animal-borne accelerometer tags, we developed predictive relationships for assessing energetic costs from swim speed, stroke rate and partial dynamic acceleration. Combining these energetic data with calculated aerobic dive limits for beluga whales (8.8 min), we found that high-speed responses to disturbance markedly reduce the whale's capacity for prolonged submergence, pushing the cetaceans to costly anaerobic performances that require prolonged recovery periods. Together, these species-specific energetic measurements for beluga whales provide two important metrics, gait-related locomotor costs and aerobic capacity limits, for identifying relative levels of physiological vulnerability to anthropogenic disturbances that have become increasingly pervasive in their Arctic habitats.

Targeting of mitochondrial fission through natural flavanones elicits anti-myeloma activity.

Mitochondrial alterations, often dependent on unbalanced mitochondrial dynamics, feature in the pathobiology of human cancers, including multiple myeloma (MM). Flavanones are natural flavonoids endowed with mitochondrial targeting activities. Herein, we investigated the capability of Hesperetin (Hes) and Naringenin (Nar), two aglycones of Hesperidin and Naringin flavanone glycosides, to selectively target Drp1, a pivotal regulator of mitochondrial dynamics, prompting anti-MM activity. Molecular docking analyses were performed on the crystallographic structure of Dynamin-1-like protein (Drp1), using Hes and Nar molecular structures. Cell viability and apoptosis were assessed in MM cell lines, or in co-culture systems with primary bone marrow stromal cells, using Cell Titer Glo and Annexin V-7AAD staining, respectively; clonogenicity was determined using methylcellulose colony assays. Transcriptomic analyses were carried out using the Ion AmpliSeq™ platform; mRNA and protein expression levels were determined by quantitative RT-PCR and western blotting, respectively. Mitochondrial architecture was assessed by transmission electron microscopy. Real time measurement of oxygen consumption was performed by high resolution respirometry in living cells. In vivo anti-tumor activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells. Hes and Nar were found to accommodate within the GTPase binding site of Drp1, and to inhibit Drp1 expression and activity, leading to hyperfused mitochondria with reduced OXPHOS. In vitro, Hes and Nar reduced MM clonogenicity and viability, even in the presence of patient-derived bone marrow stromal cells, triggering ER stress and apoptosis. Interestingly, Hes and Nar rewired MM cell metabolism through the down-regulation of master transcriptional activators (SREBF-1, c-MYC) of lipogenesis genes. An extract of Tacle, a Citrus variety rich in Hesperidin and Naringin, was capable to recapitulate the phenotypic and molecular perturbations of each flavanone, triggering anti-MM activity in vivo. Hes and Nar inhibit proliferation, rewire the metabolism and induce apoptosis of MM cells via antagonism of the mitochondrial fission driver Drp1. These results provide a framework for the development of natural anti-MM therapeutics targeting aberrant mitochondrial dependencies.

Peak oxygen consumption by smartwatches compared with cardiopulmonary exercise test in complex congenital heart disease

ObjectiveTo evaluate for correlation between exercise capacity as assessed by peak oxygen consumption (pVO2) measurement during a cardiopulmonary exercise test (CPET) and smartwatches reporting this parameter in patients with adult...

Developmental energetics in the oviparous corn snake, Pantherophis guttatus, confirms a conservative evolutionary pattern in snakes

Eggs of oviparous reptiles are ideal models for studying evolutionary patterns of embryonic metabolism since they allow tracking of energy allocation during development. Analyzing oxygen consumption of whole eggs throughout development indicates three patterns among reptiles. Embryos initially grow and consume oxygen exponentially, but oxygen consumption slows, or drops before hatching in some species. Turtles, crocodilians, and most lizards follow curves with initial exponential increases followed by declines, whereas embryonic snakes that have been studied exhibit a consistently exponential pattern. This study measured oxygen consumption of corn snake, Pantherophis guttatus, embryos to determine if this species also exhibits an exponential increase in oxygen consumption. Individual eggs, sampled weekly from oviposition to hatching, were placed in respirometry chambers for 24-h during which oxygen consumption was recorded. Embryos were staged and carcasses and yolk were weighed separately. Results indicate steady inclines in oxygen consumption during early stages of development, with a rapid increase prior to hatching. The findings support the hypothesis that embryonic oxygen consumption of snakes differs from most other non-avian reptiles. Total energy required for development was determined based on calorimetry of initial yolk compared to hatchlings and residual yolk and by integration of the area under the curve plotting oxygen consumption versus age of embryos. The cost of development estimates based on these two methods were 6.4 and 10.0 kJ, respectively. Our results emphasize the unique physiological aspects of snake embryogenesis and illustrate how the study of physiological characteristics can contribute to the broader understanding of reptilian evolution.

Taking a walk on the heart failure side: comparison of metabolic variables during walking and maximal exertion.

Although cardiopulmonary exercise testing (CPET) is the gold standard to assess exercise capacity, simpler tests (i.e., 6-min walk test, 6MWT) are also commonly used. The aim of this study was to evaluate the relationship between cardiorespiratory parameters during CPET and 6MWT in a large, multicentre, heterogeneous population. We included athletes, healthy subjects, and heart failure (HF) patients of different severity, including left ventricular assist device (LVAD) carriers, who underwent both CPET and 6MWT with oxygen consumption measurement. We enrolled 186 subjects (16 athletes, 40 healthy, 115 non-LVAD HF patients, and 15 LVAD carriers). CPET-peakV̇O2 was 41.0 [35.0-45.8], 26.2 [23.1-31.0], 12.8 [11.1-15.3], and 15.2 [13.6-15.6] ml/Kg/min in athletes, healthy, HF patients, and LVAD carriers, respectively (P<0.001). During 6MWT they used 63.5 [56.3-76.8], 72.0 [57.8-81.0], 95.5 [80.3-109], and 95.0 [92.0-99.0] % of their peakV̇O2, respectively. None of the athletes, 1 healthy (2.5%), 30 HF patients (26.1%), and 1 LVAD carrier (6.7%), reached a 6MWT-V̇O2 higher than their CPET-peakV̇O2. Both 6MWT-V̇O2 and walked distance were significantly associated with CPET-peakV̇O2 in the whole population (R2=0.637 and R2=0.533, P≤0.001) but not in the sub-groups. This was confirmed after adjustment for groups. The 6MWT can be a maximal effort especially in most severe HF patients and suggest that, in absence of prognostic studies related to 6MWT metabolic values, CPET should remain the first method of choice in the functional assessment of patients with HF as well as in sport medicine.

Validation of Indirect Calorimetry in Children Undergoing Single-Limb Non-Invasive Ventilation: A Proof of Concept, Cross-Over Study.

The accurate assessment of resting energy expenditure (REE) is essential for personalized nutrition, particularly in critically ill children. Indirect calorimetry (IC) is the gold standard for measuring REE. This methodology is based on the measurement of oxygen consumption (VO2) and carbon dioxide production (VCO2). These parameters are integrated into the Weir equation to calculate REE. Additionally, IC facilitates the determination of the respiratory quotient (RQ), offering valuable insights into a patient's carbohydrate and lipid consumption. IC validation is limited to spontaneously breathing and mechanically ventilated patients, but it is not validated in patients undergoing non-invasive ventilation (NIV). This study investigates the application of IC during NIV-CPAP (continuous positive airway pressure) and NIV-PS (pressure support). This study was conducted in the Pediatric Intensive Care Unit of IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milan, between 2019 and 2021. Children < 6 years weaning from NIV were enrolled. IC was performed during spontaneous breathing (SB), NIV-CPAP, and NIV-PS in each patient. A Bland-Altman analysis was employed to compare REE, VO2, VCO2, and RQ measured by IC. Fourteen patients (median age 7 (4; 18) months, median weight 7.7 (5.5; 9.7) kg) were enrolled. The REE, VO2, VCO2, and RQ did not differ significantly between the groups. The Limits of Agreement (LoA) and bias of REE indicated good agreement between SB and NIV-CPAP (LoA +28.2, -19.4 kcal/kg/day; bias +4.4 kcal/kg/day), and between SB and NIV-PS (LoA -22.2, +23.1 kcal/kg/day; bias 0.4 kcal/kg/day). These preliminary findings support the accuracy of IC in children undergoing NIV. Further validation in a larger cohort is warranted.

Fizička aktivnost i srčana slabost sa očuvanom ejekcionom frakcijom leve komore

Heart failure with preserved left ventricular ejection fraction (HFpEF) is a clinical syndrome in which the structure and the function of the heart are damaged, with stiffness of the heart muscle, increased pressures in diastole and in the pulmonary circulation. The latest understanding of HFpEF is that it is a systemic disease of the microcirculation with chronic inflammation and impaired cellular metabolism. The aim of this review was to determine the association between physical activity and HFpEF based on the available research. By summarizing the literature data, it was shown that a sedentary lifestyle represents a risk factor for HFpEF and that continuous physical activity prevents the occurrence of HFpEF, while in patients already suffering from HFpEF it increases functional capacity, as well as the quality of life, mainly due to the improvement of peripheral muscles oxidative metabolism. The impact of physical activity on improving the diastolic function of the heart in people with heart failure is still under research. Patients with HFpEF usually have poor exercise tolerance, thus an individualized prescription of physical activity is indicated in accordance with their capabilities, preferably a combination of aerobic training, strength exercises, balance and an adequate diet. The gold standard for the assessment of aerobic capacity is the cardiopulmonary exercise test, which allows the measurement of oxygen consumption, maximum heart rate, the first and the second anaerobic thresholds, parameters needed for dosing the intensity of physical exercise.

Spatially resolved quantification of oxygen consumption rate in ex vivo lymph node slices.

Cellular metabolism has been closely linked to activation state in cells of the immune system, and the oxygen consumption rate (OCR) in particular serves as a valuable metric for assessing metabolic activity. Several oxygen sensing assays have been reported for cells in standard culture conditions. However, none have provided a spatially resolved, optical measurement of local oxygen consumption in intact tissue samples, making it challenging to understand regional dynamics of consumption. Therefore, here we established a system to monitor the rates of oxygen consumption in ex vivo tissue slices, using murine lymphoid tissue as a case study. By integrating an optical oxygen sensor into a sealed perfusion chamber and incorporating appropriate correction for photobleaching of the sensor and of tissue autofluorescence, we were able to visualize and quantify rates of oxygen consumption in tissue. This method revealed for the first time that the rate of oxygen consumption in naïve lymphoid tissue was higher in the T cell region compared to the B cell and cortical regions. To validate the method, we measured OCR in the T cell regions of naïve lymph node slices using the optical assay and estimated the consumption rate per cell. The predictions from the optical assay were similar to reported values and were not significantly different from those of the Seahorse metabolic assay, a gold standard method for measuring OCR in cell suspensions. Finally, we used this method to quantify the rate of onset of tissue hypoxia for lymph node slices cultured in a sealed chamber and showed that continuous perfusion was sufficient to maintain oxygenation. In summary, this work establishes a method to monitor oxygen consumption with regional resolution in intact tissue explants, suitable for future use to compare tissue culture conditions and responses to stimulation.

A versatile micromodel technology to explore biofilm development in porous media flows.

Bacterial biofilms that grow in porous media are critical to ecosystem processes and applications ranging from soil bioremediation to bioreactors for treating wastewater or producing value-added products. However, understanding and engineering the complex phenomena that drive the development of biofilms in such systems remains a challenge. Here we present a novel micromodel technology to explore bacterial biofilm development in porous media flows. The technology consists of a set of modules that can be combined as required for any given experiment and conveniently tuned for specific requirements. The core module is a 3D-printed micromodel where biofilm is grown into a perfusable porous substrate. High-precision additive manufacturing, in particular stereolithography, is used to fabricate porous scaffolds with precisely controlled architectures integrating flow channels with diameters down to several hundreds of micrometers. The system is instrumented with: ultraviolet-C light-emitting diodes; on-line measurements of oxygen consumption and pressure drop across the porous medium; camera and spectrophotometric cells for the detection of biofilm detachment events at the outlet. We demonstrate how this technology can be used to study the development of Pseudomonas aeruginosa biofilm for several days within a network of flow channels. We find complex dynamics whereby oxygen consumption reaches a steady-state but not the pressure drop, which instead features a permanent regime with large fluctuations. We further use X-ray computed microtomography to image the spatial distribution of biofilms and computational fluid dynamics to link biofilm development with local flow properties. By combining the advantages of additive manufacturing for the creation of reproducible 3D porous microarchitectures with the flow control and instrumentation accuracy of microfluidics, our system provides a platform to study the dynamics of biofilm development in 3D porous media and to rapidly test new concepts in process engineering.

Evaluation of Oxygen Consumption Rates In Situ.

An analysis of the mitochondrial respiration function represented by the oxygen consumption rate is necessary to assess mitochondrial bioenergetics and redox function. This protocol describes two alternative techniques to evaluate mitochondrial respiration function in situ: (1) measure oxygen consumption rates via an electrode; (2) measure oxygen consumption rates via a seahorse instrument. These in situ approaches provide more physiological access to mitochondria to evaluate mitochondrial respiration function in a relatively integrated cellular system.

High-Resolution Respirometry for the Assessment of Teratogenic Chemicals.

Pesticides are often used in agriculture and residential areas to mitigate pests and weeds. These chemicals canenter aquatic ecosystems via runoff and rain events, exerting negative effects on aquatic species. In rapidly developing fish embryos, metabolic disruption can alter the developmental trajectory and alter ATP levels. Therefore, it is important to quantify mitochondrial integrity in organisms following exposure to pesticides. To achieve this, a high throughput method to assess pesticide effects on oxidative phosphorylation and mitochondria has been optimized for fish embryos. Fish embryos are first exposed to pesticides for 24 or 48h, and oxygen consumption rates are measured using the SeahorseXFe24/96 Flux Analyzer (formerlySeahorse Biosciences, now Agilent). The assay utilizes a single embryo and precisely measures oxygen consumption and extracellular acidification. Based upon these measurements, characteristics related to mitochondrial bioenergetics are calculated to provide information on mitochondrial integrity. Using this approach, one can identify pesticides affecting the electron transport chain and ultimately ATP production. In this chapter, we describe the mitochondrial stress test to understand mitochondrial dysfunction and metabolic shifts within the fish embryo.

New System for Simultaneous Measurement of Oxygen Consumption and Changes in Wine Color.

The design, construction and validation of a device for the accurate measurement of the dissolved oxygen content in wine and simultaneously the variation of its spectral fingerprint is presented. The novelty of this system is due to two innovative approaches. First, robustness in measurements is obtained by using cuvettes designed to simultaneously measure the dissolved oxygen and color. Secondly, automatic monitoring is performed to ensure that measurements are always taken at the same cuvette position. The fine-tuning of the device with the study of white and red wines makes it possible, on the one hand, to establish the appropriate measurement conditions and, on the other hand, to determine the amount of oxygen required to cause specific changes in the wine spectrum, information that could not be obtained until now. The preliminary results are very interesting, presenting precise data on the amount of oxygen consumed by the wine and the variations in its visible spectrum, thus reflecting the modification of the responsible phenolic compounds. This information is of great interest, since it helps to optimize the handling of the wine and, if necessary, to moderate the uptake of oxygen in each type of wine to ensure the maintenance of the color during the winemaking and conservation processes of each type of wine. The results of the experiments indicate that this new instrument is feasible and accurate for detecting oxygen changes during wine production.

Gallic acid triphenylphosphonium derivatives TPP+-C10 and TPP+-C12 inhibit mitochondrial function in Candida albicans exerting antifungal and antibiofilm effects.

To evaluate the antifungal and antibiofilm activity of gallic acid derivatives TPP+-C10 and TPP+-C12 and their effects on mitochondrial function on two Candida albicans reference strains (ATCC 90029 and ATCC 10231). First, we determined minimal inhibitory concentration (MIC) using a microdilution assay. Both compounds exerted antifungal effects, and their MICs ranged from 3.9 to 13µM, with no statistically significant differences between them (P>0.05, t-test). These concentrations served as references for following assays. Subsequently, we measured oxygen consumption with a Clark electrode. Our observations revealed that both drugs inhibited oxygen consumption in both strains with TPP+-C12 exerting a more pronounced inhibitory effect. We then employed flow cytometry with TMRE as a probe to assess mitochondrial membrane potential. For each strain assayed, the compounds induced a decay in transmembrane potential by 75%-90% compared to the control condition (P<0.05, ANOVA). Then, we measured ATP levels using a commercial kit. TPP+-C12 showed a 50% decrease of ATP content (P<0.05 ANOVA), while TPP+-C10 exhibited a less pronounced effect. Finally, we assessed the antibiofilm effect using the MTT reduction assay. Both compounds were effective, but TPP+-C12 displayed a greater potency, requiring a lower concentration to inhibit 50% of biofilms viability (P<0.05, t-test). Derivatives of gallic acid linked to a TPP+ group exert antifungal and antibiofilm activity through impairment of mitochondrial function in C. albicans.

PRRG4 regulates mitochondrial function and promotes migratory behaviors of breast cancer cells through the Src-STAT3-POLG axis

BackgroundBreast cancer is the leading cause of cancer death for women worldwide. Most of the breast cancer death are due to disease recurrence and metastasis. Increasingly accumulating evidence indicates that mitochondria play key roles in cancer progression and metastasis. Our recent study revealed that transmembrane protein PRRG4 promotes the metastasis of breast cancer. However, it is not clear whether PRRG4 can affect the migration and invasion of breast cancer cells through regulating mitochondria function.MethodsRNA-seq analyses were performed on breast cancer cells expressing control and PRRG4 shRNAs. Quantitative PCR analysis and measurements of mitochondrial ATP content and oxygen consumption were carried out to explore the roles of PRRG4 in regulating mitochondrial function. Luciferase reporter plasmids containing different lengths of promoter fragments were constructed. Luciferase activities in breast cancer cells transiently transfected with these reporter plasmids were analyzed to examine the effects of PRRG4 overexpression on promoter activity. Transwell assays were performed to determine the effects of PRRG4-regulated pathway on migratory behaviors of breast cancer cells.ResultsAnalysis of the RNA-seq data revealed that PRRG4 knockdown decreased the transcript levels of all the mitochondrial protein-encoding genes. Subsequently, studies with PRRG4 knockdown and overexpression showed that PRRG4 expression increased mitochondrial DNA (mtDNA) content. Mechanistically, PRRG4 via Src activated STAT3 in breast cancer cells. Activated STAT3 in turn promoted the transcription of mtDNA polymerase POLG through a STAT3 DNA binding site present in the POLG promoter region, and increased mtDNA content as well as mitochondrial ATP production and oxygen consumption. In addition, PRRG4-mediated activation of STAT3 also enhanced filopodia formation, migration, and invasion of breast cancer cells. Moreover, PRRG4 elevated migratory behaviors and mitochondrial function of breast cancer cells through POLG.ConclusionOur results indicate that PRRG4 via the Src-STAT3-POLG axis enhances mitochondrial function and promotes migratory behaviors of breast cancer cells.

Identification of a Novel Mechanism Fostering the Alterations of Brain Energy Metabolism: a Link Between AD and T2DM

AbstractBackgroundBrain insulin resistance (bIR) is associated with mitochondrial stress, failure of energy metabolism, synaptic loss and ultimately neurodegeneration both in Alzheimer’s disease (AD) and Type 2‐Diabetes Mellitus (T2DM). Our group previously demonstrated that loss of BVR‐A is an early event triggering bIR in AD. Hence, we tested the hypothesis that reduced BVR‐A levels link bIR and mitochondrial defects resulting in AD‐like neuropathology in T2DM.MethodAlterations of insulin signalling, autophagic flux, oxidative stress levels and AD hallmarks were analyzed in the hippocampus of wild‐type and the goto‐kakizaki (GK) model of T2DM rats. Hippocampal mitochondrial function was evaluated by measuring oxygen consumption rate (OCR), mitochondrial complexes, mitochondrial unfolded protein response (UPRmt) and oxidative stress levels. These data were correlated with peripheral metabolic measurements (fasting glucose, insulin and OGTT) and cognitive tasks (spatial memory). To confirm the role of BVR‐A, similar analyses were performed in the hippocampus of WT and BVR‐A KO mice fed a high fat diet (HFD, 60% kcal from fat) for 1, 3 or 8 weeks. Additional mechanistic insights were gained by evaluating mitochondrial function (sea‐horse) and the above‐mentioned intracellular pathways in response to insulin, in neuronal cells lacking BVR‐A.ResultGK rats displayed a diabetic phenotype and impaired spatial memory. Reduced BVR‐A levels along with IRS1 hyper‐activation and loss of Akt‐mediated inhibition of GSK3ß were observed in the hippocampus, consistent with the regulatory role for BVR‐A. As result, hyperactive GSK3ß accumulated in hippocampal mitochondria fostering their impairment characterized by reduced OCR and activation of UPRmt. Similar alterations in BVR‐A KO mice and in neuronal cells lacking BVR‐A were observed, reinforcing the role for BVR‐A in regulating mitochondrial bioenergetics in response to insulin. HFD regimen promoted worse outcomes in BVR‐A KO mice with respect to WT. Remarkably, gender‐differences were observed whereby female mice show earlier and greater alterations with respect to male mice.ConclusionThese results suggest that early BVR‐A loss triggers bIR and the hyper‐action of GSK3ß, that, in turn, drives the development of mitochondrial stress in T2DM brain. These alterations accelerate the impairment of energy metabolism and development of AD‐like neuropathology in T2DM.

Color Stability During Retail Display Differs Among Muscles in Pork

The objectives of this study were to determine differences in color stability among pork muscles and whether changes in color measurements for longissimus dorsi (LD) were related to changes for triceps brachii (TB) or psoas major (PM). The LD, TB, and PM from 20 pigs were aged in vacuum packages for 21 d, then fabricated into 3 chops per muscle. All chops from a single muscle were overwrapped, displayed for 5 d under constant lighting, and evaluated daily for instrumental color, myoglobin forms, and subjective visual discoloration. Chops were removed on day 1, 3, or 5 to measure oxygen consumption (OC) and metmyoglobin reducing activity (MRA). Data were analyzed using the MIXED procedure of SAS as repeated measures. Pearson correlation coefficients were determined using the CORR procedure. Over the 5-d display period, TB and PM chops had greater changes in a*, b*, chroma, 630/580 nm ratio, deoxymyoglobin, and oxymyoglobin compared to LD chops (display time×muscle interaction P≤0.01), but TB and PM did not differ from each other (P≥0.07). Conversely, LD had decreased OC and increased MRA on each display day compared to TB and PM (P&amp;lt;0.01). Using 20% discolored on day 5 of display as a threshold for acceptability, 85% of LD samples were considered acceptable whereas only 5% and 20% of TB and PM samples were considered acceptable, respectively. Changes in LD were correlated with TB and PM changes for redness (0.51≤r≤0.55; P≤0.02), and they trended toward a correlation with changes in TB and PM 630/580 nm ratio and hue angle (0.39≤r≤0.63). Chops from TB and PM were less color stable during display, but LD changes were not consistently correlated with changes in other muscles.

Measuring effect of mutations & conditions on microbial respiratory rates

Cellular respiration is central to a wide range of cellular processes. In microorganisms, the effect of a mutation or an environmental condition on the rate of respiration is usually determined by measuring oxygen consumption in the media. We describe this method and discuss caveats and controls for the method.

Method for depletion of mitochondria DNA in human bronchial epithelial cells

Mitochondria are increasingly recognized to play a role in the airway inflammation of asthma. Model systems to study the role of mitochondrial gene expression in bronchial epithelium are lacking. Here, we create custom bronchial epithelial cell lines that are depleted of mitochondrial DNA. One week of ethidium bromide (EtBr) treatment led to ∼95 % reduction of mtDNA copy number (mtDNA-CN) in cells, which was further reduced by addition of 25 µM 2′,3′-dideoxycytidin (ddC). Treatment for up to three weeks with EtBr and ddC led to near complete loss of mtDNA. The basal oxygen consumption rate (OCR) of mtDNA-depleted BET-1A and BEAS-2B cells dropped to near zero. Glycolysis measured by extracellular acidification rate (ECAR) increased ∼two-fold in cells when mtDNA was eliminated. BET-1A ρ0 and BEAS-2B ρ0 cells were cultured for two months, frozen and thawed, cultured for two more months, and maintained near zero mtDNA-CN. Mitochondrial DNA–depleted BET-1A ρ0 and BEAS-2B ρ0 cell lines are viable, lack the capacity for aerobic respiration, and increase glycolysis.•BET-1A and BEAS-2B cells were treated with ethidium bromide (EtBr) with or without 2′,3′-dideoxycytidine (ddC) to create cells lacking mitochondrial DNA (mtDNA).•Cells’ mtDNA copy number relative to nuclear DNA (nDNA) were verified by quantitative polymerase chain reaction (qPCR).•Cells were also assessed for oxidative phosphorylation by measures of oxygen consumption using the Seahorse analyzer.

A simple assay for inhibitors of mycobacterial oxidative phosphorylation

Oxidative phosphorylation, the combined activities of the electron transport chain (ETC) and adenosine triphosphate (ATP) synthase, has emerged as a valuable target for antibiotics to treat infection with Mycobacterium tuberculosis and related pathogens. In oxidative phosphorylation, the ETC establishes a transmembrane electrochemical proton gradient that powers ATP synthesis. Monitoring oxidative phosphorylation with luciferase-based detection of ATP synthesis or measurement of oxygen consumption can be technically challenging and expensive. These limitations reduce the utility of these methods for characterization of mycobacterial oxidative phosphorylation inhibitors. Here we show that fluorescence-based measurement of acidification of inverted membrane vesicles (IMVs) can detect and distinguish between inhibition of the ETC, inhibition of ATP synthase, and non-specific membrane uncoupling. In this assay, IMVs from M. smegmatis are acidified either through the activity of the ETC or ATP synthase, the latter modified genetically to allow it to serve as an ATP-driven proton pump. Acidification is monitored by fluorescence from 9-amino-6-chloro-2-methoxyacridine, which accumulates and quenches in acidified IMVs. Non-specific membrane uncouplers prevent both succinate- and ATP-driven IMV acidification. In contrast, the ETC Complex III2IV2 inhibitor telacebec (Q203) prevents succinate-driven acidification but not ATP-driven acidification and the ATP synthase inhibitor bedaquiline prevents ATP-driven acidification but not succinate-driven acidification. We use the assay to show that, as proposed previously, lansoprazole sulfide is an inhibitor of Complex III2IV2 while thioridazine uncouples the mycobacterial membrane non-specifically. Overall, the assay is simple, low cost, and scalable, which will make it useful for identifying and characterizing new mycobacterial oxidative phosphorylation inhibitors.

Medium Depth Influences O2 Availability and Metabolism in Human RPE Cultures.

Retinal pigment epithelium (RPE) oxidative metabolism is critical for normal retinal function and is often studied in cell culture systems. Here, we show that conventional culture media volumes dramatically impact O2 availability, limiting oxidative metabolism. We suggest optimal conditions to ensure cultured RPE is in a normoxic environment permissive to oxidative metabolism. We altered the availability of O2 to human primary and induced pluripotent stem cell-derived RPE cultures directly via a hypoxia chamber or indirectly via the amount of medium over cells. We measured oxygen consumption rates (OCRs), glucose consumption, lactate production, 13C6-glucose and 13C5-glutamine flux, hypoxia inducible factor 1α (HIF-1α) stability, intracellular lipid droplets after a lipid challenge, transepithelial electrical resistance, cell morphology, and pigmentation. Medium volumes commonly employed during RPE culture limit diffusion of O2 to cells, triggering hypoxia, activating HIF-1α, limiting OCR, and dramatically altering cell metabolism, with only minor effects on typical markers of RPE health. Media volume effects on O2 availability decrease acetyl-CoA utilization, increase glycolysis and reductive carboxylation, and alter the size and number of intracellular lipid droplets under lipid-rich conditions. Despite having little impact on visible and typical markers of RPE culture health, media volume dramatically affects RPE physiology "under the hood." As RPE-centric diseases like age-related macular degeneration involve oxidative metabolism, RPE cultures need to be optimized to study such diseases. We provide guidelines for optimal RPE culture volumes that balance ample nutrient availability from larger media volumes with adequate O2 availability seen with smaller media volumes.