MitoTracker Red for isolation of zone-specific hepatocytes and characterization of hepatic sublobular metabolism.

BACKGROUND: The liver lobule is divided into three zones or regions: periportal (PP or Zone 1) that is highly oxidative and active in ureagenesis, pericentral (PC or Zone 3) that is more glycolytic, and midzonal (MZ or Zone 2) with intermediate characteristics. AIM: Our goal was to isolate and metabolically characterize hepatocytes from specific sublobular zones. METHODS: Mice were administered rhodamine123 (Rh123) or MitoTracker Red (MTR) prior to intravital imaging, liver fixation, or hepatocyte isolation. After in vivo MTR, hepatocytes were isolated and sorted based on MTR fluorescence intensity. Alternatively, E-cadherin (Ecad) and cytochrome P450 2E1 (CYP2E1) immunolabeling was performed in fixed liver slices. Ecad and CYP2E1 gene expression in sorted hepatocytes was assessed by qPCR. Oxygen consumption rates (OCR) of sorted hepatocytes were also assessed. RESULTS: Multiphoton microscopy showed Rh123 and MTR fluorescence distributed zonally, decreasing from PP to PC in a flow-dependent fashion. In liver cross-sections, Ecad was expressed periportally and CYP2E1 pericentrally in association with high and low MTR labeling, respectively. Based on MTR fluorescence, hepatocytes were sorted into PP, MZ, and PC populations with PP and PC hepatocytes enriched in Ecad and CYP2E1, respectively. OCR of PP hepatocytes was ∼4 times that of PC hepatocytes. CONCLUSIONS: MTR treatment in vivo delineates sublobular hepatic zones and can be used to sort hepatocytes zonally. PP hepatocytes have substantially greater OCR compared to PC and MZ. The results also indicate a sharp midzonal demarcation between hepatocytes with PP characteristics (Ecad) and those with PC features (CYP2E1). This new method to sort hepatocytes in a zone-specific fashion holds the potential to shed light on sublobular hepatocyte metabolism and regulatory pathways in health and disease.

HuR and GRSF1 modulate the nuclear export and mitochondrial localization of the lncRNA RMRP.

Some mitochondrial long noncoding RNAs (lncRNAs) are encoded by nuclear DNA, but the mechanisms that mediate their transport to mitochondria are poorly characterized. Using affinity RNA pull-down followed by mass spectrometry analysis, we found two RNA-binding proteins (RBPs), HuR (human antigen R) and GRSF1 (G-rich RNA sequence-binding factor 1), that associated with the nuclear DNA-encoded lncRNA RMRP and mobilized it to mitochondria. In cultured human cells, HuR bound RMRP in the nucleus and mediated its CRM1 (chromosome region maintenance 1)-dependent export to the cytosol. After RMRP was imported into mitochondria, GRSF1 bound RMRP and increased its abundance in the matrix. Loss of GRSF1 lowered the mitochondrial levels of RMRP, in turn suppressing oxygen consumption rates and modestly reducing mitochondrial DNA replication priming. Our findings indicate that RBPs HuR and GRSF1 govern the cytoplasmic and mitochondrial localization of the lncRNA RMRP, which is encoded by nuclear DNA but has key functions in mitochondria.

Enhanced Expression of Glycolytic Enzymes and Succinate Dehydrogenase Complex Flavoprotein Subunit A by Mesothelin Promotes Glycolysis and Mitochondrial Respiration in Myeloblasts of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive malignancy characterized by rapid growth and uncontrolled proliferation of undifferentiated myeloid cells. Metabolic reprogramming is commonly observed in the bone marrow of AML patients, as leukemia cells require increased ATP supply to support disease progression. In this study, we examined the potential role of mesothelin as a metabolic modulator in myeloid cells in AML. Mesothelin is a well-known marker of solid tumors that promotes cancer cell proliferation and survival. We initially analyzed alterations in mesothelin expression in the myeloblast subpopulations, defined as SSC-Alow/CD45dim, obtained from the bone marrow of AML patients using flow cytometry. Our results showed overexpression of mesothelin in 34.8% of AML patients. Subsequently, metabolic changes in leukemia cells were evaluated by comparing the oxygen consumption rates (OCR) of bone marrow samples derived from adult AML patients. Notably, a higher OCR was observed in the mesothelin-positive compared to the mesothelin-low and non-expressing groups. Treatment with recombinant human mesothelin protein enhanced OCR and increased the mRNA expression of glycolytic enzymes and mitochondrial complex II in KG1α AML cells. Notably, siRNA targeting mesothelin in KG1α cells led to the reduction of glycolysis-related gene expression but had no effect on the mitochondrial complex gene. The collective results demonstrate that mesothelin induces metabolic changes in leukemia cells, facilitating the acquisition of a rapid supply of ATP for proliferation in AML. Therefore, the targeting of mesothelin presents a potentially promising approach to mitigating the progression of AML through the inhibition of glycolysis and mitochondrial respiration in myeloid cells.

The Impact of the Angiotensin-Converting Enzyme Inhibitor Lisinopril on Metabolic Rate in Drosophila melanogaster.

Evidence suggests that angiotensin-converting enzyme inhibitors (ACEIs) may increase metabolic rate by promoting thermogenesis, potentially through enhanced fat oxidation and improved insulin. More research is, however, needed to understand this intricate process. In this study, we used 22 lines from the Drosophila Genetic Reference Panel to assess the metabolic rate of virgin female and male flies that were either fed a standard medium or received lisinopril for one week or five weeks. We demonstrated that lisinopril affects the whole-body metabolic rate in Drosophila melanogaster in a genotype-dependent manner. However, the effects of genotypes are highly context-dependent, being influenced by sex and age. Our findings also suggest that lisinopril may increase the Drosophila metabolic rate via the accumulation of a bradykinin-like peptide, which, in turn, enhances cold tolerance by upregulating Ucp4b and Ucp4c genes. Finally, we showed that knocking down Ance, the ortholog of mammalian ACE in Malpighian/renal tubules and the nervous system, leads to opposite changes in metabolic rate, and that the effect of lisinopril depends on Ance in these systems, but in a sex- and age-specific manner. In conclusion, our results regarding D. melanogaster support existing evidence of a connection between ACEI drugs and metabolic rate while offering new insights into this relationship.

Nanoplastics Penetrate Human Bronchial Smooth Muscle and Small Airway Epithelial Cells and Affect Mitochondrial Metabolism.

Micro- and nanoplastic particles, including common forms like polyethylene and polystyrene, have been identified as relevant pollutants, potentially causing health problems in living organisms. The mechanisms at the cellular level largely remain to be elucidated. This study aims to visualize nanoplastics in bronchial smooth muscle (BSMC) and small airway epithelial cells (SAEC), and to assess the impact on mitochondrial metabolism. Healthy and asthmatic human BSMC and SAEC in vitro cultures were stimulated with polystyrene nanoplastics (PS-NPs) of 25 or 50 nm size, for 1 or 24 h. Live cell, label-free imaging by holotomography microscopy and mitochondrial respiration and glycolysis assessment were performed. Furthermore, 25 and 50 nm NPs were shown to penetrate SAEC, along with healthy and diseased BSMC, and they impaired bioenergetics and induce mitochondrial dysfunction compared to cells not treated with NPs, including changes in oxygen consumption rate and extracellular acidification rate. NPs pose a serious threat to human health by penetrating airway tissues and cells, and affecting both oxidative and glycolytic metabolism.

High-throughput method for Oxygen Consumption Rate measurement (OCR) in plant mitochondria.

BACKGROUND: Conventional methods to measure oxygen consumption, such as Clark-type electrodes, have limitations such as requiring a large amount of starting material. Moreover, commercially available kits for high-throughput methods are usually optimized for animal cells and mitochondria. Here, we present a novel method to measure the oxygen consumption rate using a high-throughput assay in isolated mitochondria of European beech seeds. To perform the measurements, we adapted the Agilent Seahorse XF Cell Mito Stress Test Kit protocol for measurements on plant mitochondria. RESULTS: The optimized protocol for OCR measurement of mitochondria isolated from beech seeds allowed the observation of storage period-dependent gradual decreases in non-phosphorylating respiration, phosphorylating respiration and maximal FCCP-stimulated respiration. The longer the seeds were stored, the greater the impairment of respiratory function. CONCLUSIONS: Thanks to this method it is possible to minimize the amount of plant material and conduct research to obtain information on the respiratory condition and activity of plant mitochondria, including the efficiency of oxidative phosphorylation and the maximum oxidative capacity of the respiratory chain. We demonstrated that the improved protocol is suitable for study of plant material.

Serum starvation affects mitochondrial metabolism of adipose-derived stem/stromal cells.

BACKGROUND AIMS: A large part of mesenchymal stromal cell (MSC) regenerative and immunomodulatory action is mediated by paracrine signaling. Hence, an increasing body of evidence acknowledges the potential of MSC secretome in a variety of preclinical and clinical scenarios. Mid-term serum deprivation is a common approach in the pipeline of MSC secretome production. Nevertheless, up to now, little is known about the impact of this procedure on the metabolic status of donor cells. METHODS: Here, through untargeted differential metabolomics, we revealed an impairment of mitochondrial metabolism in adipose-derived MSCs exposed for 72 h to serum deprivation. RESULTS: This evidence was further confirmed by the significant accumulation of reactive oxygen species and the reduction of succinate dehydrogenase activity. Probably as a repair mechanism, an upregulation of mitochondrial superoxide dismutase was also induced. CONCLUSIONS: Of note, the analysis of mitochondrial functionality indicated that, despite a significant reduction of basal respiration and ATP production, serum-starved MSCs still responded to changes in energy demand. This metabolic phenotype correlates with the obtained evidence of mitochondrial elongation and branching upon starvation.

Early dynamic changes in iPSC oxygen consumption rate predict future cardiomyocyte differentiation.

Human induced pluripotent stem cells (iPSCs) hold great promise for reducing the mortality of cardiovascular disease by cellular replacement of infarcted cardiomyocytes (CMs). CM differentiation via iPSCs is a lengthy multiweek process and is highly subject to batch-to-batch variability, presenting challenges in current cell manufacturing contexts. Real-time, label-free control quality attributes (CQAs) are required to ensure efficient iPSC-derived CM manufacturing. In this work, we report that live oxygen consumption rate measurements are highly predictive CQAs of CM differentiation outcome as early as the first 72 h of the differentiation protocol with an accuracy of 93%. Oxygen probes are already incorporated in commercial bioreactors, thus methods presented in this work are easily translatable to the manufacturing setting. Detecting deviations in the CM differentiation trajectory early in the protocol will save time and money for both manufacturers and patients, bringing iPSC-derived CM one step closer to clinical use.

Mitochondrial bioenergetic changes in systemic lupus erythematosus immune cell subsets: Contributions to pathogenesis and clinical applications.

The association of dysregulated metabolism in systemic lupus erythematosus (SLE) pathogenesis has prompted investigations into metabolic rewiring and the involvement of mitochondrial metabolism as a driver of disease through NLRP3 inflammasome activation, disruption of mitochondrial DNA maintenance, and pro-inflammatory cytokine release. The use of Agilent Seahorse Technology to gain functional in situ metabolic insights of selected cell types from SLE patients has identified key parameters that are dysregulated during disease. Mitochondrial functional assessments specifically can detect dysfunction through oxygen consumption rate (OCR), spare respiratory capacity, and maximal respiration measurements, which, when coupled with disease activity scores could show potential as markers of disease activity. CD4+ and CD8 + T cells have been assessed in this way and show that oxygen consumption rate, spare respiratory capacity, and maximal respiration are blunted in CD8 + T cells, with results not being as clear cut in CD4 + T cells. Additionally, glutamine, processed by mitochondrial substrate level phosphorylation is emerging as a key role player in the expansion and differentiation of Th1, Th17, ϒδ T cells, and plasmablasts. The role that circulating leukocytes play in acting as bioenergetic biomarkers of diseases such as diabetes suggests that this may also be a tool to detect preclinical SLE. Therefore, the metabolic characterization of immune cell subsets and the collection of metabolic data during interventions is also essential. The delineation of the metabolic tuning of immune cells in this way could lead to novel strategies in treating metabolically demanding processes characteristic of autoimmune diseases such as SLE.

Attenuation of PM2.5-induced alveolar epithelial cells and lung injury through regulation of mitochondrial fission and fusion.

BACKGROUND: Exposure to particulate matter (PM) with an aerodynamic diameter less than 2.5 µm (PM2.5) is a risk factor for developing pulmonary diseases and the worsening of ongoing disease. Mitochondrial fission and fusion are essential processes underlying mitochondrial homeostasis in health and disease. We examined the role of mitochondrial fission and fusion in PM2.5-induced alveolar epithelial cell damage and lung injury. Key genes in these processes include dystrophin-related protein 1 (DRP1) and optic atrophy 1 (OPA1) respectively. METHODS: Alveolar epithelial (A549) cells were treated with PM2.5 (32 µg/ml) in the presence and absence of Mdivi-1 (10µM, a DRP1 inhibitor) or BGP-15 (10µM, an OPA1 activator). Results were validated using DRP1-knockdown (KD) and OPA1-overexpression (OE). Mice were injected intraperitoneally with Mdivi-1 (20 mg/kg), BGP-15 (20 mg/kg) or distilled water (control) one hour before intranasal instillation of PM2.5 (7.8 mg/kg) or distilled water for two consecutive days. RESULTS: PM2.5 exposure of A549 cells caused oxidative stress, enhanced inflammation, necroptosis, mitophagy and mitochondrial dysfunction indicated by abnormal mitochondrial morphology, decreased mitochondrial membrane potential (ΔΨm), reduced mitochondrial respiration and disrupted mitochondrial fission and fusion. Regulating mitochondrial fission and fusion pharmacologically using Mdivi-1 and BGP-15 and genetically using DRP1-KD and OPA1-OE prevented PM2.5-induced celluar damage in A549 cells. Mdivi-1 and BGP-15 attenuated PM2.5-induced acute lung injury in mice. CONCLUSION: Increased mitochondrial fission and decreased mitochondrial fusion may underlie PM2.5-induced alveolar epithelial cell damage in vitro and lung injury in vivo.

Fine particulate matter exposure disturbs autophagy, redox balance and mitochondrial homeostasis via JNK activation to inhibit proliferation and promote EMT in human alveolar epithelial A549 cells.

Epidemiologic studies have demonstrated a direct correlation between fine particulate matter (FPM) exposure and the high risk of respiratory diseases. FPM can penetrate deep into the lung and deposit in the alveoli with breath, where it directly interacts with alveolar epithelial cell (APC). However, we know little about the effects nor mechanisms of FPM on APC. Here, using human APC A549 cells, we found that FPM resulted in blockade of autophagic flux, redox imbalance and oxidative stress, mitochondrial fragmentation, increased mitophagy and impaired mitochondrial respiration. Further we showed that activation of JNK signaling (c-Jun N-terminal kinase) and excessive ROS (reactive oxygen species) release contribute to these adverse effects, with the former being upstream of the latter. More importantly, we found that scavenging ROS or inhibiting JNK activation could restore those effects as well as ameliorate FPM-induced inhibition of cell proliferation, and epithelial-mesenchymal transformation (EMT) in A549 cells. Taken together, our findings indicate that FPM leads to toxicity in alveolar type II cells via JNK activation, and JNK-targeting or antioxidant strategies might be beneficial for prevention or treatment of FPM-related pulmonary diseases.

Modulation of Mitochondrial Metabolic Parameters and Antioxidant Enzymes in Healthy and Glaucomatous Trabecular Meshwork Cells with Hybrid Small Molecule SA-2.

Oxidative stress (OS)-induced mitochondrial damage is a risk factor for primary open-angle glaucoma (POAG). Mitochondria-targeted novel antioxidant therapies could unearth promising drug candidates for the management of POAG. Previously, our dual-acting hybrid molecule SA-2 with nitric oxide-donating and antioxidant activity reduced intraocular pressure and improved aqueous humor outflow in rodent eyes. Here, we examined the mechanistic role of SA-2 in trabecular meshwork (TM) cells in vitro and measured the activity of intracellular antioxidant enzymes during OS. Primary human TM cells isolated from normal (hNTM) or glaucomatous (hGTM) post-mortem donors and transformed glaucomatous TM cells (GTM-3) were used for in vitro assays. We examined the effect of SA-2 on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro using Seahorse Analyzer with or without the oxidant, tert-butyl hydroperoxide (TBHP) treatment. Concentrations of total antioxidant enzymes, catalase (CAT), malondialdehyde (MDA), and glutathione peroxidase (GPx) were measured. We observed significant protection of both hNTM and hGTM cells from TBHP-induced cell death by SA-2. Antioxidant enzymes were elevated in SA-2-treated cells compared to TBHP-treated cells. In addition, SA-2 demonstrated an increase in mitochondrial metabolic parameters. Altogether, SA-2 protected both normal and glaucomatous TM cells from OS via increasing mitochondrial energy parameters and the activity of antioxidant enzymes.

Impact of Exposure to Pyraclostrobin and to a Pyraclostrobin/Boscalid Mixture on the Mitochondrial Function of Human Hepatocytes.

Fungicides are widely used in agriculture for crop protection. Succinate dehydrogenase inhibitors (SDHIs) and strobilurins inhibit mitochondria electron transport chain (ETC) in fungi, by blocking complex II and complex III, respectively. Questions regarding their selectivity of action for fungi have been raised in the literature, and we previously showed that boscalid and bixafen (SDHIs) alter the mitochondrial function of human hepatocytes. Here, we analyzed the impact of the exposure of human hepatocytes to pyraclostrobin, a fungicide belonging to the class of strobilurins. Using electron paramagnetic resonance (EPR), we observed a decrease in oxygen consumption rate (OCR) and an increase in mitochondrial superoxide levels after 24 h exposure to 0.5 µM concentration. As a consequence, the content in ATP amount in the cells was reduced, the ratio reduced/oxidized glutathione was decreased, and a decrease in cell viability was observed using three different assays (PrestoBlue, crystal violet, and annexin V assays). In addition, as SDHIs and strobilurins are commonly associated in commercial preparations, we evaluated a potential "cocktail" toxic effect. We selected low concentrations of boscalid (0.5 µM) and pyraclostrobin (0.25 µM) that did not induce a mitochondrial dysfunction in liver cells when used separately. In sharp contrast, when both compounds were used in combination at the same concentration, we observed a decrease in OCR, an increase in mitochondrial superoxide production, a decrease in the ratio reduced/oxidized glutathione, and a decrease in cell viability in three different assays.

The first direct measurements of ventilatory flow and oxygen utilization after exhaustive exercise and voluntary feeding in a teleost fish, Oncorhynchus mykiss.

A new "less invasive" device incorporating an ultrasonic flow probe and a divided chamber, but no stitching of membranes to the fish, was employed to make the first direct measurements of ventilatory flow rate (V̇w) and % O2 utilization (%U) in juvenile rainbow trout (37 g, 8ºC) after exhaustive exercise (10-min chasing) and voluntary feeding (2.72% body mass ration). Under resting conditions, the allometrically scaled V̇w (300 ml kg-1 min-1 for a 37-g trout = 147 ml kg-1 min-1 for a 236-g trout exhibiting the same mass-specific O2 consumption rate, MO2) and the convection requirement for O2 (CR = 4.13 L mmol-1) were considerably lower, and the %U (67%) was considerably higher than in previous studies using surgically attached masks or the Fick principle. After exhaustive exercise, V̇w and MO2 approximately doubled whereas frequency (fr) and %U barely changed, so increased ventilatory stroke volume (Vsv) was the most important contributor to increased MO2. CR declined slightly. Values gradually returned to control conditions after 2-3 h. After voluntary feeding, short-term increases in V̇w, Vsv and MO2 were comparable to those after exercise, and fr again did not change. However, %U increased so CR declined even more. The initial peaks in V̇w, Vsv and MO2, similar to those after exercise, were likely influenced by the excitement and exercise component of voluntary feeding. However, in contrast to post-exercise fish, post-prandial fish exhibited second peaks in these same parameters at 1-3 h after feeding, and %U increased further, surpassing 85%, reflecting the true "specific dynamic action" response. We conclude that respiration in trout is much more efficient than previously believed.

Triazophos-induced Respiratory and Behavioral Effects and Development of Adverse Outcome Pathway (AOP) for short-term Exposed Freshwater Snail, Bellamya Bengalensis.

The physiological effects of triazophos were examined using respiratory and behavioral endpoints in Bellamya bengalensis under a 96-hour acute exposure regime. Physiological manifestation of respiratory stress was measured using the rate of oxygen consumption while behavioral toxicity was measured using crawling reflexes, touch response, and mucus production. The threshold effect values for LOEC (Lowest Observed Effect Concentration), NOEC (No Observed Effect Concentration), and MATC (Maximum Acceptable Toxicant Concentration) at 96 h were 0.40, 0.60, and 0.075 mg/l, respectively. Definitive 96 h acute exposures for both respiratory and behavioral endpoints tests were determined using a control group and concentrations ranging from 0.40 to 1.60 mg/l monitored for 24, 48, 72, and 96 h. Test organisms irrespective of exposure concentration demonstrated an initial rise in oxygen consumption rate after 24 h, followed by a progressive decrease in toxicant concentration and exposure period. The in silico structural analysis presents triazophos as having an electrophilic toxic structure similar to choline esterase inhibitors, and also capable of inducing oxidative stress. The AOP highlighted neurotoxicity and oxidative stress as plausible pathways of triazophos toxicity in mollusk species.

Simulation and prediction of the effect of aeration, recirculation and degradation on landfill temperature in aerobic operation.

Much heat is released in aerobic landfills, which leads to temperature change. Quantitative prediction of temperature change with time and space is essential for the safe aerobic operation of landfill. In this article, based on the theory of porous media seepage mechanics and heat transfer, a seepage-temperature coupling model considering aeration, recirculation and degradation was established, which included internal energy change, heat conduction, convection and heat transfer. Moreover, combined with the long-time on-site monitoring temperature data from Wuhan Jinkou Landfill, the model's reliability was preliminarily verified. Sensitivity analysis was carried out for aeration intensity, aeration temperature, recirculation intensity and recirculation temperature. Among the four factors, recirculation intensity influences the peak temperature most with a decrease of 20.11%. Compared with Borglin's and Hao's models, it is found that waste should not be assumed as a cell for temperature prediction. By comparing the results of Non-linear Ascent Stage model, Linear Ascent Stage model and Absent Ascent Stage model, it showed that the temperature difference of the three models decreases with the increase of operation time. In addition, the time point of peak temperature, t0, affects the temperature distribution. The above results provide a reference for predicting the spatial and temporal distribution of temperature and regulations for long-term aerobic landfill operations.

Extracellular flux analyses reveal differences in mitochondrial PBMC metabolism between high-fit and low-fit females.

Analyzing metabolism of peripheral blood mononuclear cells (PBMCs) can possibly serve as a cellular metabolic read-out for lifestyle factors and lifestyle interventions. However, the impact of PBMC composition on PBMC metabolism is not yet clear, neither is the differential impact of a longer-term lifestyle factor versus a short-term lifestyle intervention. We investigated the effect of aerobic fitness level and a recent exercise bout on PBMC metabolism in females. PBMCs from 31 young female adults divided into a high-fit (V̇o2peak ≥ 47 mL/kg/min, n = 15) and low-fit (V̇o2peak ≤ 37 mL/kg/min, n = 16) groups were isolated at baseline and overnight after a single bout of exercise (60 min, 70% V̇o2peak). Oxygen consumption rate (OCR) and glycolytic rate (GR) were measured using extracellular flux (XF) assays and PBMC subsets were characterized using fluorescence-activated cell sorting (FACS). Basal OCR, FCCP-induced OCR, spare respiratory capacity, ATP-linked OCR, and proton leak were significantly higher in high-fit than in low-fit females (all P < 0.01), whereas no significant differences in glycolytic rate (GR) were found (all P > 0.05). A recent exercise bout did not significantly affect GR or OCR parameters (all P > 0.05). The overall PBMC composition was similar between high-fit and low-fit females. Mitochondrial PBMC function was significantly higher in PBMCs from high-fit than from low-fit females, which was unrelated to PBMC composition and not impacted by a recent bout of exercise. Our study reveals a link between PBMC metabolism and levels of aerobic fitness, increasing the relevance of PBMC metabolism as a marker to study the impact of lifestyle factors on human health.NEW & NOTEWORTHY Mitochondrial metabolism was significantly higher in PBMCs from high-fit than from low-fit females. This was unrelated to PBMC composition and not impacted by a recent bout of exercise. Our study reveals a link between PBMC metabolism and levels of aerobic fitness, increasing the relevance of PBMC metabolism as a marker to study the impact of lifestyle factors on human health.

Optical imaging detects metabolic signatures associated with oocyte quality†.

Oocyte developmental potential is intimately linked to metabolism. Existing approaches to measure metabolism in the cumulus oocyte complex (COC) do not provide information on the separate cumulus and oocyte compartments. Development of an assay that achieves this may lead to an accurate diagnostic for oocyte quality. Optical imaging of the autofluorescent cofactors reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD) provides a spatially resolved indicator of metabolism via the optical redox ratio (FAD/[NAD(P)H + FAD]). This may provide an assessment of oocyte quality. Here, we determined whether the optical redox ratio is a robust methodology for measuring metabolism in the cumulus and oocyte compartments compared with oxygen consumption in the whole COC. We also determined whether optical imaging could detect metabolic differences associated with poor oocyte quality (etomoxir-treated). We used confocal microscopy to measure NAD(P)H and FAD, and extracellular flux to measure oxygen consumption. The optical redox ratio accurately reflected metabolism in the oocyte compartment when compared with oxygen consumption (whole COC). Etomoxir-treated COCs showed significantly lower levels of NAD(P)H and FAD compared to control. We further validated this approach using hyperspectral imaging, which is clinically compatible due to its low energy dose. This confirmed lower NAD(P)H and FAD in etomoxir-treated COCs. When comparing hyperspectral imaged vs non-imaged COCs, subsequent preimplantation development and post-transfer viability were comparable. Collectively, these results demonstrate that label-free optical imaging of metabolic cofactors is a safe and sensitive assay for measuring metabolism and has potential to assess oocyte developmental competence.

Breathing versus feeding in the Pacific hagfish.

Hagfish represent the oldest extant connection to the ancestral vertebrates, but their physiology is not well understood. Using behavioural (video), physiological (respirometry, flow measurements), classical morphological (dissection, silicone injection) and modern imaging approaches (micro-MRI, DICE micro-CT), we examined the interface between feeding and the unique breathing mechanism (nostril opening, high-frequency velum contraction, low-frequency gill pouch contraction and pharyngo-cutaneous duct contraction) in the Pacific hagfish, Eptatretus stoutii. A video tour via micro-MRI is presented through the breathing and feeding passages. We have reconciled an earlier disagreement as to the position of the velum chamber, which powers inhalation through the nostril, placing it downstream of the merging point of the food and water passage, such that the oronasal septum terminates at the anterior end of the velum chamber. When feeding occurs by engulfment of large chunks by the dental plates, food movement through the chamber may transiently interfere with breathing. Swallowing is accelerated by peristaltic body undulation involving the ventral musculature, and is complete within 5 s. After a large meal (anchovy, 20% body mass), hagfish remain motionless, defaecating bones and scales at 1.7 days and an intestinal peritrophic membrane at 5 days. O2 consumption rate approximately doubles within 1 h of feeding, remaining elevated for 12-24 h. This is achieved by combinations of elevated O2 utilization and ventilatory flow, the latter caused by varying increases in velar contraction frequency and stroke volume. Additional imaging casts light on the reasons for the trend for greater O2 utilization by more posterior pouches and the pharyngo-cutaneous duct in fasted hagfish.

Pyrroloquinoline quinone (PQQ) improves pulmonary hypertension by regulating mitochondrial and metabolic functions.

Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) and endothelial cells (PAECs), inflammation, as well as mitochondrial and metabolic dysregulation, contributes to the development of pulmonary hypertension (PH). Pyrroloquinoline quinone (PQQ), a potent natural antioxidant with anti-diabetic, neuroprotective, and cardioprotective properties, is known to promote mitochondrial biogenesis. However, its effect on cellular proliferation, apoptosis resistance, mitochondrial and metabolic alterations associated with PH remains unexplored. The current study was designed to investigate the effect of PQQ in the treatment of PH. Human pulmonary artery smooth muscle cells (HPASMCs), endothelial cells (PAECs), and primary cultured cardiomyocytes were subjected to hypoxia to induce PH-like phenotype. Furthermore, Sprague Dawley (SD) rats injected with monocrotaline (MCT) (60 mg/kg, SC, once) progressively developed pulmonary hypertension. PQQ treatment (2 mg/kg, PO, for 35 days) attenuated cellular proliferation and promoted apoptosis via a mitochondrial-dependent pathway. Furthermore, PQQ treatment in HPASMCs prevented mitochondrial and metabolic dysfunctions, improved mitochondrial bioenergetics while preserving respiratory complexes, and reduced insulin resistance. In addition, PQQ treatment (preventive and curative) significantly attenuated the increase in right ventricle pressure and hypertrophy as well as reduced endothelial dysfunction and pulmonary artery remodeling in MCT-treated rats. PQQ also prevented cardiac fibrosis and improved cardiac functions as well as reduced inflammation in MCT-treated rats. Altogether, the above findings demonstrate that PQQ can attenuate mitochondrial as well as metabolic abnormalities in PASMCs and also prevent the development of PH in MCT treated rats; hence PQQ may act as a potential therapeutic agent for the treatment of PH.