Respirometry System Research Articles

Evaluation of methodology for measuring standard metabolic rates of crayfishes using the red swamp crayfish Procambarus clarkii (Girard, 1852) (Decapoda: Astacidea: Cambaridae)

Abstract The importance of energy budgets in understanding the ecology, conservation and production of crayfishes has long been recognized. Standard metabolic rate (SMR) estimates the minimum metabolic rate required for basic maintenance of an organism while at rest, and is a critical parameter for investigating energy balance and metabolism. Estimating SMR involves quantifying oxygen uptake under specific conditions. Standard methodology for estimating SMR has been described and evaluated for fishes, but not thoroughly investigated for crayfishes. We adapted a recommended protocol developed for fishes in order to determine appropriate methodologies for measuring SMR in crayfishes. Study animals consisted of 18 individuals of Procambarus clarkii (Girard, 1852) collected in Alabama, USA. Respiration rates were measured using an optical respirometry system (Loligo Systems®; Viborg, Denmark) and intermittent respirometry techniques. Crayfish respiration stabilized the morning after initiation of the trial indicating that a 12 h overnight period was sufficient to acclimate crayfish to respirometry chambers. After 12 h of daylight, respiration of acclimated crayfish typically exhibited a short spike when lights were turned off, indicating data collected within ~2 h following a light change should be excluded from the dataset used to calculate SMR. When calculating SMR, a quantile approach was typically more appropriate than the mean of the lowest normal distribution approach. SMR calculated during the day was only marginally higher than SMR calculated during the night, indicating that SMR can be estimated during either period if shelters are provided in the respiration chambers. Due to the wide diversity of crayfish species and ranges, our recommendations may not be appropriate for every crayfish species or subpopulation. The recommendations can serve, however, as a valuable starting point and the described methodology provides a standardized approach for determining appropriate protocols to measure SMR of crayfish species of interest.

Effect of temperature on growth, metabolism, and gas exchange in Hermetia illucens larvae reared under commercial and laboratory conditions

Abstract The world’s socio-economic development is continuously increasing the demand for efficient production of food, feed, and energy from the agricultural sector. In this respect, the emerging production of black soldier fly larvae (BSFL) represents a promising system to upscale low-quality resources to higher-quality resources usable as feed or biodiesel. To optimize BSFL production it is critical to establish methods that are relevant to examine how rearing conditions, including temperature, affect growth, metabolism, and body composition at large scale. In a first set of experiments, we therefore compared how small-scale laboratory respirometry chambers (600 larvae) support gas exchange, growth, and metabolism in BSFL with similar measurements obtained from large-scale commercial conditions (>12 million larvae) at ∼34-35 °C. Having observed that small- and large-scale systems generated comparable measurements of growth and metabolism, we used our small-scale respirometry system to examine in detail how substrate rearing temperature ranging from 27.0 to 41.9 °C influenced growth, metabolism, and body composition in BSFL. Here we found that 7-day growth of BSFL was stable and high at 27, 35, and 39 °C, while a rearing temperature of ∼42 °C caused a severe, but sublethal, depression of growth and metabolism. Despite the general similarities in total growth at 27, 35, and 39 °C, we found considerable acceleration of metabolism and development at the two higher rearing temperatures (35 and 39 °C). In contrast, the lower rearing temperature (27 °C) resulted in reduced CO2 production per kg larvae produced and the was associated with a higher crude protein to crude lipid ratio in larvae after the 7-day growth period. Based on these findings we discuss how continued monitoring of gas exchange in production systems holds a potential to optimize production in the emerging industry of large-scale commercial insect production.

Metabolic Rate of Goldfish (Carassius auratus) in the Face of Common Aquaculture Challenges.

This study examined the metabolic rate (MO2, oxygen consumption) of goldfish (Carassius auratus) under normal management conditions in aquaculture. Using an intermittent respirometry system, we assessed daily variations and the effects of feeding, handling, temperature increase, and anesthetics. MO2 exhibited a daily rhythm, with higher values during day. Feeding to satiety produced a 35% increase in MO2 compared to fasted animals, with a maximum peak after 3 h and returning to baseline after 7 h. Handling stress (5 min) produced a 140% MO2 peak (from 180 to 252 mg O2 kg-1 h-1), returning to the routine MO2 after 2.5 h. An increase in water temperature (+0.1 °C min-1) up to 30 °C caused MO2 to peak at 200% after 2.5 h from the start of the temperature increase. The use of common anesthetics in aquaculture (MS-222, 2-phenoxyethanol and clove oil in deep anesthesia concentration) affects MO2 during the first few minutes after anesthetic recovery, but also during the following 4 h. It can be concluded that the metabolic rate is a good indicator of the goldfish's response to aquaculture practices involving energy expenditure and stress. Thus, intermittent respirometry is a valuable non-invasive tool for understanding and improving fish welfare in aquaculture.

Changes of oxygen consumption rates in response to various environmental factors and different anesthetic methods in juvenile hybrid sturgeon, Acipenser baeri♀×Acipenser schrencki♂

Live fish transportation plays a crucial role in realizing the value of aquaculture in sturgeons. However, hypoxia and hypercapnia are the major issues during the high-density transportation of live fish. Therefore, monitoring the changing environmental factors (such as water temperature, salinity, and pH) or anesthesia methods (MS-222, dissolved CO2, or low-voltage direct electrical current), which can potentially alter the oxygen consumption of fish, are crucial for a better understanding of the necessary conditions for live fish transportation. A semi-open respirometry system was used to quantify the oxygen consumption and respiration frequency of hybrid sturgeon (Acipenser baeri♀×Acipenser schrencki♂). The results indicate that the oxygen consumption rate and respiration frequency of hybrid sturgeon increased linearly with water temperature from 1.4 °C to 25°C. The oxygen consumption rate of hybrid sturgeon increased with the increase of salinity in the range from 3 to 10 ‰ in 9 hours (P < 0.05). Respiration frequency decreased but did not affect the oxygen consumption rate when pH was from 8.1 to 9.8. Exposure to low-voltage direct electrical current ranging from 0.4 to 1.0 V/cm significantly increased the respiration frequency (P < 0.05). The oxygen consumption rate maintained below 1 mg/(g·h) when CO2 concentration ranges from 8.5 to 67.85 mg/L. Compared with the change of oxygen consumption rate and respiration frequency with MS-222 at the range from 5 to 30 mg/L, immersion of fish into more than 8.5–67.85 mg/L dissolved CO2 in water could as an alternative anesthetic method for hybrid sturgeon. These findings in the study may contribute to the culture, live fish transportation, and basic research in hybrid sturgeon.

A practical and robust method to evaluate metabolic fluxes in primary pancreatic islets

ObjectiveEvaluation of mitochondrial oxygen consumption and ATP production is important to investigate pancreatic islet pathophysiology. Most studies use cell lines due to difficulties in measuring primary islet respiration, which requires specific equipment and consumables, is expensive and poorly reproducible. Our aim was to establish a practical method to assess primary islet metabolic fluxes using standard commercial consumables. MethodsPancreatic islets were isolated from mice/rats, dispersed with trypsin, and adhered to pre-coated standard Seahorse or Resipher microplates. Oxygen consumption was evaluated using a Seahorse Extracellular Flux Analyzer or a Resipher Real-time Cell Analyzer. ResultsWe provide a detailed protocol with all steps to optimize islet isolation with high yield and functionality. Our method requires a few islets per replicate; both rat and mouse islets present robust basal respiration and proper response to mitochondrial modulators and glucose. The technique was validated by other functional assays, which show these cells present conserved calcium influx and insulin secretion in response to glucose. We also show that our dispersed islets maintain robust basal respiration levels, in addition to maintaining up to 89% viability after five days in dispersed cultures. Furthermore, OCRs can be measured in Seahorse analyzers and in other plate respirometry systems, using standard materials. ConclusionsOverall, we established a practical and robust method to assess islet metabolic fluxes and oxidative phosphorylation, a valuable tool to uncover basic β-cell metabolic mechanisms as well as for translational investigations, such as pharmacological candidate discovery and islet transplantation protocols.

Effects of temperature on metabolic rate during metamorphosis in the alfalfa leafcutting bee.

Spring conditions, especially in temperate regions, may fluctuate abruptly and drastically. Environmental variability can expose organisms to temperatures outside of their optimal thermal ranges. For ectotherms, sudden changes in temperature may cause short- and long-term physiological effects, including changes in respiration, morphology, and reproduction. Exposure to variable temperatures during active development, which is likely to occur for insects developing in spring, can cause detrimental effects. Using the alfalfa leafcutting bee, Megachile rotundata, we aimed to determine if oxygen consumption could be measured using a new system and to test the hypothesis that female and male M. rotundata have a thermal performance curve with a wide optimal range. Oxygen consumption of M. rotundata pupae was measured across a large range of temperatures (6-48°C) using an optical oxygen sensor in a closed respirometry system. Absolute and mass-specific metabolic rates were calculated and compared between bees that were extracted from their brood cells and those remaining in the brood cell to determine whether pupae could be accurately measured inside their brood cells. The metabolic response to temperature was non-linear, which is an assumption of a thermal performance curve; however, the predicted negative slope at higher temperatures was not observed. Despite sexual dimorphism in body mass, sex differences only occurred in mass-specific metabolic rates. Higher metabolic rates in males may be attributed to faster development times, which could explain why there were no differences in absolute metabolic rate measurements. Understanding the physiological and ecological effects of thermal environmental variability on M. rotundata will help to better predict their response to climate change.

Combining the oxygen sensor based respirometry and 16S rRNA amplicon sequencing for the analysis of microbiota in commercial mince products

In this study, rapid respirometric microbial testing was combined with 16S rRNA amplicon sequencing, to assess the composition of microbiota in a total of 64 samples of commercial beef, turkey, lamb and pork mince. The O2 sensor-based respirometry system, while producing the anticipated total aerobic viable counts (TVC) data and patterns for most samples, also revealed unusual (linear) respiration profiles for some samples, mostly lamb and pork mince. The TVC values for beef mince, produced by respirometry and calculated using the available calibration equation, correlated well with the conventional plate counting method, ISO 4833-1:2013, 2013, while for the other species the correlation was less good. These effects, not observed in previous studies employing various food matrices, require further investigation. Using the same samples (crude homogenates) as in respirometry, the whole microbiome was also analysed by 16S rRNA amplicon sequencing for each mince-type. The sequencing showed an overall decrease in alpha diversity over shelf-life, with lamb and pork mince maintaining a proportion of rare taxa. Some taxa exhibited significant changes in abundance over shelf-life and after the respirometric analysis, with beef mince exhibiting a decrease in aerobic bacteria and an increase in facultative anaerobes. Beta diversity was also seen to depend on mince-type. Thus, the combined use of respirometry and sequencing techniques shows promise as a useful and unique analytical approach for food quality and safety evaluation, However, more data points and in-depth analysis are required to back up the findings of this initial study.

Anthelmintic nitazoxanide protects against experimental pulmonary fibrosis.

Nitazoxanide is a therapeutic anthelmintic drug. Our previous studies found that nitazoxanide and its metabolite tizoxanide activated adenosine 5'-monophosphate-activated protein kinase (AMPK) and inhibited signal transducer and activator of transcription 3 (STAT3) signals. As AMPK activation and/or STAT3 inhibition are targets for treating pulmonary fibrosis, we hypothesized that nitazoxanide would be effective in experimental pulmonary fibrosis. The mitochondrial oxygen consumption rate of cells was measured by using the high-resolution respirometry system Oxygraph-2K. The mitochondrial membrane potential of cells was evaluated by tetramethyl rhodamine methyl ester (TMRM) staining. The target protein levels were measured by using western blotting. The mice pulmonary fibrosis model was established through intratracheal instillation of bleomycin. The examination of the lung tissues changes were carried out using haematoxylin and eosin (H&E), and Masson staining. Nitazoxanide and tizoxanide activated AMPK and inhibited STAT3 signalling in human lung fibroblast cells (MRC-5 cells). Nitazoxanide and tizoxanide inhibited transforming growth factor-β1 (TGF-β1)-induced proliferation and migration of MRC-5 cells, collagen-I and α-smooth muscle cell actin (α-SMA) expression, and collagen-I secretion from MRC-5 cells. Nitazoxanide and tizoxanide inhibited epithelial-mesenchymal transition (EMT) and inhibited TGF-β1-induced Smad2/3 activation in mouse lung epithelial cells (MLE-12 cells). Oral administration of nitazoxanide reduced the bleomycin-induced mice pulmonary fibrosis and, in the established bleomycin-induced mice, pulmonary fibrosis. Delayed nitazoxanide treatment attenuated the fibrosis progression. Nitazoxanide improves the bleomycin-induced pulmonary fibrosis in mice, suggesting a potential application of nitazoxanide for pulmonary fibrosis treatment in the clinic.

Biodegradable Plastics: Biodegradation Percentage and Potential Microplastic Contamination in Seawater

Increasing plastic production, which causes the problem of plastic garbage polluting the oceans, has increased the use of biodegradable plastics to address the problem. However, it is still debatable how much microplastic contamination it can cause. So, this study aims to determine the proportion of biodegradable plastics in the marine environment, identify the microplastics it produces, and analyze the relationship between the two. Seawater sampling is located in the Bangka Strait. The research was conducted in the Genetics and Biotechnology Laboratory, Department of Biology, Sriwijaya University. The biodegradable plastic test material used was made from a mixture of polyhydroxyalkanoates (PHA) and starch. Biodegradation test method using standard ASTM D6691-17 with respirometry system design. The stage of microplastic identification is carried out through filtration with a 4.75 mm-size filter; density separation using ZnCl2 solution and Whatman No. 1 filter paper; as well as visual observation of microplastics under a microscope. last Pearson Correlation analysis with bootstrap to see the relationship of the percentage of biodegradation with microplastics. The results obtained in this study were the percentage of biodegradable plastic (26.5±1.4%) and positive control kraft paper (33.2±4.2%) for 70 days, which produced 9 microplastic particles from biodegradable plastic with fragment and film types. Correlation analysis concluded that there was no relationship between the percentage of biodegradation and the microplastics produced.

HAMLET effect on cell death and mitochondrial respiration in colorectal cancer cell lines with KRAS/BRAF mutations

PurposeTreatment of advanced colorectal cancer (CRC) depends on the correct selection of personalized strategies. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a natural proteolipid milk compound that might serve as a novel cancer prevention and therapy candidate. Our purpose was to investigate HAMLET effect on viability, death pathway and mitochondrial bioenergetics of CRC cells with different KRAS/BRAF mutational status in vitro.MethodsWe treated three cell lines (Caco-2, LoVo, WiDr) with HAMLET to evaluate cell metabolic activity and viability, flow cytometry of apoptotic and necrotic cells, pro- and anti-apoptotic genes, and protein expressions. Mitochondrial respiration (oxygen consumption) rate was recorded by high-resolution respirometry system Oxygraph-2 k.ResultsThe HAMLET complex was cytotoxic to all investigated CRC cell lines and this effect is irreversible. Flow cytometry revealed that HAMLET induces necrotic cell death with a slight increase in an apoptotic cell population. WiDr cell metabolism, clonogenicity, necrosis/apoptosis level, and mitochondrial respiration were affected significantly less than other cells.ConclusionHAMLET exhibits irreversible cytotoxicity on human CRC cells in a dose-dependent manner, leading to necrotic cell death and inhibiting the extrinsic apoptosis pathway. BRAF-mutant cell line is more resistant than other type lines. HAMLET decreased mitochondrial respiration and ATP synthesis in CaCo-2 and LoVo cell lines but did not affect WiDr cells’ respiration. Pretreatment of cancer cells with HAMLET has no impact on mitochondrial outer and inner membrane permeability.

SDA coefficient is temperature dependent in rainbow trout (Oncorhynchus mykiss, Walbaum 1792) in a practical approach using group respirometry.

Rising global temperatures have raised the need for detailed knowledge of the effects of rising temperatures on the physiology of animals used in aquaculture. Here we used a multifactorial bioenergetic approach using groups of rainbow trout (Oncorhynchus mykiss) with an average single fish weight of 183.75g±0.65g to investigate the interactions of feeding and temperature with key metabolic variables. We used a recirculating aquaculture respirometry system (RARS) to test three ration sizes (0.65; 0.975; 1.3% of live body weight (BW)) over a range of three consecutive temperatures (14; 17; 20°C). The fish were fed once per day for 6days at each temperature and subsequently starved for 5days to return to standard metabolic rate (SMR). This study aimed to answer the highly discussed topic of the temperature dependency of key metabolic specific dynamic action (SDA)-variables SDAcoef and SDAdur. We were able to provide evidence, that in rainbow trout the SDAcoef is highly dependent on the environmental temperature in the first ever approach to assess these variables in a group respirometer with this species. We compared the results of this study with a sophisticated bioenergetic model by Elliot and Hurley (2002) and thereby provide evidence for the practicability of group respirometry as a method to assess bioenergetic data under culture like conditions.

Development of a Mobile Open-Circuit Respiration Head Hood System for Measuring Gas Exchange in Camelids in the Andean Plateau.

Peru has the largest inventory of alpacas worldwide. Despite their importance as a source of net income for rural communities living at the Andean Plateau, data on energy requirements and methane (CH4) emissions for alpacas are particularly lacking. In 2019, the International Panel on Climate Change (IPCC; 2006, and Refinement 2019) outlined methods for estimating CH4 emissions from enteric fermentation and no methane (CH4) conversion factors were reported for camelids. IPCC has since updated its guidelines for estimating CH4 emissions from the enteric fermentation of livestock at a national scale. For greenhouse gas (GHG) inventory purposes, conversion factors were developed for ruminants but not for domestic South American camelids (SAC), with this category including alpacas. A mobile open-circuit respirometry system (head hood) for the rapid determination of CH4 and CO2 production, O2 consumption, and thereafter, heat production (HP) for camelids was built and validated. In addition, an experimental test with eight alpacas was conducted for validation purposes. The average HP measured by indirect calorimetry (respiratory quotient (RQ) method) was close to the average HP determined from the carbon-nitrogen balance (CN method); 402 kJ/kg BW0.75 and 398 kJ/kg BW0.75, respectively. Fasting HP was determined by the RQ method and 250 kJ/kg BW0.75 was obtained. The metabolizable energy requirement for maintenance (MEm) was calculated to be 323 kJ/kg BW0.75 with an efficiency of energy utilization of 77%. When intake was adjusted to zero energy retention by linear regression, the MEm requirement increased to 369 kJ/kg BW0.75 and the efficiency decreased up to 68%. The CH4 conversion factor (Ym) was 5.5% on average. Further research is required to gain a better understanding of the energy requirements and CH4 emissions of alpacas in conditions of the Andean Plateau and to quantify them with greater accuracy.

Piezo1 channel activation stimulates ATP production through enhancing mitochondrial respiration and glycolysis in vascular endothelial cells.

Piezo1 channels are mechanosensitive cationic channels that are activated by mechanical stretch or shear stress. Endothelial Piezo1 activation by shear stress caused by blood flow induces ATP release from endothelial cells; however, the link between shear stress and endothelial ATP production is unclear. The mitochondrial respiratory function of cells was measured by using high-resolution respirometry system Oxygraph-2k. The intracellular Ca2+ concentration was evaluated by using Fluo-4/AM and mitochondrial Ca2+ concentration by Rhod-2/AM. The specific Piezo1 channel activator Yoda1 or its analogue Dooku1 increased [Ca2+ ]i in human umbilical vein endothelial cells (HUVECs), and both Yoda1 and Dooku1 increased mitochondrial oxygen consumption rates (OCRs) and mitochondrial ATP production in HUVECs and primary cultured rat aortic endothelial cells (RAECs). Knockdown of Piezo1 inhibited Yoda1- and Dooku1-induced increases of mitochondrial OCRs and mitochondrial ATP production in HUVECs. The shear stress mimetics, Yoda1 and Dooku1, and the Piezo1 knock-down technique also demonstrated that Piezo1 activation increased glycolysis in HUVECs. Chelating extracellular Ca2+ with EGTA or chelating cytosolic Ca2+ with BAPTA-AM did not affect Yoda1- and Dooku1-induced increases of mitochondrial OCRs and ATP production, but chelating cytosolic Ca2+ inhibited Yoda1- and Dooku1-induced increase of glycolysis. Confocal microscopy showed that Piezo1 channels are present in mitochondria of endothelial cells, and Yoda1 and Dooku1 increased mitochondrial Ca2+ in endothelial cells. Piezo1 channel activation stimulates ATP production through enhancing mitochondrial respiration and glycolysis in vascular endothelial cells, suggesting a novel role of Piezo1 channel in endothelial ATP production.

Use of Solar Panels for Shade for Holstein Heifers.

Animal Agrivoltaics combines electric energy generation, animal thermal comfort, and sustainable production at the same time. This model of production can foster the sustainable intensification of dairy production in tropical areas where solar irradiance is high and nearly constant throughout the year. In this study, we propose Animal Agrivoltaics as an alternative practice to reduce the heat load and eCH4 emissions from dairy heifers in tropical areas. To attest this hypothesis, (1) the meteorological data and the behavioral and physiological responses of the animals were integrated in order to determine the benefits provided by the shade from the solar panels on the thermoregulation of the dairy heifers, and (2) measurements of the enteric methane emissions were taken to determine the potential of the solar panels to offset the GHG. Seven crossbred Holstein heifers (7/8, Holstein × Gyr) with a mean body weight of 242 kg (SD = 53.5) were evaluated in a paddock shaded with ten modules of solar panels. Miniature temperature loggers were used to record the body surface, skin and vaginal temperatures of the heifers every five minutes. The respiratory rate and the shade-use behavior were also monitored by two observers. These measurements were taken from 08:00 to 17:00 h for 18 consecutive days. After completing the field study, the heifers underwent for assessments of the daily oscillations of eCH4 emission using a flow-through respirometry system. The use of shade by the heifers was progressively increased (p < 0.01) with an increasing level of solar irradiance. Lying and ruminating were more likely (p < 0.01) to occur when the heifers were in the shade, especially when the solar irradiance exceeded 500 W m-2. Between 10:00 and 14:00 h, the heifers benefited from the shade produced by the solar panels, with a reduction of 40% in the radiant heat load. With an increasing intensity of solar irradiance, body surface temperature, skin temperature and respiratory rate of the heifers in the shade were lower (p < 0.01) compared to when they were exposed to the sun. The heifers had a daily methane emission total of 63.5 g per animal-1 or 1.7 kg of CO2-eq. Based on this emission rate and the amount of CO2-eq that was not emitted to the atmosphere due to the electricity generated by solar panels, 4.1 m2 of panels per animal (nominal power = 335 W) would be expected to obtain a net-zero eCH4 emission. Over a period of one year (from September 2018 to August 2019), a set of ten photovoltaic panels used in the study produced 4869.4 kWh of electricity, thereby saving US $970.00 or US $48.00 per m2 of solar panel. Based on the results of this study, it can be concluded that use of Animal Agrivoltaics, in addition to producing electricity, has significant potential benefit in providing better thermal comfort to cattle, as well as offsetting the enteric methane emissions released into the environment. In addition, the system would provide extra income to farmers, as well as a potential source of energy micro-generation.

Energy budgets of captive Chinese pangolins (Manis pentadactyla).

The Chinese pangolin is an endangered species, and ex situ conservation and captive rescue are important conservation measures. This requires reliable information on nutritional energy requirements and expenditure characteristics. However, we lack sufficient knowledge of their energy physiology to determine their energy requirements for maintenance and growth. An open-flow respirometry system was used to measure the resting metabolic rate (RMR) and the daily energy expenditure (DEE) of Chinese pangolins (Manis pentadactyla), and the dietary digestive energy was measured. The average RMR in Chinese pangolins was 3.23mlO2kg-1min-1 at an ambient temperature (Ta) of 24.5-30°C, which was only 73.0% of the expected value based on body mass (BM). The average DEE values were 744.9kJday-1 in animals with BM >3kg and 597.3kJday-1 in those with BM <3kg, which were only 52.4% and 60.6% of the predicted values, respectively. The RMR and DEE levels of the Chinese pangolin were lower than those of similar-sized eutherian mammals and close to those of anteaters. These characteristics suggest that the Chinese pangolin has a low demand for energy in its diet. Although metabolic level data alone cannot be used to calculate the energy requirements of each Chinese pangolin, we believe they can provide a tangible reference for the relocation of Chinese pangolins. These results provide a scientific basis for future research on the physiology and ecology of endangered wildlife such as the Chinese pangolin.

Axolotl Metabolism: Measuring Metabolic Rate.

Deciphering how metabolic processes contribute to control of stem cell proliferation and differentiation is essential for understanding the mechanisms of regeneration. However, much is still unknown about axolotls' metabolism, which has not been studied in detail over their lifespan or under varied experimental conditions. We summarize the theoretical underpinnings of metabolism and respirometry, and describe a closed respirometry system to investigate metabolic energetics in axolotls as a specific aspect of metabolism. Placement of post-absorptive, fairly inactive animals in the multiple-probe respirometer for 24-48h allows us to measure changes in concentrations of respiratory gases: oxygen (atmospheric and dissolved) and carbon dioxide, while monitoring the temperature and salinity (conductivity) of the chamber's water. Respirometry data are used to calculate oxygen intake and carbon dioxide output to estimate animal's metabolic energy dynamics during the observation periods. This method creates opportunities for study of potential fluctuations in axolotls' metabolic rate as it pertains to respiratory gases' dynamics during 24-h circadian cycle, as well as examination of changes in metabolic energy management during aging, under varied environmental temperatures, during post-amputation regeneration and many other circumstances.

Repurposing nitazoxanide as a novel anti-atherosclerotic drug based on mitochondrial uncoupling mechanisms.

The anthelmintic drug nitazoxanide has a mitochondrial uncoupling effect. Mitochondrial uncouplers have been proven to inhibit smooth muscle cell proliferation and migration, inhibit NLRP3 inflammasome activation of macrophages and improve dyslipidaemia. Therefore, we aimed to demonstrate that nitazoxanide would protect against atherosclerosis. The mitochondrial oxygen consumption of cells was measured by using the high-resolution respirometry system, Oxygraph-2K. The proliferation and migration of A10 cells were measured by using Edu immunofluorescence staining, wound-induced migration and the Boyden chamber assay. Protein levels were measured by using the western blot technique. ApoE (-/-) mice were fed with a Western diet to establish an atherosclerotic model in vivo. The in vitro experiments showed that nitazoxanide and tizoxanide had a mitochondrial uncoupling effect and activated cellular AMPK. Nitazoxanide and tizoxanide inhibited serum- and PDGF-induced proliferation and migration of A10 cells. Nitazoxanide and tizoxanide inhibited NLRP3 inflammasome activation in RAW264.7 macrophages, the mechanism by which involved the AMPK/IκBα/NF-κB pathway. Nitazoxanide and tizoxanide also induced autophagy in A10 cells and RAW264.7 macrophages. The in vivo experiments demonstrated that oral administration of nitazoxanide reduced the increase in serum IL-1β and IL-6 levels and suppressed atherosclerosis in Western diet-fed ApoE (-/-) mice. Nitazoxanide inhibits the formation of atherosclerotic plaques in ApoE (-/-) mice fed on a Western diet. In view of nitazoxanide being an antiprotozoal drug already approved by the FDA, we propose it as a novel anti-atherosclerotic drug with clinical translational potential.

Individual variation in heat substitution: is activity in the cold energetically cheaper for some individuals than others?

In many endotherms, a potentially important yet often overlooked mechanism to save energy is the use of the heat generated by active skeletal muscles to replace heat that would have been generated by thermogenesis (i.e. 'activity-thermoregulatory heat substitution'). While substitution has been documented numerous times, the extent of individual variation in substitution has never been quantified. Here, we used a home-cage respirometry system to repeatedly measure substitution through the concomitant monitoring of metabolic rate (MR) and locomotor activity in 46 female white-footed mice (Peromyscus leucopus). A total of 117 measures of substitution were taken by quantifying the difference in the slope of the relationship between MR and locomotor activity speed at two different ambient temperatures. Consistency repeatability (±s.e.) of substitution was 0.313 (±0.131); hence, about a third of the variation in substitution occurs at the among-individual level. Body length and heart mass were positively correlated with substitution whereas surface area was negatively correlated with substitution. These three sub-organismal traits accounted for the majority of the among-individual variation (i.e. individual differences in substitution were not significant after accounting for these traits). Overall, our results imply that the energetic cost of activity below the thermoneutral zone is consistently cheaper from some individuals than others, and that the energy saved from substitution might be available to invest in fitness-enhancing activities.

The Pharmacological Effects of Silver Nanoparticles Functionalized with Eptifibatide on Platelets and Endothelial Cells.

PurposeIn the search for new drug delivery platforms for cardiovascular diseases and coating of medical devices, we synthesized eptifibatide-functionalized silver nanoparticles (AgNPs-EPI) and examined the pharmacological activity of AgNPs-EPI on platelets and endothelial cells in vitro and ex vivo.MethodsSpherical AgNPs linked to eptifibatide were synthesized and characterized. Cytotoxicity was measured in microvascular endothelial cells (HMEC-1), platelets and red blood cells. Platelet mitochondrial respiration was measured using the Oxygraph-2k, a high-resolution modular respirometry system. The effect of AgNPs-EPI on the aggregation of washed platelets was measured by light aggregometry and the ex vivo occlusion time was determined using a reference laboratory method. The surface amount of platelet receptors such as P-selectin and GPIIb/IIIa was measured. The influence of AgNPS-EPI on blood coagulation science was assessed. Finally, the effect of AgNPs-EPI on endothelial cells was measured by the levels of 6-keto-PGF1alpha, tPa, cGMP and vWF.ResultsWe describe the synthesis of AgNPs using eptifibatide as the stabilizing ligand. The molecules of this drug are directly bonded to the surface of the nanoparticles. The synthesized AgNPs-EPI did not affect the viability of platelets, endothelial cells and erythrocytes. Preincubation of platelets with AgNPs-EPI protected by mitochondrial oxidative phosphorylation capacity. AgNPs-EPI inhibited aggregation-induced P-selectin expression and GPIIb/IIIa conformational changes in platelets. AgNPs-EPI caused prolongation of the occlusion time in the presence of collagen/ADP and collagen/adrenaline. AgNPs-EPI regulated levels of 6-keto-PGF1alpha, tPa, vWf and cGMP produced in thrombin stimulated HMEC-1 cells.ConclusionAgNPs-EPI show anti-aggregatory activity at concentrations lower than those required by the free drug acting via regulation of platelet aggregation, blood coagulation, and endothelial cell activity. Our results provide proof-of-principle evidence that AgNPs may be used as an effective delivery platform for antiplatelet drugs.

How the green crab Carcinus maenas copes physiologically with a range of salinities.

To evaluate the physiological ability to adjust to environmental variations of salinity, Carcinus maenas were maintained in 10, 20, 32 (control), 40, and 50ppt (13.8 ± 0.6°C) for 7days. Closed respirometry systems were used to evaluate oxygen consumption ([Formula: see text]), ammonia excretion (Jamm), urea-N excretion (Jurea-N) and diffusive water fluxes (with 3H2O). Ions, osmolality, metabolites, and acid-base status were determined in the hemolymph and seawater, and transepithelial potential (TEP) was measured. At the lowest salinity, there were marked increases in [Formula: see text] and Jamm, greater reliance on N-containing fuels to support aerobic metabolism, and a state of internal metabolic alkalosis (increased [HCO3-]) despite lower seawater pH. At higher salinities, an activation of anaerobic metabolism and a state of metabolic acidosis (decreased [HCO3-] and increased [lactate]), in combination with respiratory compensation (decreased PCO2), were detected. TEP became more negative with decreasing salinity. Osmoregulation and osmoconformation occurred at low and high salinities, respectively, with complex patterns in individual ions; hemolymph [Mg2+] was particularly well regulated at levels well below the external seawater at all salinities. Diffusive water flux rates increased at higher salinities. Our results show that C. maenas exhibits wide plasticity of physiological responses when acclimated to different salinities and tolerates substantial disturbances of physiological parameters, illustrating that this species is well adapted to invade and survive in diverse habitats.