Measurements of glycolysis and mitochondrial function are required to quantify energy metabolism in a wide variety of cellular contexts. In human pluripotent stem cells (hPSCs) and their ...differentiated progeny, this analysis can be challenging because of the unique cell properties, growth conditions and expense required to maintain these cell types. Here we provide protocols for analyzing energy metabolism in hPSCs and their early differentiated progenies that are generally applicable to mature cell types as well. Our approach has revealed distinct energy metabolism profiles used by hPSCs, differentiated cells, a variety of cancer cells and Rho-null cells. The protocols measure or estimate glycolysis on the basis of the extracellular acidification rate, and they measure or estimate oxidative phosphorylation on the basis of the oxygen consumption rate. Assays typically require 3 h after overnight sample preparation. Companion methods are also discussed and provided to aid researchers in developing more sophisticated experimental regimens for extended analyses of cellular bioenergetics.
An important issue concerning tracking detectors is that they undergo deformations, which, if not negligible, may worsen the detector intrinsic resolution. The objective of this paper is to assess ...the accuracy of Fibre Bragg Grating (FBG) technology to monitor the deformations of a silicon microstrip detector under stationary thermal conditions. For this purpose, a detector module was employed, of the same type as those composing the vertex detector of the FINUDA apparatus at DAΦNE. Two bare FBG sensors were glued close to the border of the silicon microstrip detectors and their responses were compared with the responses of two CCD laser displacement sensors over a time period of 24 days. A calibration method to determine the quantitative relationship between the Bragg wavelengths of the gratings and the lateral deflection of the microstrip module was developed. This method does not require either knowledge of the exact position or calibration of the individual FBG sensor. The lateral deflection of the microstrip detector could be reconstructed with an accuracy of approximately 10μm. The results obtained suggest that a better accuracy could be achieved by employing a larger number of FBG sensors.
•There is a clear relationship between string stability and hysteresis.•String unstable platoons are hysteretic both for simulations and observed trajectories.•A new mechanism that causes hysteresis ...is investigated.•A new criterion for hysteresis partly explaining the connection to string stability.•The most string stable platoons produced poor flows during the acceleration phase.
The existence, source, and characteristics of hysteresis loops in traffic oscillations have been extensively investigated in the past decades. At the same time, the string stability of vehicle platoons has also attracted significant attention and has been considered the main cause of the spontaneously emerging oscillations. However, the relationship between these two seemingly related properties remains underexplored, leading to deficits in knowledge about the effect of microscopic controllers’ operations on macroscopic traffic characteristics. Filling this gap becomes important with the advent of driving automation and the possibility to make future road transport not only safer but also more efficient. This would however require setting proper requirements to their operations, able to ensure that they would generate an efficient traffic flow without limiting too much the freedom of the system developers.
By analyzing both simulation and real-world data, the present work has confirmed the existence of a strong correlation between hysteresis and string instability. In particular, string unstable platoons are found to be also hysteretic, while only a limited number of string stable platoons were hysteretic. Further, for a simple linear car following model, hysteresis loops are shown to emerge in the absence of inter-driver and/or intra-driver heterogeneity, which have been suggested as the main sources of hysteresis. This implies a new mechanism that can lead to hysteresis, emerging as a characteristic of the driving behavior. An analytical investigation of this mechanism shows a link to string instability, at least for the simple linear model used. Although further research will be needed in order to generalize the findings of this paper, the present results strongly suggest that introducing a requirement for string stability may lead to a significant reduction in the occurrence and the negative effects of traffic oscillations.
Two adolescent males presented within 3 days after the first and second dose of the BNT162b2 vaccine with chest pain. Elevated troponin levels, ST segment elevation, and enhancement of the myocardium ...in cardiac MRI suggested myocarditis. Left ventricular function remained normal, symptoms resolved, and patients were discharged in 4 days. BNT162b2 vaccine may be associated with self-limited myocarditis in youth.
Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate ...carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. We sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells.
Multiple signaling pathways in the adult
enterocyte sense cellular damage or stress and signal to intestinal stem cells (ISCs) to undergo proliferation and differentiation, thereby maintaining ...intestinal homeostasis. Here we show that misregulation of mitochondrial pyruvate metabolism in enterocytes can stimulate ISC proliferation and differentiation. Our studies focus on the Mitochondrial Pyruvate Carrier (MPC), which is an evolutionarily-conserved protein complex that resides in the inner mitochondrial membrane and transports cytoplasmic pyruvate into the mitochondrial matrix. Loss of
function in enterocytes induces Unpaired cytokine expression, which activates the JAK/STAT pathway in ISCs, promoting their proliferation. Upd3 and JNK signaling are required in enterocytes for ISC proliferation, indicating that this reflects a canonical non-cell autonomous damage response. Disruption of lactate dehydrogenase in enterocytes has no effect on ISC proliferation but it suppresses the proliferative response to a loss of enterocyte MPC function, suggesting that lactate contributes to this pathway. These studies define an important role for cellular pyruvate metabolism in differentiated enterocytes to maintain stem cell proliferation rates.
Although metabolic adaptations have been demonstrated to be essential for tumor cell proliferation, the metabolic underpinnings of tumor initiation are poorly understood. We found that the earliest ...stages of colorectal cancer (CRC) initiation are marked by a glycolytic metabolic signature, including downregulation of the mitochondrial pyruvate carrier (MPC), which couples glycolysis and glucose oxidation through mitochondrial pyruvate import. Genetic studies in Drosophila suggest that this downregulation is required because hyperplasia caused by loss of the Apc or Notch tumor suppressors in intestinal stem cells can be completely blocked by MPC overexpression. Moreover, in two distinct CRC mouse models, loss of Mpc1 prior to a tumorigenic stimulus doubled the frequency of adenoma formation and produced higher grade tumors. MPC loss was associated with a glycolytic metabolic phenotype and increased expression of stem cell markers. These data suggest that changes in cellular pyruvate metabolism are necessary and sufficient to promote cancer initiation.
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•Intestinal tumors exhibit low MPC expression•MPC inactivation is sufficient to promote intestinal tumor formation in mice and flies•MPC overexpression in the fly is sufficient to prevent oncogene-induced tumorigenesis•MPC expression correlates negatively with expression of Wnt/β-catenin target genes
A hallmark of cancer is altered metabolism in tumor cells; however, it is unclear whether cancer initiation also requires metabolic changes. Bensard et al. demonstrate that constitutive enforcement of a glycolytic program through inactivation of the mitochondrial pyruvate carrier (MPC) is necessary and sufficient to drive intestinal tumor formation.
Background
The ADCK proteins are predicted mitochondrial kinases. Most studies of these proteins have focused on the Abc1/Coq8 subfamily, which contributes to Coenzyme Q biosynthesis. In contrast, ...little is known about ADCK1 despite its evolutionary conservation in yeast, Drosophila, Caenorhabditis elegans and mammals.
Results
We show that Drosophila ADCK1 mutants die as second instar larvae with double mouth hooks and tracheal breaks. Tissue‐specific genetic rescue and RNAi studies show that ADCK1 is necessary and sufficient in the trachea for larval viability. In addition, tracheal‐rescued ADCK1 mutant adults have reduced lifespan, are developmentally delayed, have reduced body size, and normal levels of basic metabolites.
Conclusion
The larval lethality and double mouth hooks seen in ADCK1 mutants are often associated with reduced levels of the steroid hormone ecdysone, suggesting that this gene could contribute to controlling ecdysone levels or bioavailability. Similarly, the tracheal defects in these animals could arise from defects in intracellular lipid trafficking. These studies of ADCK1 provide a new context to define the physiological functions of this poorly understood member of the ADCK family of predicted mitochondrial proteins.
Key Findings
This study presents a functional analysis of the predicted mitochondrial kinase encoded by ADCK1 in Drosophila. We show that ADCK1 mutants die as second instar larvae with double mouth hooks and tracheal breaks. Tissue‐specific genetic studies show that ADCK1 is necessary and sufficient in the trachea for larval viability. Tracheal‐rescued ADCK1 mutant adults have reduced lifespan, are developmentally delayed, and have reduced body size. These studies provide a new context to define the physiological functions of this poorly understood member of the ADCK family.
There are few data on the role of nebulised magnesium sulphate (MgSO4) in the management of acute asthma in children. Those studies that have been published are underpowered, and use different ...methods, interventions and comparisons. Thus, no firm conclusions can be drawn.
Does the use of nebulised MgSO4, when given as an adjunct to standard therapy in acute severe asthma in children, result in a clinical improvement when compared with standard treatment alone?
Patients were randomised to receive three doses of MgSO4 or placebo, each combined with salbutamol and ipratropium bromide, for 1 hour. The Yung Asthma Severity Score (ASS) was measured at baseline, randomisation, and at 20, 40, 60 (T60), 120, 180 and 240 minutes after randomisation.
Emergency departments and children's assessment units at 30 hospitals in the UK.
Children aged 2-15 years with acute severe asthma.
Patients were randomised to receive nebulised salbutamol 2.5 mg (ages 2-5 years) or 5 mg (ages ≥ 6 years) and ipratropium bromide 0.25 mg mixed with either 2.5 ml of isotonic MgSO4 (250 mmol/l, tonicity 289 mOsm; 151 mg per dose) or 2.5 ml of isotonic saline on three occasions at approximately 20-minute intervals.
The primary outcome measure was the ASS after 1 hour of treatment. Secondary measures included 'stepping down' of treatment at 1 hour, number and frequency of additional salbutamol administrations, length of stay in hospital, requirement for intravenous bronchodilator treatment, and intubation and/or admission to a paediatric intensive care unit. Data on paediatric quality of life, time off school/nursery, health-care resource usage and time off work were collected 1 month after randomisation.
A total of 508 children were recruited into the study; 252 received MgSO4 and 256 received placebo along with the standard treatment. There were no differences in baseline characteristics. There was a small, but statistically significant difference in ASS at T60 in those children who received nebulised MgSO4 {0.25 95% confidence interval (CI) 0.02 to 0.48; p = 0.034} and this difference was sustained for up to 240 minutes 0.20 (95% CI 0.01 to 0.40), p = 0.042. The clinical significance of this gain is uncertain. Assessing treatment-covariate interactions, there is evidence of a larger effect in those children with more severe asthma exacerbations ( p = 0.034) and those with a shorter duration of symptoms ( p = 0.049). There were no significant differences in the secondary outcomes measured. Adverse events (AEs) were reported in 19% of children in the magnesium group and 20% in the placebo group. There were no clinically significant serious AEs in either group. The results of the base-case economic analyses are accompanied by considerable uncertainty, but suggest that, from an NHS and Personal Social Services perspective, the addition of magnesium to standard treatment may be cost-effective compared with standard treatment only. The results of economic evaluation show that the probability of magnesium being cost-effective is over 60% at cost-effectiveness thresholds of £1000 per unit decrement in ASS and £20,000 per quality-adjusted life-year (QALY) gained, respectively; it is noted that for some parameter variations this probability is much lower, reflecting the labile nature of the cost-effectiveness ratio in light of the small differences in benefits and costs shown in the trial and the relation between the main outcome measure (ASS) and preference based measures of quality of life used in cost-utility analysis (European Quality of Life-5 Dimensions; EQ-5D).
This study supports the use of nebulised isotonic MgSO4 at the dose of 151 mg given three times in the first hour of treatment as an adjuvant to standard treatment when a child presents with an acute episode of severe asthma. No harm is done by adding magnesium to salbutamol and ipratropium bromide, and in some individuals it may be clinically helpful. The response is likely to be more marked in those children with more severe attacks and with a shorter duration of exacerbation. Although the study was not powered to demonstrate this fully, the data certainly support the hypotheses that nebulised magnesium has a greater clinical effect in children who have more severe exacerbation with shorter duration of symptoms.
Current Controlled Trials ISRCTN81456894.
The National Institute for Health Research Health Technology Assessment programme.
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