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.
Homeostatic maintenance of cellular mitochondria requires a dynamic balance between fission and fusion, and disruptions in this balance have been implicated in multiple pathological conditions, ...including Charcot-Marie-Tooth, Parkinson's, and Alzheimer's diseases. Whereas deregulated fission and fusion can be detrimental to health and survival, controlled changes in morphology are important for processes like cellular division and apoptosis. Specifically, regulated mitochondrial fission occurs closely with cytochrome c release during apoptosis and upon entry into mitosis during the cell cycle. Using cell culture-based assays, microscopy, and fly genetics, we examine how changes in the mitochondrial network are mediated at the molecular level during apoptosis and the cell cycle.
First, we report that the fly protein Reaper induces mitochondrial fragmentation in mammalian cells, likely through inhibition of the mitochondrial fusion protein Mfn2. Reaper colocalizes with and binds to Mfn2 and its fly orthologue dMFN, and the colocalization of the two proteins is necessary for Reaper-induced mitochondrial fission. Moreover, the overexpression of dMFN inhibits Reaper-induced killing both in vitro and in vivo.
Our data and work in a number of experimental systems demonstrate a requirement for mitochondrial fragmentation during apoptosis that is conserved from worms to flies to mammals. Our findings indicate that Reaper may function to inactivate mitochondrial metabolic function and/or to facilitate mitochondrial elimination during apoptosis.
Secondly, we characterize Drp1 degradation by the APC/C during mitotic exit and interphase. We provide evidence that APC/CCdh1-mediated degradation of Drp1 underlies both the morphological changes that occur during progression through the cell cycle and changes in mitochondrial metabolism during interphase. Inhibition of Cdh1-mediated Drp1 ubiquitylation and proteasomal degradation during interphase prevents the normal regrowth of mitochondrial networks after mitosis, prevents cyclin E accumulation, and alters the profile of lipid-derived metabolites. Our findings describe a novel role for APC/CCdh1-mediated Drp1 degradation in cell cycle-dependent changes in mitochondrial morphology and metabolic function and suggest that the APC/CCdh1complex may regulate the distinct bioenergetic needs of a growing cell during synthetic phases of the cell cycle.
Dissertation
mothur aims to be a comprehensive software package that allows users to use a single piece of software to analyze community sequence data. It builds upon previous tools to provide a flexible and ...powerful software package for analyzing sequencing data. As a case study, we used mothur to trim, screen, and align sequences; calculate distances; assign sequences to operational taxonomic units; and describe the α and β diversity of eight marine samples previously characterized by pyrosequencing of 16S rRNA gene fragments. This analysis of more than 222,000 sequences was completed in less than 2 h with a laptop computer.
The irreversible ErbB family tyrosine kinase inhibitor (TKI) afatinib plus the EGFR monoclonal antibody cetuximab was previously shown to overcome resistance to EGFR TKIs. We studied whether the ...combination of afatinib plus cetuximab compared with afatinib alone would improve progression-free survival (PFS) in patients with treatment-naive
-mutant non-small-cell lung cancer (NSCLC) by preventing or delaying resistance.
Patients with
-mutant NSCLC without prior treatment of advanced disease were enrolled in this phase II, multicenter trial and randomly assigned to receive afatinib 40 mg orally daily plus cetuximab 500 mg/m
intravenously every 2 weeks or afatinib alone. The primary end point was PFS.
Between March 25, 2015 and April 23, 2018, 174 patients were randomly assigned, and 168 (83 on afatinib + cetuximab and 85 on afatinib) were eligible. There was no improvement in PFS in patients receiving afatinib plus cetuximab compared with afatinib alone (hazard ratio HR, 1.01; 95% CI, 0.72 to 1.43;
= .94; median, 11.9 months
13.4 months). Similarly, there was no difference in response rate (67%
74%;
= .38) or overall survival (HR, 0.82; 95% CI, 0.50 to 1.36;
= .44). Toxicity was greater with the combination: grade ≥ 3 adverse events related to treatment occurred in 72% of patients receiving afatinib plus cetuximab compared with 40% of those receiving afatinib alone, most commonly rash and diarrhea. Dose reductions were more common in patients receiving the combination, and 30% of patients in this arm discontinued cetuximab due to toxicity. At interim analysis, there was insufficient evidence to support continued accrual, and the trial was closed.
The addition of cetuximab to afatinib did not improve outcomes in previously untreated
-mutant NSCLC, despite recognized activity in the acquired resistance setting.
The aim of this consensus paper is to discuss the roles of the cerebellum in human gait, as well as its assessment and therapy. Cerebellar vermis is critical for postural control. The cerebellum ...ensures the mapping of sensory information into temporally relevant motor commands. Mental imagery of gait involves intrinsically connected fronto-parietal networks comprising the cerebellum. Muscular activities in cerebellar patients show impaired timing of discharges, affecting the patterning of the synergies subserving locomotion. Ataxia of stance/gait is amongst the first cerebellar deficits in cerebellar disorders such as degenerative ataxias and is a disabling symptom with a high risk of falls. Prolonged discharges and increased muscle coactivation may be related to compensatory mechanisms and enhanced body sway, respectively. Essential tremor is frequently associated with mild gait ataxia. There is growing evidence for an important role of the cerebellar cortex in the pathogenesis of essential tremor. In multiple sclerosis, balance and gait are affected due to cerebellar and spinal cord involvement, as a result of disseminated demyelination and neurodegeneration impairing proprioception. In orthostatic tremor, patients often show mild-to-moderate limb and gait ataxia. The tremor generator is likely located in the posterior fossa. Tandem gait is impaired in the early stages of cerebellar disorders and may be particularly useful in the evaluation of pre-ataxic stages of progressive ataxias. Impaired inter-joint coordination and enhanced variability of gait temporal and kinetic parameters can be grasped by wearable devices such as accelerometers. Kinect is a promising low cost technology to obtain reliable measurements and remote assessments of gait. Deep learning methods are being developed in order to help clinicians in the diagnosis and decision-making process. Locomotor adaptation is impaired in cerebellar patients. Coordinative training aims to improve the coordinative strategy and foot placements across strides, cerebellar patients benefiting from intense rehabilitation therapies. Robotic training is a promising approach to complement conventional rehabilitation and neuromodulation of the cerebellum. Wearable dynamic orthoses represent a potential aid to assist gait. The panel of experts agree that the understanding of the cerebellar contribution to gait control will lead to a better management of cerebellar ataxias in general and will likely contribute to use gait parameters as robust biomarkers of future clinical trials.
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•Copper resinate is a mixed abietate/acetate copper salt dissolved in resin.•Its green color comes from the blue organic copper salt and the yellow resin.•Mild basic solutions convert ...copper resinate to orange-brown CuO.
Copper resinate was a green pigment used through the Renaissance when it was largely discontinued due to its tendency to discolor to brown over time. Historically, this pigment was made by grinding copper(II) acetate, or verdigris, in a tree resin rich in abietic acid. Commercial copper resinate and copper(II) abietate mixtures were analyzed by electrospray ionization mass spectrometry, infrared and visible spectroscopy. The pigment consists of mixed organic copper salts with bridged acetate (Ac) and abietate (Ab) ligands (i.e, Cu2Ab4, Cu2Ab3Ac, and Cu2Ab2Ac2) based upon sodiated ions detected in mass spectra. The lipophilic abietate groups allow the molecules to dissolve in the resin and oil binders for a transparent glaze free of pigment particles. A paddlewheel bridged-dimer structure of Cu2Ab4 modeled by density functional theory has similar bond distances to copper(II) acetate. Experimental results suggest that copper(II) abietate lacks the axial water ligands found in copper(II) acetate. The green color of copper resinate originates from a combination of the blue typical for organic copper salts and the yellow of free abietate/abietic acid. Solutions of both verdigris and copper resinate quickly discolor to brown upon the addition of a dilute basic solution. Slow conversion of organic copper salts in the paint layer to brown copper(II) oxide, possibly enhanced by environmental conditions or cleaning with alkaline materials, may be an alternate discoloration mechanism in cultural artifacts. Copper detected by portable energy dispersive X-ray fluorescence (XRF) spectroscopy and scanning electron microscopy with energy dispersive X-ray (SEM-EDX) in discolored paint layers of an Italian Renaissance painting by Tintoretto is consistent with this artist’s established use of copper resinate.
Abstract Background and aims Cardiovascular disease (CVD) is among the leading causes of morbidity and mortality worldwide. Traditional risk factors predict 75–80% of an individual's risk of incident ...CVD. However, the role of early life experiences in future disease risk is gaining attention. The Barker hypothesis proposes fetal origins of adult disease, with consistent evidence demonstrating the deleterious consequences of birth weight outside the normal range. In this study, we investigate the role of birth weight in CVD risk prediction. Methods and results The Women's Health Initiative (WHI) represents a large national cohort of post-menopausal women with 63,815 participants included in this analysis. Univariable proportional hazards regression analyses evaluated the association of 4 self-reported birth weight categories against 3 CVD outcome definitions, which included indicators of coronary heart disease, ischemic stroke, coronary revascularization, carotid artery disease and peripheral arterial disease. The role of birth weight was also evaluated for prediction of CVD events in the presence of traditional risk factors using 3 existing CVD risk prediction equations: one body mass index (BMI)-based and two laboratory-based models. Low birth weight (LBW) (<6 lbs.) was significantly associated with all CVD outcome definitions in univariable analyses (HR = 1.086, p = 0.009). LBW was a significant covariate in the BMI-based model (HR = 1.128, p < 0.0001) but not in the lipid-based models. Conclusion LBW (<6 lbs.) is independently associated with CVD outcomes in the WHI cohort. This finding supports the role of the prenatal and postnatal environment in contributing to the development of adult chronic disease.
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