RESUMO
The "Cell Painting" technology utilizes multiplexed fluorescent staining of various cell organelles, to produce high-content microscopy images of cells for multidimensional phenotype assessment. The phenotypic profiles extracted from those images can be analyzed upon perturbations with biologically active molecules to annotate the mode of action or biological activity by comparison with reference profiles of already known mechanisms of action, ultimately enabling the determination of on-target and off-target effects. This approach is already described in various human cell cultures, the most commonly used being the U2OS cell line, yet allows broad applications in additional areas of chemical-biological research. Here we describe for the first time the application and adaptation of Cell Painting to an insect cell line, the Sf9 cells from Spodoptera frugiperda. By adjusting image acquisition and analysis models, specific phenotypic profiles were obtained in a dose-dependent manner for 20 reference compounds, including representatives for the most relevant insecticidal modes of action categories (nerve & muscle, respiration and growth & development). Through a dimensionality-reduction method, both calculations of phenotypic half maximal inhibition concentration (IC50) values as well as similarity analysis of the obtained profiles by hierarchical clustering were performed. By Cell Painting effects on the phenotype could be obtained at higher sensitivity than in other assay formats, such as cytotoxicity assessments. More importantly, these analyses provide insight into mechanistic determinants of biological activity. Compounds with similar modes of action showed a high degree of proximity in a hierarchical clustering analysis while being distinct from actives with an unrelated mode of action. In essence, we provide strong evidence on the impact of Cell Painting mechanistic understanding of insecticides with regards to determinants of efficacy and safety utilizing an insect cell model system.
Assuntos
Inseticidas , Spodoptera , Animais , Spodoptera/efeitos dos fármacos , Spodoptera/citologia , Inseticidas/farmacologia , Inseticidas/toxicidade , Células Sf9RESUMO
Automated disassembly is increasingly in focus for Recycling, Re-use, and Remanufacturing (Re-X) activities. Trends in digitalization, in particular digital twin (DT) technologies and the digital product passport, as well as recently proposed European legislation such as the Net Zero and the Critical materials Acts will accelerate digitalization of product documentation and factory processes. In this contribution we look beyond these activities by discussing digital information for stakeholders at the Re-X segment of the value-chain. Furthermore, we present an approach to automated product disassembly based on different levels of available product information. The challenges for automated disassembly and the subsequent requirements on modeling of disassembly processes and product states for electronic waste are examined. The authors use a top-down (e.g., review of existing standards and process definitions) methodology to define an initial data model for disassembly processes. An additional bottom-up approach, whereby 5 exemplary electronics products were manually disassembled, was employed to analyze the efficacy of the initial data model and to offer improvements. This paper reports on our suggested informal data models for automatic electronics disassembly and the associated robotic skills.
RESUMO
Mitochondria are central hubs of cellular metabolism and are tightly connected to signaling pathways. The dynamic plasticity of mitochondria to fuse, divide, and contact other organelles to flux metabolites is central to their function. To ensure bona fide functionality and signaling interconnectivity, diverse molecular mechanisms evolved. An ancient and long-overlooked mechanism is the generation of mitochondrial-derived vesicles (MDVs) that shuttle selected mitochondrial cargoes to target organelles. Just recently, we gained significant insight into the mechanisms and functions of MDV transport, ranging from their role in mitochondrial quality control to immune signaling, thus demonstrating unexpected and diverse physiological aspects of MDV transport. This review highlights the origin of MDVs, their biogenesis, and their cargo selection, with a specific focus on the contribution of MDV transport to signaling across cell and organ barriers. Additionally, the implications of MDVs in peroxisome biogenesis, neurodegeneration, metabolism, aging, and cancer are discussed.
Assuntos
Mitocôndrias , Peroxissomos , Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Transporte BiológicoRESUMO
The ongoing COVID-19 pandemic constitutes a critical phase for the transnationalization of public spheres. Against this backdrop, we ask how transnational COVID-19 related online discourse has been throughout the EU over the first year of the pandemic. Which events triggered higher transnational coherence or national structuration of this specific issue public on Twitter? In order to study these questions, we rely on Twitter data obtained from the TBCOV database, i.e., a dataset for multilingual, geolocated COVID-19 related Twitter communication. We selected corpora for the 27 member states of the EU plus the United Kingdom. We defined three research periods representing different phases of the pandemic, namely April (1st wave), August (interim) and December 2020 (2nd wave) resulting in a set of 51,893,966 unique tweets for comparative analysis. In order to measure the level and temporal variation of transnational discursive linkages, we conducted a spatiotemporal network analysis of so-called Heterogeneous Information Networks (HINs). HINs allow for the integration of multiple, heterogeneous network entities (hashtags, retweets, @-mentions, URLs and named entities) to better represent the complex discursive structures reflected in social media communication. Therefrom, we obtained an aggregate measure of transnational linkages on a daily base by relating these linkages back to their geolocated authors. We find that the share of transnational discursive linkages increased over the course of the pandemic, indicating effects of adaptation and learning. However, stringent political measures of crisis management at the domestic level (such as lockdown decisions) caused stronger national structuration of COVID-19 related Twitter discourse.
RESUMO
This research note introduces the EPINetz Twitter Politicians Dataset, a comprehensive dataset of 2449 Twitter accounts of German parliamentarians, minsters, state secretaries, parties, and ministries on a state, federal, and European Union level for the year 2021. This hand-curated dataset not only provides up-to-date information on elected officials, but it also includes additional variables such as their party affiliation, age, and gender. Furthermore, it provides linkages to additional data sources by providing the accounts' Wikidata and Abgeordnetenwatch (Parliamentwatch) IDs. While it does not provide actual tweet data, the dataset will be a valuable resource for researchers by providing easy access to elected German politicians. We demonstrate some of the dataset's uses with an analysis of the 2021 German Federal Elections. The full dataset can be accessed via 10.7802/2415. Supplementary Information: The online version of this article (10.1007/s11615-022-00405-7) contains supplementary material, which is available to authorized users.
RESUMO
The accumulation of sphingolipid species in the cell contributes to the development of obesity and neurological disease. However, the subcellular localization of sphingolipid-synthesizing enzymes is unclear, limiting the understanding of where and how these lipids accumulate inside the cell and why they are toxic. Here, we show that SPTLC2, a subunit of the serine palmitoyltransferase (SPT) complex, catalyzing the first step in de novo sphingolipid synthesis, localizes dually to the ER and the outer mitochondrial membrane. We demonstrate that mitochondrial SPTLC2 interacts and forms a complex in trans with the ER-localized SPT subunit SPTLC1. Loss of SPTLC2 prevents the synthesis of mitochondrial sphingolipids and protects from palmitate-induced mitochondrial toxicity, a process dependent on mitochondrial ceramides. Our results reveal the in trans assembly of an enzymatic complex at an organellar membrane contact site, providing novel insight into the localization of sphingolipid synthesis and the composition and function of ER-mitochondria contact sites.
Assuntos
Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Serina C-Palmitoiltransferase/metabolismo , Transporte Biológico , Complexos Multienzimáticos/metabolismoRESUMO
Mitochondrial-derived vesicles (MDVs) are implicated in diverse physiological processes-for example, mitochondrial quality control-and are linked to various neurodegenerative diseases. However, their specific cargo composition and complex molecular biogenesis are still unknown. Here we report the proteome and lipidome of steady-state TOMM20+ MDVs. We identified 107 high-confidence MDV cargoes, which include all ß-barrel proteins and the TOM import complex. MDV cargoes are delivered as fully assembled complexes to lysosomes, thus representing a selective mitochondrial quality control mechanism for multi-subunit complexes, including the TOM machinery. Moreover, we define key biogenesis steps of phosphatidic acid-enriched MDVs starting with the MIRO1/2-dependent formation of thin membrane protrusions pulled along microtubule filaments, followed by MID49/MID51/MFF-dependent recruitment of the dynamin family GTPase DRP1 and finally DRP1-dependent scission. In summary, we define the function of MDVs in mitochondrial quality control and present a mechanistic model for global GTPase-driven MDV biogenesis.
Assuntos
Vesículas Citoplasmáticas/fisiologia , Dinaminas/metabolismo , Dinâmica Mitocondrial/fisiologia , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Lipidômica , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial/metabolismo , Doenças Neurodegenerativas/patologia , Fatores de Alongamento de Peptídeos/metabolismo , Ácidos Fosfatídicos/metabolismo , Proteoma/genética , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Schuler et al. (2021) demonstrate that mitochondrial-derived compartments protect cells from amino acid toxicity by activation of amino acid catabolism through the Ehrlich pathway, thus highlighting the incredible plasticity of mitochondria in rewiring cellular metabolism.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Álcoois , Mitocôndrias , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Heart development relies on PTMs that control cardiomyocyte proliferation, differentiation and cardiac morphogenesis. We generated a map of phosphorylation sites during the early stages of cardiac postnatal development in mice; we quantified over 10,000 phosphorylation sites and 5000 proteins that were assigned to different pathways. Analysis of mitochondrial proteins led to the identification of PGC-1- and ERR-induced regulator in muscle 1 (PERM1), which is specifically expressed in skeletal muscle and heart tissue and associates with the outer mitochondrial membrane. We demonstrate PERM1 is subject to rapid changes mediated by the UPS through phosphorylation of its PEST motif by casein kinase 2. Ablation of Perm1 in mice results in reduced protein expression of lipin-1 accompanied by accumulation of specific phospholipid species. Isolation of Perm1-deficient mitochondria revealed significant downregulation of mitochondrial transport proteins for amino acids and carnitines, including SLC25A12/13/29/34 and CPT2. Consistently, we observed altered levels of various lipid species, amino acids, and acylcarnitines in Perm1-/- mitochondria. We conclude that the outer mitochondrial membrane protein PERM1 regulates homeostasis of lipid and amino acid metabolites in mitochondria.
Assuntos
Proteínas de Membrana/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Fosfoproteínas/metabolismo , Proteômica , Animais , Coração/embriologia , Metabolismo dos Lipídeos , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Proteínas Musculares/genética , Organogênese/genética , Proteômica/métodosRESUMO
Disturbances in the morphology and function of mitochondria cause neurological diseases, which can affect the central and peripheral nervous system. The i-AAA protease YME1L ensures mitochondrial proteostasis and regulates mitochondrial dynamics by processing of the dynamin-like GTPase OPA1. Mutations in YME1L cause a multi-systemic mitochondriopathy associated with neurological dysfunction and mitochondrial fragmentation but pathogenic mechanisms remained enigmatic. Here, we report on striking cell-type-specific defects in mice lacking YME1L in the nervous system. YME1L-deficient mice manifest ocular dysfunction with microphthalmia and cataracts and develop deficiencies in locomotor activity due to specific degeneration of spinal cord axons, which relay proprioceptive signals from the hind limbs to the cerebellum. Mitochondrial fragmentation occurs throughout the nervous system and does not correlate with the degenerative phenotype. Deletion of Oma1 restores tubular mitochondria but deteriorates axonal degeneration in the absence of YME1L, demonstrating that impaired mitochondrial proteostasis rather than mitochondrial fragmentation causes the observed neurological defects.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/deficiência , Metaloendopeptidases/deficiência , Doenças Mitocondriais/patologia , Doenças Mitocondriais/fisiopatologia , Doenças do Sistema Nervoso/patologia , Doenças do Sistema Nervoso/fisiopatologia , Animais , Catarata/etiologia , Catarata/patologia , Modelos Animais de Doenças , GTP Fosfo-Hidrolases/metabolismo , Transtornos Neurológicos da Marcha/etiologia , Transtornos Neurológicos da Marcha/patologia , Camundongos , Microftalmia/etiologia , Microftalmia/patologia , Proteínas Mitocondriais/deficiência , Medula Espinal/patologiaRESUMO
Intramembrane-cleaving peptidases of the rhomboid family regulate diverse cellular processes that are critical for development and cell survival. The function of the rhomboid protease PARL in the mitochondrial inner membrane has been linked to mitophagy and apoptosis, but other regulatory functions are likely to exist. Here, we identify the START domain-containing protein STARD7 as an intramitochondrial lipid transfer protein for phosphatidylcholine. We demonstrate that PARL-mediated cleavage during mitochondrial import partitions STARD7 to the cytosol and the mitochondrial intermembrane space. Negatively charged amino acids in STARD7 serve as a sorting signal allowing mitochondrial release of mature STARD7 upon cleavage by PARL On the other hand, membrane insertion of STARD7 mediated by the TIM23 complex promotes mitochondrial localization of mature STARD7. Mitochondrial STARD7 is necessary and sufficient for the accumulation of phosphatidylcholine in the inner membrane and for the maintenance of respiration and cristae morphogenesis. Thus, PARL preserves mitochondrial membrane homeostasis via STARD7 processing and is emerging as a critical regulator of protein localization between mitochondria and the cytosol.
Assuntos
Proteínas de Transporte/metabolismo , Citosol/metabolismo , Metaloproteases/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Sequência de Aminoácidos , Apoptose , Células HEK293 , Células HeLa , Humanos , Mitofagia , Homologia de SequênciaRESUMO
Mutations in mitochondrial acylglycerol kinase (AGK) cause Sengers syndrome, which is characterized by cataracts, hypertrophic cardiomyopathy, and skeletal myopathy. AGK generates phosphatidic acid and lysophosphatidic acid, bioactive phospholipids involved in lipid signaling and the regulation of tumor progression. However, the molecular mechanisms of the mitochondrial pathology remain enigmatic. Determining its mitochondrial interactome, we have identified AGK as a constituent of the TIM22 complex in the mitochondrial inner membrane. AGK assembles with TIMM22 and TIMM29 and supports the import of a subset of multi-spanning membrane proteins. The function of AGK as a subunit of the TIM22 complex does not depend on its kinase activity. However, enzymatically active AGK is required to maintain mitochondrial cristae morphogenesis and the apoptotic resistance of cells. The dual function of AGK as lipid kinase and constituent of the TIM22 complex reveals that disturbances in both phospholipid metabolism and mitochondrial protein biogenesis contribute to the pathogenesis of Sengers syndrome.
Assuntos
Cardiomiopatias/enzimologia , Catarata/enzimologia , Mitocôndrias/enzimologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Translocador 1 do Nucleotídeo Adenina/metabolismo , Antiporters/metabolismo , Apoptose , Proteínas de Ligação ao Cálcio/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/patologia , Catarata/genética , Catarata/patologia , Predisposição Genética para Doença , Células HEK293 , Células HeLa , Humanos , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos , Mutação , Fenótipo , Fosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Transporte Proteico , Fatores de Tempo , TransfecçãoRESUMO
PURPOSE: In interstitial high-dose rate brachytherapy, liver cancer is treated by internal radiation, requiring percutaneous placement of applicators within or close to the tumor. To maximize utility, the optimal applicator configuration is pre-planned on magnetic resonance images. The pre-planned configuration is then implemented via a magnetic resonance-guided intervention. Mapping the pre-planning information onto interventional data would reduce the radiologist's cognitive load during the intervention and could possibly minimize discrepancies between optimally pre-planned and actually placed applicators. METHODS: We propose a fast and robust two-step registration framework suitable for interventional settings: first, we utilize a multi-resolution rigid registration to correct for differences in patient positioning (rotation and translation). Second, we employ a novel iterative approach alternating between bias field correction and Markov random field deformable registration in a multi-resolution framework to compensate for non-rigid movements of the liver, the tumors and the organs at risk. In contrast to existing pre-correction methods, our multi-resolution scheme can recover bias field artifacts of different extents at marginal computational costs. RESULTS: We compared our approach to deformable registration via B-splines, demons and the SyN method on 22 registration tasks from eleven patients. Results showed that our approach is more accurate than the contenders for liver as well as for tumor tissues. We yield average liver volume overlaps of 94.0 ± 2.7% and average surface-to-surface distances of 2.02 ± 0.87 mm and 3.55 ± 2.19 mm for liver and tumor tissue, respectively. The reported distances are close to (or even below) the slice spacing (2.5 - 3.0 mm) of our data. Our approach is also the fastest, taking 35.8 ± 12.8 s per task. CONCLUSION: The presented approach is sufficiently accurate to map information available from brachytherapy pre-planning onto interventional data. It is also reasonably fast, providing a starting point for computer-aidance during intervention.
Assuntos
Artefatos , Braquiterapia/métodos , Neoplasias Hepáticas/radioterapia , Fígado/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Radioterapia Assistida por Computador/métodos , Humanos , Neoplasias Hepáticas/diagnóstico , MasculinoRESUMO
Human embryonic stem cells can replicate indefinitely while maintaining their undifferentiated state and, therefore, are immortal in culture. This capacity may demand avoidance of any imbalance in protein homeostasis (proteostasis) that would otherwise compromise stem cell identity. Here we show that human pluripotent stem cells exhibit enhanced assembly of the TRiC/CCT complex, a chaperonin that facilitates the folding of 10% of the proteome. We find that ectopic expression of a single subunit (CCT8) is sufficient to increase TRiC/CCT assembly. Moreover, increased TRiC/CCT complex is required to avoid aggregation of mutant Huntingtin protein. We further show that increased expression of CCT8 in somatic tissues extends Caenorhabditis elegans lifespan in a TRiC/CCT-dependent manner. Ectopic expression of CCT8 also ameliorates the age-associated demise of proteostasis and corrects proteostatic deficiencies in worm models of Huntington's disease. Our results suggest proteostasis is a common principle that links organismal longevity with hESC immortality.
Assuntos
Caenorhabditis elegans/fisiologia , Chaperonina com TCP-1/metabolismo , Longevidade , Células-Tronco Pluripotentes/metabolismo , Proteostase , Animais , Diferenciação Celular , Técnicas de Silenciamento de Genes , Células HEK293 , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mutação/genética , Fenótipo , Agregados Proteicos , Subunidades Proteicas/metabolismo , Estresse FisiológicoRESUMO
The SPFH (stomatin, prohibitin, flotillin, HflC/K) superfamily is composed of scaffold proteins that form ring-like structures and locally specify the protein-lipid composition in a variety of cellular membranes. Stomatin-like protein 2 (SLP2) is a member of this superfamily that localizes to the mitochondrial inner membrane (IM) where it acts as a membrane organizer. Here, we report that SLP2 anchors a large protease complex composed of the rhomboid protease PARL and the i-AAA protease YME1L, which we term the SPY complex (for SLP2-PARL-YME1L). Association with SLP2 in the SPY complex regulates PARL-mediated processing of PTEN-induced kinase PINK1 and the phosphatase PGAM5 in mitochondria. Moreover, SLP2 inhibits the stress-activated peptidase OMA1, which can bind to SLP2 and cleaves PGAM5 in depolarized mitochondria. SLP2 restricts OMA1-mediated processing of the dynamin-like GTPase OPA1 allowing stress-induced mitochondrial hyperfusion under starvation conditions. Together, our results reveal an important role of SLP2 membrane scaffolds for the spatial organization of IM proteases regulating mitochondrial dynamics, quality control, and cell survival.
Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Metaloproteases/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Proteínas Sanguíneas/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Metaloproteases/genética , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Peptídeo Hidrolases/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , ProteóliseRESUMO
Mutations in subunits of mitochondrial m-AAA proteases in the inner membrane cause neurodegeneration in spinocerebellar ataxia (SCA28) and hereditary spastic paraplegia (HSP7). m-AAA proteases preserve mitochondrial proteostasis, mitochondrial morphology, and efficient OXPHOS activity, but the cause for neuronal loss in disease is unknown. We have determined the neuronal interactome of m-AAA proteases in mice and identified a complex with C2ORF47 (termed MAIP1), which counteracts cell death by regulating the assembly of the mitochondrial Ca2+ uniporter MCU. While MAIP1 assists biogenesis of the MCU subunit EMRE, the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU. Loss of the m-AAA protease results in accumulation of constitutively active MCU-EMRE channels lacking gatekeeper subunits in neuronal mitochondria and facilitates mitochondrial Ca2+ overload, mitochondrial permeability transition pore opening, and neuronal death. Together, our results explain neuronal loss in m-AAA protease deficiency by deregulated mitochondrial Ca2+ homeostasis.
Assuntos
Canais de Cálcio/metabolismo , Cerebelo/metabolismo , Corpo Estriado/metabolismo , Hipocampo/metabolismo , Metaloendopeptidases/genética , Mitocôndrias/metabolismo , Neurônios/metabolismo , Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Morte Celular , Cerebelo/patologia , Corpo Estriado/patologia , Regulação da Expressão Gênica , Células HEK293 , Hipocampo/patologia , Homeostase/genética , Humanos , Transporte de Íons , Metaloendopeptidases/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Neurônios/patologia , Mapeamento de Interação de Proteínas , Transdução de SinaisRESUMO
The mitochondrial matrix protease CLPP plays a central role in the activation of the mitochondrial unfolded protein response (UPR(mt)) in Caenorhabditis elegans Far less is known about mammalian UPR(mt) signaling, although similar roles were assumed for central players, including CLPP To better understand the mammalian UPR(mt) signaling, we deleted CLPP in hearts of DARS2-deficient animals that show robust induction of UPR(mt) due to strong dysregulation of mitochondrial translation. Remarkably, our results clearly show that mammalian CLPP is neither required for, nor it regulates the UPR(mt) in mammals. Surprisingly, we demonstrate that a strong mitochondrial cardiomyopathy and diminished respiration due to DARS2 deficiency can be alleviated by the loss of CLPP, leading to an increased de novo synthesis of individual OXPHOS subunits. These results question our current understanding of the UPR(mt) signaling in mammals, while introducing CLPP as a possible novel target for therapeutic intervention in mitochondrial diseases.
Assuntos
Cardiomiopatias/genética , Endopeptidase Clp/deficiência , Mitocôndrias Cardíacas/genética , Transdução de Sinais , Animais , Aspartato-tRNA Ligase/deficiência , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Feminino , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Estresse FisiológicoRESUMO
BACKGROUND: Ensuring adequate vitamin D status in older adults may reduce the risk of osteoporosis. The serum 25-hydroxyvitamin D [25(OH)D] concentration is the recommended biomarker of vitamin D status, but the optimal serum 25(OH)D concentration for bone health in postmenopausal women remains unclear. OBJECTIVE: The aim of this study was to apply the highly sensitive (41)Ca skeletal labeling technique and the measurement of urinary (41)Ca:(40)Ca ratios to determine the serum 25(OH)D concentration that has greatest benefit on bone calcium flux in postmenopausal women. METHODS: We administered a mean intravenous (41)Ca dose of 870 pmol to healthy postmenopausal women [n = 24, age (mean ± SD): 64 ± 6.0 y] without osteoporosis. After 6 mo, at the nadir of their wintertime serum 25(OH)D status, each of the women sequentially consumed daily oral cholecalciferol supplements of 10, 25, and 50 µg/d (in this order), each for 3 mo. We assessed serum 25(OH)D concentrations monthly and urinary (41)Ca:(40)Ca ratios biweekly. (41)Ca:(40)Ca ratios were measured with low-energy accelerator mass spectrometry. With the use of pharmacokinetic analysis, we determined the effect of varying serum 25(OH)D concentrations on (41)Ca transfer rates. RESULTS: At baseline, the mean (95% CI) serum 25(OH)D concentration was 16.2 (13.5, 18.8) µg/L. After the first, second, and third intervention periods, mean (95% CI) serum 25(OH)D increased to 29.8 (27.2, 32.4), 36.9 (34.2, 39.7), and 46.6 (41.2, 52.0) µg/L, respectively. Supplementation was associated with a downward shift in the urinary (41)Ca:(40)Ca ratio compared with the predicted (41)Ca:(40)Ca ratio without vitamin D supplementation. In the model, the most likely site of action of the increase in serum 25(OH)D was transfer from the central compartment to a fast exchanging compartment. At this transfer rate, predicted values were a concentration with half-maximal effect of 2.33 µg/L and an estimate of the maximal effect of 31.7%. After the first, second, and third intervention periods, the mean changes in this transfer rate were +18.0%, +25.7%, and +28.5%, respectively. CONCLUSION: In healthy postmenopausal women, increasing serum 25(OH)D primarily affects calcium transfer from the central compartment to a fast exchanging compartment; it is possible that this represents transfer from the extracellular space to the surface of bone. A serum 25(OH)D concentration of ~40 µg/L achieves ~90% of the expected maximal effect on this transfer rate. This trial was registered at clinicaltrials.gov as NCT01053481.
Assuntos
Conservadores da Densidade Óssea/uso terapêutico , Remodelação Óssea , Calcifediol/sangue , Colecalciferol/uso terapêutico , Suplementos Nutricionais , Regulação para Baixo , Osteoporose Pós-Menopausa/prevenção & controle , 25-Hidroxivitamina D 2/sangue , Idoso , Biomarcadores/sangue , Biomarcadores/urina , Conservadores da Densidade Óssea/administração & dosagem , Cálcio/urina , Radioisótopos de Cálcio , Colecalciferol/administração & dosagem , Estudos de Coortes , Feminino , Humanos , Cinética , Pessoa de Meia-Idade , Osteoporose Pós-Menopausa/epidemiologia , Osteoporose Pós-Menopausa/metabolismo , Osteoporose Pós-Menopausa/fisiopatologia , Fatores de Risco , Estações do Ano , Suíça/epidemiologiaRESUMO
Stomatin-like protein 2 (SLP-2) is a mainly mitochondrial protein that is widely expressed and is highly conserved across evolution. We have previously shown that SLP-2 binds the mitochondrial lipid cardiolipin and interacts with prohibitin-1 and -2 to form specialized membrane microdomains in the mitochondrial inner membrane, which are associated with optimal mitochondrial respiration. To determine how SLP-2 functions, we performed bioenergetic analysis of primary T cells from T cell-selective Slp-2 knockout mice under conditions that forced energy production to come almost exclusively from oxidative phosphorylation. These cells had a phenotype characterized by increased uncoupled mitochondrial respiration and decreased mitochondrial membrane potential. Since formation of mitochondrial respiratory chain supercomplexes (RCS) may correlate with more efficient electron transfer during oxidative phosphorylation, we hypothesized that the defect in mitochondrial respiration in SLP-2-deficient T cells was due to deficient RCS formation. We found that in the absence of SLP-2, T cells had decreased levels and activities of complex I-III2 and I-III2-IV(1-3) RCS but no defects in assembly of individual respiratory complexes. Impaired RCS formation in SLP-2-deficient T cells correlated with significantly delayed T cell proliferation in response to activation under conditions of limiting glycolysis. Altogether, our findings identify SLP-2 as a key regulator of the formation of RCS in vivo and show that these supercomplexes are required for optimal cell function.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/fisiologia , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Linfócitos T/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Potencial da Membrana Mitocondrial , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , Proteínas do Tecido Nervoso/genética , Fosforilação Oxidativa , FenótipoRESUMO
PURPOSE: Diagnosis of neuromuscular diseases in ultrasonography is a challenging task since experts are often unable to discriminate between healthy and pathological cases. A computer-aided diagnosis (CAD) system for skeletal muscle ultrasonography was developed and tested for myositis detection in ultrasound images of biceps brachii. METHODS: Several types of features were extracted from rectangular and polygonal image regions-of-interest (ROIs), including first-order statistics, wavelet-based features, and Haralick's features. Features were chosen that are sensitive to the change in contrast and structure for pathological ultrasound images of neuromuscular diseases. The number of features was reduced by applying different sequential feature selection strategies followed by a supervised principal component analysis. For classification, two linear approaches were investigated: Fisher's classifier and the linear support vector machine (SVM) as well as the nonlinear [Formula: see text]-nearest neighbor approach. The CAD system was benchmarked on datasets of 18 subjects, seven of which were healthy, while 11 were affected by myositis. Three expert radiologists provided pre-classification and testing interpretations. RESULTS: Leave-one-out cross-validation on the training data revealed that the linear SVM was best suited for discriminating healthy and pathological muscle tissue, achieving 85/87 % accuracy, 90 % sensitivity, and 83/85 % specificity, depending on the radiologist. CONCLUSION: A muscle ultrasonography CAD system was developed, allowing a classification of an ultrasound image by one-click positioning of rectangular ROIs with minimal user effort. The applicability of the system was demonstrated with the challenging example of myositis detection, showing highly accurate results that were robust to imprecise user input.