Publications by authors named "Ilka Wittig"

165 Publications

Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples.

J Biol Chem 2021 Sep 17;297(4):101204. Epub 2021 Sep 17.

Division of Neonatology, Department of Pediatrics, Columbia University Medical Center, New York, New York, USA; Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, New York, USA. Electronic address:

Impairments in mitochondrial energy metabolism have been implicated in human genetic diseases associated with mitochondrial and nuclear DNA mutations, neurodegenerative and cardiovascular disorders, diabetes, and aging. Alteration in mitochondrial complex I structure and activity has been shown to play a key role in Parkinson's disease and ischemia/reperfusion tissue injury, but significant difficulty remains in assessing the content of this enzyme complex in a given sample. The present study introduces a new method utilizing native polyacrylamide gel electrophoresis in combination with flavin fluorescence scanning to measure the absolute content of complex I, as well as α-ketoglutarate dehydrogenase complex, in any preparation. We show that complex I content is 19 ± 1 pmol/mg of protein in the brain mitochondria, whereas varies up to 10-fold in different mouse tissues. Together with the measurements of NADH-dependent specific activity, our method also allows accurate determination of complex I catalytic turnover, which was calculated as 10 min for NADH:ubiquinone reductase in mouse brain mitochondrial preparations. α-ketoglutarate dehydrogenase complex content was determined to be 65 ± 5 and 123 ± 9 pmol/mg protein for mouse brain and bovine heart mitochondria, respectively. Our approach can also be extended to cultured cells, and we demonstrated that about 90 × 10 complex I molecules are present in a single human embryonic kidney 293 cell. The ability to determine complex I content should provide a valuable tool to investigate the enzyme status in samples after in vivo treatment in mutant organisms, cells in culture, or human biopsies.
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http://dx.doi.org/10.1016/j.jbc.2021.101204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8503622PMC
September 2021

Complexome Profiling: Assembly and Remodeling of Protein Complexes.

Int J Mol Sci 2021 Jul 21;22(15). Epub 2021 Jul 21.

Institute for Cardiovascular Physiology, Goethe University, 60590 Frankfurt, Germany.

Many proteins have been found to operate in a complex with various biomolecules such as proteins, nucleic acids, carbohydrates, or lipids. Protein complexes can be transient, stable or dynamic and their association is controlled under variable cellular conditions. Complexome profiling is a recently developed mass spectrometry-based method that combines mild separation techniques, native gel electrophoresis, and density gradient centrifugation with quantitative mass spectrometry to generate inventories of protein assemblies within a cell or subcellular fraction. This review summarizes applications of complexome profiling with respect to assembly ranging from single subunits to large macromolecular complexes, as well as their stability, and remodeling in health and disease.
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http://dx.doi.org/10.3390/ijms22157809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8346016PMC
July 2021

The biochemical basis of mitochondrial dysfunction in Zellweger Spectrum Disorder.

EMBO Rep 2021 10 5;22(10):e51991. Epub 2021 Aug 5.

Howard Hughes Medical Institute, Salt Lake City, UT, USA.

Peroxisomal biogenesis disorders (PBDs) are genetic disorders of peroxisome biogenesis and metabolism that are characterized by profound developmental and neurological phenotypes. The most severe class of PBDs-Zellweger spectrum disorder (ZSD)-is caused by mutations in peroxin genes that result in both non-functional peroxisomes and mitochondrial dysfunction. It is unclear, however, how defective peroxisomes contribute to mitochondrial impairment. In order to understand the molecular basis of this inter-organellar relationship, we investigated the fate of peroxisomal mRNAs and proteins in ZSD model systems. We found that peroxins were still expressed and a subset of them accumulated on the mitochondrial membrane, which resulted in gross mitochondrial abnormalities and impaired mitochondrial metabolic function. We showed that overexpression of ATAD1, a mitochondrial quality control factor, was sufficient to rescue several aspects of mitochondrial function in human ZSD fibroblasts. Together, these data suggest that aberrant peroxisomal protein localization is necessary and sufficient for the devastating mitochondrial morphological and metabolic phenotypes in ZSDs.
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http://dx.doi.org/10.15252/embr.202051991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8490991PMC
October 2021

Increased presence of nuclear DNAJA3 and upregulation of cytosolic STAT1 and of nucleic acid sensors trigger innate immunity in the ClpP-null mouse.

Neurogenetics 2021 10 3;22(4):297-312. Epub 2021 Aug 3.

Experimental Neurology, Medical School, Goethe University, 60590, Frankfurt, Germany.

Mitochondrial dysfunction may activate innate immunity, e.g. upon abnormal handling of mitochondrial DNA in TFAM mutants or in altered mitophagy. Recent reports showed that also deletion of mitochondrial matrix peptidase ClpP in mice triggers transcriptional upregulation of inflammatory factors. Here, we studied ClpP-null mouse brain at two ages and mouse embryonal fibroblasts, to identify which signaling pathways are responsible, employing mass spectrometry, subcellular fractionation, immunoblots, and reverse transcriptase polymerase chain reaction. Several mitochondrial unfolded protein response factors showed accumulation and altered migration in blue-native gels, prominently the co-chaperone DNAJA3. Its mitochondrial dysregulation increased also its extra-mitochondrial abundance in the nucleus, a relevant observation given that DNAJA3 modulates innate immunity. Similar observations were made for STAT1, a putative DNAJA3 interactor. Elevated expression was observed not only for the transcription factors Stat1/2, but also for two interferon-stimulated genes (Ifi44, Gbp3). Inflammatory responses were strongest for the RLR pattern recognition receptors (Ddx58, Ifih1, Oasl2, Trim25) and several cytosolic nucleic acid sensors (Ifit1, Ifit3, Oas1b, Ifi204, Mnda). The consistent dysregulation of these factors from an early age might influence also human Perrault syndrome, where ClpP loss-of-function leads to early infertility and deafness, with subsequent widespread neurodegeneration.
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http://dx.doi.org/10.1007/s10048-021-00657-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8426249PMC
October 2021

N471D WASH complex subunit strumpellin knock-in mice display mild motor and cardiac abnormalities and BPTF and KLHL11 dysregulation in brain tissue.

Neuropathol Appl Neurobiol 2021 Jul 27. Epub 2021 Jul 27.

Institute of Neuropathology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany.

Aims: We investigated N471D WASH complex subunit strumpellin (Washc5) knock-in and Washc5 knock-out mice as models for hereditary spastic paraplegia type 8 (SPG8).

Methods: We generated heterozygous and homozygous N471D Washc5 knock-in mice and subjected them to a comprehensive clinical, morphological and laboratory parameter screen, and gait analyses. Brain tissue was used for proteomic analysis. Furthermore, we generated heterozygous Washc5 knock-out mice. WASH complex subunit strumpellin expression was determined by qPCR and immunoblotting.

Results: Homozygous N471D Washc5 knock-in mice showed mild dilated cardiomyopathy, decreased acoustic startle reactivity, thinner eye lenses, increased alkaline phosphatase and potassium levels and increased white blood cell counts. Gait analyses revealed multiple aberrations indicative of locomotor instability. Similarly, the clinical chemistry, haematology and gait parameters of heterozygous mice also deviated from the values expected for healthy animals, albeit to a lesser extent. Proteomic analysis of brain tissue depicted consistent upregulation of BPTF and downregulation of KLHL11 in heterozygous and homozygous knock-in mice. WASHC5-related protein interaction partners and complexes showed no change in abundancies. Heterozygous Washc5 knock-out mice showing normal WASHC5 levels could not be bred to homozygosity.

Conclusions: While biallelic ablation of Washc5 was prenatally lethal, expression of N471D mutated WASHC5 led to several mild clinical and laboratory parameter abnormalities, but not to a typical SPG8 phenotype. The consistent upregulation of BPTF and downregulation of KLHL11 suggest mechanistic links between the expression of N471D mutated WASHC5 and the roles of both proteins in neurodegeneration and protein quality control, respectively.
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http://dx.doi.org/10.1111/nan.12750DOI Listing
July 2021

The Consequences of Soluble Epoxide Hydrolase Deletion on Tumorigenesis and Metastasis in a Mouse Model of Breast Cancer.

Int J Mol Sci 2021 Jul 1;22(13). Epub 2021 Jul 1.

Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, 60590 Frankfurt, Germany.

Epoxides and diols of polyunsaturated fatty acids (PUFAs) are bioactive and can influence processes such as tumor cell proliferation and angiogenesis. Studies with inhibitors of the soluble epoxide hydrolase (sEH) in animals overexpressing cytochrome P450 enzymes or following the systemic administration of specific epoxides revealed a markedly increased incidence of tumor metastases. To determine whether PUFA epoxides increased metastases in a model of spontaneous breast cancer, sEH mice were crossed onto the polyoma middle T oncogene (PyMT) background. We found that the deletion of the sEH accelerated the growth of primary tumors and increased both the tumor macrophage count and angiogenesis. There were small differences in the epoxide/diol content of tumors, particularly in epoxyoctadecamonoenic acid versus dihydroxyoctadecenoic acid, and marked changes in the expression of proteins linked with cell proliferation and metabolism. However, there was no consequence of sEH inhibition on the formation of metastases in the lymph node or lung. Taken together, our results confirm previous reports of increased tumor growth in animals lacking sEH but fail to substantiate reports of enhanced lymph node or pulmonary metastases.
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http://dx.doi.org/10.3390/ijms22137120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8269362PMC
July 2021

Prediction and analysis of redox-sensitive cysteines using machine learning and statistical methods.

Biol Chem 2021 Jul 6;402(8):925-935. Epub 2021 Jan 6.

Molecular Bioinformatics Group, Institute of Computer Science, Goethe-University, Robert-Mayer-Str. 11-15, 60325, Frankfurt am Main, Germany.

Reactive oxygen species are produced by a number of stimuli and can lead both to irreversible intracellular damage and signaling through reversible post-translational modification. It is unclear which factors contribute to the sensitivity of cysteines to redox modification. Here, we used statistical and machine learning methods to investigate the influence of different structural and sequence features on the modifiability of cysteines. We found several strong structural predictors for redox modification. Sensitive cysteines tend to be characterized by higher exposure, a lack of secondary structure elements, and a high number of positively charged amino acids in their close environment. Our results indicate that modified cysteines tend to occur close to other post-translational modifications, such as phosphorylated serines. We used these features to create models and predict the presence of redox-modifiable cysteines in human mitochondrial complex I as well as make novel predictions regarding redox-sensitive cysteines in proteins.
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http://dx.doi.org/10.1515/hsz-2020-0321DOI Listing
July 2021

Reduced exploratory behavior in neuronal nucleoredoxin knockout mice.

Redox Biol 2021 09 23;45:102054. Epub 2021 Jun 23.

Institute of Clinical Pharmacology, Goethe-University, Medical Faculty, Frankfurt, Germany. Electronic address:

Nucleoredoxin is a thioredoxin-like redoxin that has been recognized as redox modulator of WNT signaling. Using a Yeast-2-Hybrid screen, we identified calcium calmodulin kinase 2a, Camk2a, as a prominent prey in a brain library. Camk2a is crucial for nitric oxide dependent processes of neuronal plasticity of learning and memory. Therefore, the present study assessed functions of NXN in neuronal Nestin-NXN deficient mice. The NXN-Camk2a interaction was confirmed by coimmunoprecipitation, and by colocalization in neuropil and dendritic spines. Functionally, Camk2a activity was reduced in NXN deficient neurons and restored with recombinant NXN. Proteomics revealed reduced oxidation in the hippocampus of Nestin-NXN deficient mice, including Camk2a, further synaptic and mitochondrial proteins, and was associated with a reduction of mitochondrial respiration. Nestin-NXN mice were healthy and behaved normally in behavioral tests of anxiety, activity and sociability. They had no cognitive deficits in touchscreen based learning & memory tasks, but omitted more trials showing a lower interest in the reward. They also engaged less in rewarding voluntary wheel running, and in exploratory behavior in IntelliCages. Accuracy was enhanced owing to the loss of exploration. The data suggested that NXN maintained the oxidative state of Camk2a and thereby its activity. In addition, it supported oxidation of other synaptic and mitochondrial proteins, and mitochondrial respiration. The loss of NXN-dependent pro-oxidative functions manifested in a loss of exploratory drive and reduced interest in reward in behaving mice.
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http://dx.doi.org/10.1016/j.redox.2021.102054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8254043PMC
September 2021

Genetic deletion of Nox4 enhances cancerogen-induced formation of solid tumors.

Proc Natl Acad Sci U S A 2021 03;118(11)

Institute for Cardiovascular Physiology, Goethe University, 60590 Frankfurt am Main, Germany;

Reactive oxygen species (ROS) can cause cellular damage and promote cancer development. Besides such harmful consequences of overproduction of ROS, all cells utilize ROS for signaling purposes and stabilization of cell homeostasis. In particular, the latter is supported by the NADPH oxidase 4 (Nox4) that constitutively produces low amounts of HO By that mechanism, Nox4 forces differentiation of cells and prevents inflammation. We hypothesize a constitutive low level of HO maintains basal activity of cellular surveillance systems and is unlikely to be cancerogenic. Utilizing two different murine models of cancerogen-induced solid tumors, we found that deletion of Nox4 promotes tumor formation and lowers recognition of DNA damage. Nox4 supports phosphorylation of H2AX (γH2AX), a prerequisite of DNA damage recognition, by retaining a sufficiently low abundance of the phosphatase PP2A in the nucleus. The underlying mechanism is continuous oxidation of AKT by Nox4. Interaction of oxidized AKT and PP2A captures the phosphatase in the cytosol. Absence of Nox4 facilitates nuclear PP2A translocation and dephosphorylation of γH2AX. Simultaneously AKT is left phosphorylated. Thus, in the absence of Nox4, DNA damage is not recognized and the increased activity of AKT supports proliferation. The combination of both events results in genomic instability and promotes tumor formation. By identifying Nox4 as a protective source of ROS in cancerogen-induced cancer, we provide a piece of knowledge for understanding the role of moderate production of ROS in preventing the initiation of malignancies.
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http://dx.doi.org/10.1073/pnas.2020152118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980388PMC
March 2021

Sulforaphane exposure impairs contractility and mitochondrial function in three-dimensional engineered heart tissue.

Redox Biol 2021 05 31;41:101951. Epub 2021 Mar 31.

Institute of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany; DZHK (German Center for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany. Electronic address:

Sulforaphane (SFN) is a phytochemical compound extracted from cruciferous plants, like broccoli or cauliflower. Its isothiocyanate group renders SFN reactive, thus allowing post-translational modification of cellular proteins to regulate their function with the potential for biological and therapeutic actions. SFN and stabilized variants recently received regulatory approval for clinical studies in humans for the treatment of neurological disorders and cancer. Potential unwanted side effects of SFN on heart function have not been investigated yet. The present study characterizes the impact of SFN on cardiomyocyte contractile function in cardiac preparations from neonatal rat, adult mouse and human induced-pluripotent stem cell-derived cardiomyocytes. This revealed a SFN-mediated negative inotropic effect, when administered either acutely or chronically, with an impairment of the Frank-Starling response to stretch activation. A direct effect of SFN on myofilament function was excluded in chemically permeabilized mouse trabeculae. However, SFN pretreatment increased lactate formation and enhanced the mitochondrial production of reactive oxygen species accompanied by a significant reduction in the mitochondrial membrane potential. Transmission electron microscopy revealed disturbed sarcomeric organization and inflated mitochondria with whorled membrane shape in response to SFN exposure. Interestingly, administration of the alternative energy source l-glutamine to the medium that bypasses the uptake route of pyruvate into the mitochondrial tricarboxylic acid cycle improved force development in SFN-treated EHTs, suggesting indeed mitochondrial dysfunction as a contributor of SFN-mediated contractile dysfunction. Taken together, the data from the present study suggest that SFN might impact negatively on cardiac contractility in patients with cardiovascular co-morbidities undergoing SFN supplementation therapy. Therefore, cardiac function should be monitored regularly to avoid the onset of cardiotoxic side effects.
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http://dx.doi.org/10.1016/j.redox.2021.101951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056268PMC
May 2021

Nox4-dependent upregulation of S100A4 after peripheral nerve injury modulates neuropathic pain processing.

Free Radic Biol Med 2021 05 28;168:155-167. Epub 2021 Mar 28.

Institute of Pharmacology and Clinical Pharmacy, Goethe University, 60438 Frankfurt am Main, Germany. Electronic address:

Previous studies suggested that reactive oxygen species (ROS) produced by NADPH oxidase 4 (Nox4) affect the processing of neuropathic pain. However, mechanisms underlying Nox4-dependent pain signaling are incompletely understood. In this study, we aimed to identify novel Nox4 downstream interactors in the nociceptive system. Mice lacking Nox4 specifically in sensory neurons were generated by crossing Advillin-Cre mice with Nox4 mice. Tissue-specific deletion of Nox4 in sensory neurons considerably reduced mechanical hypersensitivity and neuronal action potential firing after peripheral nerve injury. Using a proteomic approach, we detected various proteins that are regulated in a Nox4-dependent manner after injury, including the small calcium-binding protein S100A4. Immunofluorescence staining and Western blot experiments confirmed that S100A4 expression is massively up-regulated in peripheral nerves and dorsal root ganglia after injury. Furthermore, mice lacking S100A4 showed increased mechanical hypersensitivity after peripheral nerve injury and after delivery of a ROS donor. Our findings suggest that S100A4 expression is up-regulated after peripheral nerve injury in a Nox4-dependent manner and that deletion of S100A4 leads to an increased neuropathic pain hypersensitivity.
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http://dx.doi.org/10.1016/j.freeradbiomed.2021.03.021DOI Listing
May 2021

Loss of Mitochondrial Protease CLPP Activates Type I IFN Responses through the Mitochondrial DNA-cGAS-STING Signaling Axis.

J Immunol 2021 04 17;206(8):1890-1900. Epub 2021 Mar 17.

Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M University Health Science Center, Bryan, TX 77807;

Caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP) is a serine protease that degrades damaged or misfolded mitochondrial proteins. CLPP-null mice exhibit growth retardation, deafness, and sterility, resembling human Perrault syndrome, but also display immune system alterations. However, the molecular mechanisms and signaling pathways underlying immunological changes in CLPP-null mice remain unclear. In this study, we report the steady-state activation of type I IFN signaling and antiviral gene expression in CLPP-deficient cells and tissues, resulting in marked resistance to RNA and DNA virus infection. Depletion of the cyclic GMP-AMP (cGAS)-stimulator of IFN genes (STING) DNA sensing pathway reduces steady-state IFN-I signaling and abrogates the broad antiviral phenotype of CLPP-null cells. Moreover, we report that CLPP deficiency leads to mitochondrial DNA (mtDNA) instability and packaging alterations. Pharmacological and genetic approaches to deplete mtDNA or inhibit cytosolic release markedly reduce antiviral gene expression, implicating mtDNA stress as the driver of IFN-I signaling in CLPP-null mice. Our work places the cGAS-STING-IFN-I innate immune pathway downstream of CLPP and may have implications for understanding Perrault syndrome and other human diseases involving CLPP dysregulation.
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http://dx.doi.org/10.4049/jimmunol.2001016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026707PMC
April 2021

CEDAR, an online resource for the reporting and exploration of complexome profiling data.

Biochim Biophys Acta Bioenerg 2021 07 17;1862(7):148411. Epub 2021 Mar 17.

Center for Molecular and Biomolecular Informatics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands. Electronic address:

Complexome profiling is an emerging 'omics' approach that systematically interrogates the composition of protein complexes (the complexome) of a sample, by combining biochemical separation of native protein complexes with mass-spectrometry based quantitation proteomics. The resulting fractionation profiles hold comprehensive information on the abundance and composition of the complexome, and have a high potential for reuse by experimental and computational researchers. However, the lack of a central resource that provides access to these data, reported with adequate descriptions and an analysis tool, has limited their reuse. Therefore, we established the ComplexomE profiling DAta Resource (CEDAR, www3.cmbi.umcn.nl/cedar/), an openly accessible database for depositing and exploring mass spectrometry data from complexome profiling studies. Compatibility and reusability of the data is ensured by a standardized data and reporting format containing the "minimum information required for a complexome profiling experiment" (MIACE). The data can be accessed through a user-friendly web interface, as well as programmatically using the REST API portal. Additionally, all complexome profiles available on CEDAR can be inspected directly on the website with the profile viewer tool that allows the detection of correlated profiles and inference of potential complexes. In conclusion, CEDAR is a unique, growing and invaluable resource for the study of protein complex composition and dynamics across biological systems.
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http://dx.doi.org/10.1016/j.bbabio.2021.148411DOI Listing
July 2021

Long Non-coding RNA Aerrie Controls DNA Damage Repair via YBX1 to Maintain Endothelial Cell Function.

Front Cell Dev Biol 2020 11;8:619079. Epub 2021 Jan 11.

Department of Physiology, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands.

Aging is accompanied by many physiological changes. These changes can progressively lead to many types of cardiovascular diseases. During this process blood vessels lose their ability to maintain vascular homeostasis, ultimately resulting in hypertension, stroke, or myocardial infarction. Increase in DNA damage is one of the hallmarks of aging and can be repaired by the DNA signaling and repair system. In our study we show that long non-coding RNA Aerrie (linc01013) contributes to the DNA signaling and repair mechanism. Silencing of Aerrie in endothelial cells impairs angiogenesis, migration, and barrier function. Aerrie associates with YBX1 and together they act as important factors in DNA damage signaling and repair. This study identifies Aerrie as a novel factor in genomic stability and as a binding partner of YBX1 in responding to DNA damage.
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http://dx.doi.org/10.3389/fcell.2020.619079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829583PMC
January 2021

Impaired complex I repair causes recessive Leber's hereditary optic neuropathy.

J Clin Invest 2021 03;131(6)

IRCCS Istituto delle Scienze Neurologiche di Bologna, Bologna, Italy.

Leber's hereditary optic neuropathy (LHON) is the most frequent mitochondrial disease and was the first to be genetically defined by a point mutation in mitochondrial DNA (mtDNA). A molecular diagnosis is achieved in up to 95% of cases, the vast majority of which are accounted for by 3 mutations within mitochondrial complex I subunit-encoding genes in the mtDNA (mtLHON). Here, we resolve the enigma of LHON in the absence of pathogenic mtDNA mutations. We describe biallelic mutations in a nuclear encoded gene, DNAJC30, in 33 unsolved patients from 29 families and establish an autosomal recessive mode of inheritance for LHON (arLHON), which to date has been a prime example of a maternally inherited disorder. Remarkably, all hallmarks of mtLHON were recapitulated, including incomplete penetrance, male predominance, and significant idebenone responsivity. Moreover, by tracking protein turnover in patient-derived cell lines and a DNAJC30-knockout cellular model, we measured reduced turnover of specific complex I N-module subunits and a resultant impairment of complex I function. These results demonstrate that DNAJC30 is a chaperone protein needed for the efficient exchange of complex I subunits exposed to reactive oxygen species and integral to a mitochondrial complex I repair mechanism, thereby providing the first example to our knowledge of a disease resulting from impaired exchange of assembled respiratory chain subunits.
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http://dx.doi.org/10.1172/JCI138267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954600PMC
March 2021

Enriched conditioning expands the regenerative ability of sensory neurons after spinal cord injury via neuronal intrinsic redox signaling.

Nat Commun 2020 12 21;11(1):6425. Epub 2020 Dec 21.

Division of Neuroscience, Department of Brain Sciences, Imperial College London, London, UK.

Overcoming the restricted axonal regenerative ability that limits functional repair following a central nervous system injury remains a challenge. Here we report a regenerative paradigm that we call enriched conditioning, which combines environmental enrichment (EE) followed by a conditioning sciatic nerve axotomy that precedes a spinal cord injury (SCI). Enriched conditioning significantly increases the regenerative ability of dorsal root ganglia (DRG) sensory neurons compared to EE or a conditioning injury alone, propelling axon growth well beyond the spinal injury site. Mechanistically, we established that enriched conditioning relies on the unique neuronal intrinsic signaling axis PKC-STAT3-NADPH oxidase 2 (NOX2), enhancing redox signaling as shown by redox proteomics in DRG. Finally, NOX2 conditional deletion or overexpression respectively blocked or phenocopied enriched conditioning-dependent axon regeneration after SCI leading to improved functional recovery. These studies provide a paradigm that drives the regenerative ability of sensory neurons offering a potential redox-dependent regenerative model for mechanistic and therapeutic discoveries.
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http://dx.doi.org/10.1038/s41467-020-20179-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7752916PMC
December 2020

Mapping the Endothelial Cell -Sulfhydrome Highlights the Crucial Role of Integrin Sulfhydration in Vascular Function.

Circulation 2021 Mar 14;143(9):935-948. Epub 2020 Dec 14.

Institute for Vascular Signalling (S-I.B., J.H., J.W., M.K.D., V.R., F.D.L., B.F., S.Z., A.K., A.F.O.J., I.F.), Goethe University, Frankfurt am Main, Germany.

Background: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (HS), that exert their biological actions via cysteine -sulfhydration of target proteins. This study set out to map the "-sulfhydrome" (ie, the spectrum of proteins targeted by HS) in human endothelial cells.

Methods: Liquid chromatography with tandem mass spectrometry was used to identify -sulfhydrated cysteines in endothelial cell proteins and β3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction.

Results: Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing HS donor, SG1002. The endothelial cell "-sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on β3 integrin in detail we found that -sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the β leg. β3 integrin -sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of -sulfhydration impaired interactions between β3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low HS generation, impaired flow-induced dilatation, and failure to detect β3 integrin -sulfhydration, all of which were rescued after the administration of an HS supplement.

Conclusions: Vascular disease is associated with marked changes in the -sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term HS supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.051877DOI Listing
March 2021

Platelet-derived calpain cleaves the endothelial protease-activated receptor 1 to induce vascular inflammation in diabetes.

Basic Res Cardiol 2020 12 1;115(6):75. Epub 2020 Dec 1.

Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Theodor-Stern-Kai 7, 60596, Frankfurt am Main, Germany.

Diabetes mellitus is a major risk factor for cardiovascular disease. Platelets from diabetic patients are hyperreactive and release microparticles that carry activated cysteine proteases or calpains. Whether platelet-derived calpains contribute to the development of vascular complications in diabetes is unknown. Here we report that platelet-derived calpain1 (CAPN1) cleaves the protease-activated receptor 1 (PAR-1) on the surface of endothelial cells, which then initiates a signaling cascade that includes the activation of the tumor necrosis factor (TNF)-α converting enzyme (TACE). The latter elicits the shedding of the endothelial protein C receptor and the generation of TNF-α, which in turn, induces intracellular adhesion molecule (ICAM)-1 expression to promote monocyte adhesion. All of the effects of CAPN1 were mimicked by platelet-derived microparticles from diabetic patients or from wild-type mice but not from CAPN1 mice, and were not observed in PAR-1-deficient endothelial cells. Importantly, aortae from diabetic mice expressed less PAR-1 but more ICAM-1 than non-diabetic mice, effects that were prevented by treating diabetic mice with a calpain inhibitor as well as by the platelet specific deletion of CAPN1. Thus, platelet-derived CAPN1 contributes to the initiation of the sterile vascular inflammation associated with diabetes via the cleavage of PAR-1 and the release of TNF-α from the endothelial cell surface.
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http://dx.doi.org/10.1007/s00395-020-00833-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716944PMC
December 2020

Mitochondrial Complexome Profiling.

Methods Mol Biol 2021 ;2192:269-285

Functional Proteomics, ZBC, Goethe-University, Frankfurt am Main, Germany.

Complexome profiling combines blue native gel electrophoresis (BNE) and quantitative mass spectrometry to define an entire protein interactome of a cell, an organelle, or a biological membrane preparation. The method allows the identification of protein assemblies with low abundance and detects dynamic processes of protein complex assembly. Applications of complexome profiling range from the determination of complex subunit compositions, assembly of single protein complexes, and supercomplexes to comprehensive differential studies between patients or disease models. This chapter describes the workflow of complexome profiling from sample preparation, mass spectrometry to data analysis with a bioinformatics tool.
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http://dx.doi.org/10.1007/978-1-0716-0834-0_19DOI Listing
March 2021

Characterization of the novel OXA-213-like β-lactamase OXA-822 from Acinetobacter calcoaceticus.

J Antimicrob Chemother 2021 02;76(3):626-634

Institute for Medical Microbiology and Infection Control, Hospital of the Goethe University, Frankfurt am Main, Germany.

Objectives: This study analysed the novel carbapenem-hydrolysing class D β-lactamase OXA-822 identified in the clinical Acinetobacter calcoaceticus isolate AC_2117.

Methods: WGS was employed for identification of β-lactamases. Micro-broth dilution was used for evaluation of antibiotic susceptibility of AC_2117 and transformants containing blaOXA-822. After heterologous purification of OXA-822, OXA-359 and OXA-213, enzyme kinetics were determined using spectrometry. The effect of OXA-822 upon meropenem treatment was analysed in the Galleria mellonella in vivo infection model.

Results: OXA-822 is a member of the intrinsic OXA-213-like family found in A. calcoaceticus and Acinetobacter pittii. Amino acid sequence similarity to the nearest related OXA-359 was 97%. Production of OXA-822, OXA-359 and OXA-213 in Acinetobacter baumannii ATCC® 19606T resulted in elevated MICs for carbapenems (up to 16-fold). Penicillinase activity of the purified OXA-822 revealed high KM values, in the millimolar range, combined with high turnover numbers. OXA-822 showed the highest affinity to carbapenems, but affinity to imipenem was ∼10-fold lower compared with other carbapenems. Molecular modelling revealed that imipenem does not interact with a negatively charged side chain of OXA-822, as doripenem does, leading to the lower affinity. Presence of OXA-822 decreased survival of infected Galleria mellonella larvae after treatment with meropenem. Only 52.7% ± 7.7% of the larvae survived after 24 h compared with 90.9% ± 3.7% survival in the control group.

Conclusions: The novel OXA-822 from a clinical A. calcoaceticus isolate displayed penicillinase and carbapenemase activity in vitro, elevated MICs in different species and decreased carbapenem susceptibility in A. baumannii in vivo.
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http://dx.doi.org/10.1093/jac/dkaa488DOI Listing
February 2021

Bi-allelic pathogenic variants in NDUFC2 cause early-onset Leigh syndrome and stalled biogenesis of complex I.

EMBO Mol Med 2020 11 24;12(11):e12619. Epub 2020 Sep 24.

Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK.

Leigh syndrome is a progressive neurodegenerative disorder, most commonly observed in paediatric mitochondrial disease, and is often associated with pathogenic variants in complex I structural subunits or assembly factors resulting in isolated respiratory chain complex I deficiency. Clinical heterogeneity has been reported, but key diagnostic findings are developmental regression, elevated lactate and characteristic neuroimaging abnormalities. Here, we describe three affected children from two unrelated families who presented with Leigh syndrome due to homozygous variants (c.346_*7del and c.173A>T p.His58Leu) in NDUFC2, encoding a complex I subunit. Biochemical and functional investigation of subjects' fibroblasts confirmed a severe defect in complex I activity, subunit expression and assembly. Lentiviral transduction of subjects' fibroblasts with wild-type NDUFC2 cDNA increased complex I assembly supporting the association of the identified NDUFC2 variants with mitochondrial pathology. Complexome profiling confirmed a loss of NDUFC2 and defective complex I assembly, revealing aberrant assembly intermediates suggestive of stalled biogenesis of the complex I holoenzyme and indicating a crucial role for NDUFC2 in the assembly of the membrane arm of complex I, particularly the ND2 module.
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http://dx.doi.org/10.15252/emmm.202012619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645371PMC
November 2020

MIC26 and MIC27 cooperate to regulate cardiolipin levels and the landscape of OXPHOS complexes.

Life Sci Alliance 2020 10 11;3(10). Epub 2020 Aug 11.

Institute of Biochemistry and Molecular Biology I, Heinrich Heine University Düsseldorf, Medical Faculty, Düsseldorf, Germany

Homologous apolipoproteins of MICOS complex, MIC26 and MIC27, show an antagonistic regulation of their protein levels, making it difficult to deduce their individual functions using a single gene deletion. We obtained single and double knockout (DKO) human cells of and and found that DKO show more concentric onion-like cristae with loss of CJs than any single deletion indicating overlapping roles in formation of CJs. Using a combination of complexome profiling, STED nanoscopy, and blue-native gel electrophoresis, we found that MIC26 and MIC27 are dispensable for the stability and integration of the remaining MICOS subunits into the complex suggesting that they assemble late into the MICOS complex. MIC26 and MIC27 are cooperatively required for the integrity of respiratory chain (super) complexes (RCs/SC) and the FF-ATP synthase complex and integration of F subunits into the monomeric FF-ATP synthase. While cardiolipin was reduced in DKO cells, overexpression of cardiolipin synthase in DKO restores the stability of RCs/SC. Overall, we propose that MIC26 and MIC27 are cooperatively required for global integrity and stability of multimeric OXPHOS complexes by modulating cardiolipin levels.
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http://dx.doi.org/10.26508/lsa.202000711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425215PMC
October 2020

Redox Regulation of PPAR in Polarized Macrophages.

PPAR Res 2020 1;2020:8253831. Epub 2020 Jul 1.

Institute of Biochemistry I, Goethe-University Frankfurt, 60590 Frankfurt am Main, Germany.

The peroxisome proliferator-activated receptor (PPAR) is a central mediator of cellular lipid metabolism and immune cell responses during inflammation. This is facilitated by its role as a transcription factor as well as a DNA-independent protein interaction partner. We addressed how the cellular redox milieu in the cytosol and the nucleus of lipopolysaccharide (LPS)/interferon-- (IFN-) and interleukin-4- (IL4-) polarized macrophages (M) initiates posttranslational modifications of PPAR, that in turn alter its protein function. Using the redox-sensitive GFP2 (roGFP2), we validated oxidizing and reducing conditions following classical and alternative activation of M, while the redox status of PPAR was determined via mass spectrometry. Cysteine residues located in the zinc finger regions (amino acid fragments AA 90-115, AA 116-130, and AA 160-167) of PPAR were highly oxidized, accompanied by phosphorylation of serine 82 in response to LPS/IFN, whereas IL4-stimulation provoked minor serine 82 phosphorylation and less cysteine oxidation, favoring a reductive milieu. Mutating these cysteines to alanine to mimic a redox modification decreased PPAR-dependent reporter gene transactivation supporting a functional shift of PPAR associated with the M phenotype. These data suggest distinct mechanisms for regulating PPAR function based on the redox state of M.
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http://dx.doi.org/10.1155/2020/8253831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350077PMC
July 2020

NADPH oxidase subunit NOXO1 is a target for emphysema treatment in COPD.

Nat Metab 2020 06 8;2(6):532-546. Epub 2020 Jun 8.

Department of Surgery, Justus-Liebig University, Giessen, Germany.

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and death worldwide. Peroxynitrite, formed from nitric oxide, which is derived from inducible nitric oxide synthase, and superoxide, has been implicated in the development of emphysema, but the source of the superoxide was hitherto not characterized. Here, we identify the non-phagocytic NADPH oxidase organizer 1 (NOXO1) as the superoxide source and an essential driver of smoke-induced emphysema and pulmonary hypertension development in mice. NOXO1 is consistently upregulated in two models of lung emphysema, Cybb (also known as NADPH oxidase 2, Nox2)-knockout mice and wild-type mice with tobacco-smoke-induced emphysema, and in human COPD. Noxo1-knockout mice are protected against tobacco-smoke-induced pulmonary hypertension and emphysema. Quantification of superoxide, nitrotyrosine and multiple NOXO1-dependent signalling pathways confirm that peroxynitrite formation from nitric oxide and superoxide is a driver of lung emphysema. Our results suggest that NOXO1 may have potential as a therapeutic target in emphysema.
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http://dx.doi.org/10.1038/s42255-020-0215-8DOI Listing
June 2020

Human platelets are a source of collagen I.

Haematologica 2021 03 1;106(3):899-902. Epub 2021 Mar 1.

Institute for Vascular Signalling, Centre of Molecular Medicine, Goethe University, Frankfurt am Main

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http://dx.doi.org/10.3324/haematol.2020.255612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928007PMC
March 2021

Multiple pathways coordinate assembly of human mitochondrial complex IV and stabilization of respiratory supercomplexes.

EMBO J 2020 07 8;39(14):e103912. Epub 2020 Jun 8.

Instituto de Investigación Hospital 12 de Octubre (i+12), Madrid, Spain.

Mitochondrial respiratory chain complexes I, III, and IV can associate into larger structures termed supercomplexes or respirasomes, thereby generating structural interdependences among the individual complexes yet to be understood. In patients, nonsense mutations in complex IV subunit genes cause severe encephalomyopathies randomly associated with pleiotropic complex I defects. Using complexome profiling and biochemical analyses, we have explored the structural rearrangements of the respiratory chain in human cell lines depleted of the catalytic complex IV subunit COX1 or COX2. In the absence of a functional complex IV holoenzyme, several supercomplex I+III species coexist, which differ in their content of COX subunits and COX7A2L/HIGD2A assembly factors. The incorporation of an atypical COX1-HIGD2A submodule attenuates supercomplex I+III turnover rate, indicating an unexpected molecular adaptation for supercomplexes stabilization that relies on the presence of COX1 independently of holo-complex IV formation. Our data set the basis for complex I structural dependence on complex IV, revealing the co-existence of alternative pathways for the biogenesis of "supercomplex-associated" versus individual complex IV, which could determine physiological adaptations under different stress and disease scenarios.
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http://dx.doi.org/10.15252/embj.2019103912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360963PMC
July 2020

Long non-coding RNA LASSIE regulates shear stress sensing and endothelial barrier function.

Commun Biol 2020 05 26;3(1):265. Epub 2020 May 26.

Dept. of Physiology, Amsterdam Cardiovascular Sciences (ACS), Amsterdam UMC, VU University Medical Center, Amsterdam, The Netherlands.

Blood vessels are constantly exposed to shear stress, a biomechanical force generated by blood flow. Normal shear stress sensing and barrier function are crucial for vascular homeostasis and are controlled by adherens junctions (AJs). Here we show that AJs are stabilized by the shear stress-induced long non-coding RNA LASSIE (linc00520). Silencing of LASSIE in endothelial cells impairs cell survival, cell-cell contacts and cell alignment in the direction of flow. LASSIE associates with junction proteins (e.g. PECAM-1) and the intermediate filament protein nestin, as identified by RNA affinity purification. The AJs component VE-cadherin showed decreased stabilization, due to reduced interaction with nestin and the microtubule cytoskeleton in the absence of LASSIE. This study identifies LASSIE as link between nestin and VE-cadherin, and describes nestin as crucial component in the endothelial response to shear stress. Furthermore, this study indicates that LASSIE regulates barrier function by connecting AJs to the cytoskeleton.
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http://dx.doi.org/10.1038/s42003-020-0987-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251106PMC
May 2020

Loss of mitochondrial ClpP, Lonp1, and Tfam triggers transcriptional induction of Rnf213, a susceptibility factor for moyamoya disease.

Neurogenetics 2020 07 28;21(3):187-203. Epub 2020 Apr 28.

Experimental Neurology, Goethe University Medical School, 60590, Frankfurt am Main, Germany.

Human RNF213, which encodes the protein mysterin, is a known susceptibility gene for moyamoya disease (MMD), a cerebrovascular condition with occlusive lesions and compensatory angiogenesis. Mysterin mutations, together with exposure to environmental trigger factors, lead to an elevated stroke risk since childhood. Mysterin is induced during cell stress, to function as cytosolic AAA+ ATPase and ubiquitylation enzyme. Little knowledge exists, in which context mysterin is needed. Here, we found that genetic ablation of several mitochondrial matrix factors, such as the peptidase ClpP, the transcription factor Tfam, as well as the peptidase and AAA+ ATPase Lonp1, potently induces Rnf213 transcript expression in various organs, in parallel with other components of the innate immune system. Mostly in mouse fibroblasts and human endothelial cells, the Rnf213 levels showed prominent upregulation upon Poly(I:C)-triggered TLR3-mediated responses to dsRNA toxicity, as well as upon interferon gamma treatment. Only partial suppression of Rnf213 induction was achieved by C16 as an antagonist of PKR (dsRNA-dependent protein kinase). Since dysfunctional mitochondria were recently reported to release immune-stimulatory dsRNA into the cytosol, our results suggest that mysterin becomes relevant when mitochondrial dysfunction or infections have triggered RNA-dependent inflammation. Thus, MMD has similarities with vasculopathies that involve altered nucleotide processing, such as Aicardi-Goutières syndrome or systemic lupus erythematosus. Furthermore, in MMD, the low penetrance of RNF213 mutations might be modified by dysfunctions in mitochondria or the TLR3 pathway.
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http://dx.doi.org/10.1007/s10048-020-00609-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283203PMC
July 2020

Aging-regulated anti-apoptotic long non-coding RNA Sarrah augments recovery from acute myocardial infarction.

Nat Commun 2020 04 27;11(1):2039. Epub 2020 Apr 27.

Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam, the Netherlands.

Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.
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http://dx.doi.org/10.1038/s41467-020-15995-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184724PMC
April 2020
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