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| 2. |
- Grandfield, Kathryn, et al.
(författare)
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Bone response to free form fabricated hydroxyapatite and zirconia scaffolds : a transmission electron microscopy study in the human maxilla
- 2012
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Ingår i: Clinical Implant Dentistry and Related Research. - : Wiley. - 1523-0899 .- 1708-8208. ; 14:3, s. 461-469
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Tidskriftsartikel (refereegranskat)abstract
- Background: Understanding the interfacial reactions to synthetic bone regenerative scaffolds in vivo is fundamental for improving osseointegration and osteogenesis. Using transmission electron microscopy, it is possible to study the biological response of hydroxyapatite (HA) and zirconia (ZrO2) scaffolds at the nanometer scale.Purpose: In this study, the bone-bonding abilities of HA and ZrO2 scaffolds produced by free-form fabrication were evaluated in the human maxilla at 3 months and 7 months.Materials and Methods: HA and ZrO2 scaffolds (ø: 3 mm) were implanted in the human maxilla, removed with surrounding bone, embedded in resin, and sectioned. A novel focused ion beam (FIB) sample preparation technique enabled the production of thin lamellae for study by scanning transmission electron microscopy.Results: Interface regions were investigated using high-angle annular dark-field imaging and energy-dispersive X-ray spectroscopy analysis. Interfacial apatite layers of 80 nm and 50 nm thickness were noted in the 3- and 7-month HA samples, respectively, and bone growth was discovered in micropores up to 10 µm into the samples.Conclusions: The absence of an interfacial layer in the ZrO2 samples suggest the formation of a direct contact with bone, while HA, which bonds through an apatite layer, shows indications of resorption with increasing implantation time. This study demonstrates the potential of HA and ZrO2 scaffolds for use as bone regenerative materials.
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| 3. |
- Oldfors Hedberg, Carola, 1969, et al.
(författare)
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COX deficiency and leukoencephalopathy due to a novel homozygous APOPT1/COA8 mutation
- 2020
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Ingår i: Neurology-Genetics. - : Ovid Technologies (Wolters Kluwer Health). - 2376-7839. ; 6:4
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Tidskriftsartikel (refereegranskat)abstract
- Objective To describe the long-term follow-up and pathogenesis in a child with leukoencephalopathy and cytochrome c oxidase (COX) deficiency due to a novel homozygous nonsense mutation in APOPT1/COA8. Methods The patient was clinically investigated at 3, 5, 9, and 25 years of age. Brain MRI, repeat muscle biopsies with biochemical, morphologic, and protein expression analyses were performed, and whole-genome sequencing was used for genetic analysis. Results Clinical investigation revealed dysarthria, dysphagia, and muscle weakness following pneumonia at age 3 years. There was clinical regression leading to severe loss of ambulation, speech, swallowing, hearing, and vision. The clinical course stabilized after 2.5 years and improved over time. The MRI pattern in the patient demonstrated cavitating leukoencephalopathy, and muscle mitochondrial investigations showed COX deficiency with loss of complex IV subunits and ultrastructural abnormalities. Genetic analysis revealed a novel homozygous mutation in the APOPT1/COA8 gene, c.310T>C; p.(Gln104*). Conclusions We describe a novel nonsense mutation in APOPT1/COA8 and provide additional experimental evidence for a COX assembly defect in human muscle causing the complex IV deficiency. The long-term outcome of the disease seems in general to be favorable, and the characteristic MRI pattern with cavitating leukoencephalopathy in combination with COX deficiency should prompt for testing of the APOPT1/COA8 gene.
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| 4. |
- Oldfors Hedberg, Carola, 1969, et al.
(författare)
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Respiratory chain dysfunction in perifascicular muscle fibres in patients with dermatomyositis is associated with mitochondrial DNA depletion
- 2022
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Ingår i: Neuropathology and Applied Neurobiology. - : Wiley. - 0305-1846 .- 1365-2990. ; 48:7
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Tidskriftsartikel (refereegranskat)abstract
- Aims Patients with dermatomyositis (DM) suffer from reduced aerobic metabolism contributing to impaired muscle function, which has been linked to cytochrome c oxidase (COX) deficiency in muscle tissue. This mitochondrial respiratory chain dysfunction is typically seen in perifascicular regions, which also show the most intense inflammatory reaction along with capillary loss and muscle fibre atrophy. The objective of this study was to investigate the pathobiology of the oxidative phosphorylation deficiency in DM. Methods Muscle biopsy specimens with perifascicular COX deficiency from five juveniles and seven adults with DM were investigated. We combined immunohistochemical analyses of subunits in the respiratory chain including complex I (subunit NDUFB8), complex II (succinate dehydrogenase, subunit SDHB) and complex IV (COX, subunit MTCO1) with in situ hybridisation, next generation deep sequencing and quantitative polymerase chain reaction (PCR). Results There was a profound deficiency of complexes I and IV in the perifascicular regions with enzyme histochemical COX deficiency, whereas succinate dehydrogenase activity and complex II were preserved. In situ hybridisation of mitochondrial RNA showed depletion of mitochondrial DNA (mtDNA) transcripts in the perifascicular regions. Analysis of mtDNA by next generation deep sequencing and quantitative PCR in affected muscle regions showed an overall reduction of mtDNA copy number particularly in the perifascicular regions. Conclusion The respiratory chain dysfunction in DM muscle is associated with mtDNA depletion causing deficiency of complexes I and IV, which are partially encoded by mtDNA, whereas complex II, which is entirely encoded by nuclear DNA, is preserved. The depletion of mtDNA indicates a perturbed replication of mtDNA explaining the muscle pathology and the disturbed aerobic metabolism.
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| 5. |
- Roos, Sara, 1979, et al.
(författare)
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Mitochondrial complex IV deficiency caused by a novel frameshift variant in MT-CO2 associated with myopathy and perturbed acylcarnitine profile
- 2019
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Ingår i: European Journal of Human Genetics. - : Springer Science and Business Media LLC. - 1018-4813 .- 1476-5438. ; 27:2, s. 331-335
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondrial myopathies are a heterogeneous group of disorders associated with a wide range of clinical phenotypes. We present a 16-year-old girl with a history of exercise intolerance since childhood. Acylcarnitine species suggestive of multiple acyl-CoA dehydrogenase deficiency were found in serum, however genetic analysis did not reveal variants in genes associated with this disorder. Biochemical analyses of skeletal muscle mitochondria revealed an isolated and extremely low activity of cytochrome c oxidase (COX). This finding was confirmed by enzyme histochemistry, which demonstrated an almost complete absence of fibers with normal COX activity. Whole-exome sequencing revealed a single base-pair deletion (m.8088delT) in MT-CO2, which encodes subunit 2 of COX, resulting in a premature stop codon. Restriction fragment length polymorphism-analysis confirmed mtDNA heteroplasmy with high mutant load in skeletal muscle, the only clinically affected tissue, but low levels in other investigated tissues. Single muscle fiber analysis showed segregation of the mutant genotype with respiratory chain dysfunction. Immuno-histochemical studies indicated that the truncating variant in COX2 has an inhibitory effect on the assembly of the COX holoenzyme.
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| 6. |
- Svec, David, et al.
(författare)
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Identification of inhibitors regulating cell proliferation and FUS-DDIT3 expression in myxoid liposarcoma using combined DNA, mRNA, and protein analyses.
- 2018
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Ingår i: Laboratory investigation; a journal of technical methods and pathology. - : Elsevier BV. - 1530-0307. ; 98, s. 957-967
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Tidskriftsartikel (refereegranskat)abstract
- FUS-DDIT3 belongs to the FET (FUS, EWSR1, and TAF15) family of fusion oncogenes, which collectively are considered to be key players in tumor development. Even though over 90% of all myxoid liposarcomas (MLS) have a FUS-DDIT3 gene fusion, there is limited understanding of the signaling pathways that regulate its expression. In order to study cell proliferation and FUS-DDIT3 regulation at mRNA and protein levels, we first developed a direct cell lysis approach that allows DNA, mRNA, and protein to be analyzed in the same sample using quantitative PCR, reverse transcription quantitative qPCR and proximity ligation assay, respectively. We screened 70 well-characterized kinase inhibitors and determined their effects on cell proliferation and expression of FUS-DDIT3 and FUS at both mRNA and protein levels in the MLS 402-91 cell line, where twelve selected inhibitors were evaluated further in two additional MLS cell lines. Both FUS-DDIT3 and FUS mRNA expression correlated with cell proliferation and both transcripts were co-regulated in most conditions, indicating that the common 5' FUS promotor is important in transcriptional regulation. In contrast, FUS-DDIT3 and FUS protein levels displayed more cell line dependent expression. Furthermore, most JAK inhibitors caused FUS-DDIT3 downregulation at both mRNA and protein levels. In conclusion, defining factors that regulate FUS-DDIT3 expression opens new means to understand MLS development at the molecular level.
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| 7. |
- Thomsen, Christer, 1977, et al.
(författare)
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Proteomic characterisation of polyglucosan bodies in skeletal muscle in RBCK1 deficiency
- 2022
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Ingår i: Neuropathology and Applied Neurobiology. - : Wiley. - 0305-1846 .- 1365-2990. ; 48:1
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Tidskriftsartikel (refereegranskat)abstract
- Aims Several neurodegenerative and neuromuscular disorders are characterised by storage of polyglucosan, consisting of proteins and amylopectin-like polysaccharides, which are less branched than in normal glycogen. Such diseases include Lafora disease, branching enzyme deficiency, glycogenin-1 deficiency, polyglucosan body myopathy type 1 (PGBM1) due to RBCK1 deficiency and others. The protein composition of polyglucosan bodies is largely unknown. Methods We combined quantitative mass spectrometry, immunohistochemical and western blot analyses to identify the principal protein components of polyglucosan bodies in PGBM1. Histologically stained tissue sections of skeletal muscle from four patients were used to isolate polyglucosan deposits and control regions by laser microdissection. Prior to mass spectrometry, samples were labelled with tandem mass tags that enable quantitative comparison and multiplexed analysis of dissected samples. To study the distribution and expression of the accumulated proteins, immunohistochemical and western blot analyses were performed. Results Accumulated proteins were mainly components of glycogen metabolism and protein quality control pathways. The majority of fibres showed depletion of glycogen and redistribution of key enzymes of glycogen metabolism to the polyglucosan bodies. The polyglucosan bodies also showed accumulation of proteins involved in the ubiquitin-proteasome and autophagocytosis systems and protein chaperones. Conclusions The sequestration of key enzymes of glycogen metabolism to the polyglucosan bodies may explain the glycogen depletion in the fibres and muscle function impairment. The accumulation of components of the protein quality control systems and other proteins frequently found in protein aggregate disorders indicates that protein aggregation may be an essential part of the pathobiology of polyglucosan storage.
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| 8. |
- Visuttijai, Kittichate, et al.
(författare)
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Glycogenin is dispensable for glycogen synthesis in human muscle and glycogenin deficiency causes polyglucosan storage.
- 2020
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Ingår i: The Journal of clinical endocrinology and metabolism. - : The Endocrine Society. - 1945-7197 .- 0021-972X. ; 105:2, s. 557-566
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Tidskriftsartikel (refereegranskat)abstract
- To investigate the importance of glycogenin-1 and glycogenin-2 for glycogen synthesis in skeletal and cardiac muscle.Glycogenin-1 and glycogenin-2 expression was analyzed by western blot, mass spectrometry, and immunohistochemistry in liver, heart, and skeletal muscle from controls and in skeletal and cardiac muscle from patients with glycogenin-1 deficiency.Both glycogenin-1 and glycogenin-2 were found to be expressed in liver, but only glycogenin-1 was identified in heart and skeletal muscle from controls. In patients with truncating GYG1 mutations, neither glycogenin-1 nor glycogenin-2 was expressed in skeletal muscle. However, non-functional glycogenin-1 but not glycogenin-2 was identified in cardiac muscle from patients with cardiomyopathy due to GYG1 missense mutations. By immunohistochemistry, the mutated glycogenin-1 co-localized with the storage of glycogen and polyglucosan in cardiomyocytes.Glycogen can be synthesised in the absence of glycogenin, and glycogenin-1 deficiency is not compensated for by upregulation of functional glycogenin-2. Absence of glycogenin-1 leads to focal accumulation of glycogen and polyglucosan in skeletal muscle fibers. Expression of mutated glycogenin-1 in the heart is deleterious, and it leads to storage of abnormal glycogen and cardiomyopathy.
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