137 research outputs found
RT-PCR tests for SARS-CoV-2 processed at a large Italian Hospital and false negative results among COVID-19 confirmed cases
No full textGenetics of coenzyme q10 deficiency.
No full textCoenzyme Q10 (CoQ10) is an essential component of eukaryotic cells and is involved in crucial biochemical reactions such as the production of ATP in the mitochondrial respiratory chain, the biosynthesis of pyrimidines, and the modulation of apoptosis. CoQ10 requires at least 13 genes for its biosynthesis. Mutations in these genes cause primary CoQ10 deficiency, a clinically and genetically heterogeneous disorder. To date mutations in 8 genes (PDSS1, PDSS2, COQ2, COQ4, COQ6, ADCK3, ADCK4, and COQ9) have been associated with CoQ10 deficiency presenting with a wide variety of clinical manifestations. Onset can be at virtually any age, although pediatric forms are more common. Symptoms include those typical of respiratory chain disorders (encephalomyopathy, ataxia, lactic acidosis, deafness, retinitis pigmentosa, hypertrophic cardiomyopathy), but some (such as steroid-resistant nephrotic syndrome) are peculiar to this condition. The molecular bases of the clinical diversity of this condition are still unknown. It is of critical importance that physicians promptly recognize these disorders because most patients respond to oral administration of CoQ10
Molecular characterization of the human COQ5 C-methyltransferase in coenzyme Q10 biosynthesis.
No full textCoq5 catalyzes the only C-methylation involved in the biosynthesis of coenzymeQ(Q or ubiquinone) in humans and
yeast Saccharomyces cerevisiae. As one of eleven polypeptides required for Q production in yeast, Coq5 has also been
shown to assemble with the multi-subunit complex termed the CoQ-synthome. In humans, mutations in several
COQ genes cause primary Q deficiency, and a decrease in Q biosynthesis is associated withmitochondrial, cardiovascular,
kidney and neurodegenerative diseases. In this study, we characterize the human COQ5 polypeptide and examine
its complementation of yeast coq5 point and null mutants.Weshow that human COQ5 RNA is expressed in all
tissues and that the COQ5 polypeptide is associatedwith the mitochondrial innermembrane on thematrix side. Previouswork
inyeasthas shownthatpointmutations within or adjacent to conserved COQ5 methyltransferasemotifs
result in a loss of Coq5 function but not Coq5 steady state levels. Here, we show that stabilization of the CoQsynthome
within coq5 pointmutants or by over-expression of COQ8 in coq5 nullmutants permits the human COQ5 homolog
to partially restore coq5 mutant growth on respiratory media and Q6 content. Immunoblotting against the
human COQ5 polypeptide in isolated yeast mitochondria shows that the human Coq5 polypeptide migrates in twodimensional
blue-native/SDS-PAGE at the same high molecularmass as other yeast Coq proteins. The results presented
suggest that human and Escherichia coli Coq5 homologs expressed in yeast retain C-methyltransferase activity but are
capable of rescuing the coq5 yeast mutants only when the CoQ-synthome is assembled
Chemically driven tetragonal-to-monoclinic polymorphic transformation in retrieved ZTA femoral heads from dual mobility hip implants
No full textTwo short-term (two and nine months) retrieved zirconia-toughened alumina (ZTA) femoral heads and nine pristine femoral heads from the same manufacturer have been investigated with respect to their surface stability by means of confocal Raman spectroscopy. Quantitative estimations of monoclinic volume fraction have been carried out in both non-wear and main wear zones of the retrieved heads, which invariantly showed high volume fractions of monoclinic polymorph. In-depth (sub-surface) profiles, non-destructively collected in the main wear zones with the Raman probe in confocal configuration, indeed confirmed that polymorphic transformation was extended down to 100 mu m below the bearing surface of the femoral heads. Acceleration of tetragonal-to-monoclinic transformation rate leads to unexpectedly high fractions of monoclinic phase within very short-term in-vivo exposures. Phase transformation in-vivo is much more marked than what one could actually predict according to simply simulating a hydrothermal environment in-vitro and could not be simply ascribed to the mechanical stress fields generated during normal service at the bearing surface. Instead, the chemical consequences of metal contamination on the ZTA femoral head surface are shown to play the most detrimental role in phase destabilization. (C) 2015 Elsevier Ltd. All rights reserved
COX16 is required for assembly of cytochrome c oxidase in human cells and is involved in copper delivery to COX2
No full textCytochrome c oxidase (COX), complex IV of the mitochondrial respiratory chain, is comprised of 14 structural subunits, several prosthetic groups and metal cofactors, among which copper. Its biosynthesis involves a number of ancillary proteins, encoded by the COX-assembly genes that are required for the stabilization and membrane insertion of the nascent polypeptides, the synthesis of the prosthetic groups, and the delivery of the metal cofactors, in particular of copper. Recently, a modular model for COX assembly has been proposed, based on the sequential incorporation of different assembly modules formed by specific subunits. We have cloned and characterized the human homologue of yeast COX16. We show that human COX16 encodes a small mitochondrial transmembrane protein that faces the intermembrane space and is highly expressed in skeletal and cardiac muscle. Its knockdown in C. elegans produces COX deficiency, and its ablation in HEK293 cells impairs COX assembly. Interestingly, COX16 knockout cells retain significant COX activity, suggesting that the function of COX16 is partially redundant. Analysis of steady-state levels of COX subunits and of assembly intermediates by Blue-Native gels shows a pattern similar to that reported in cells lacking COX18, suggesting that COX16 is required for the formation of the COX2 subassembly module. Moreover, COX16 co-immunoprecipitates with COX2. Finally, we found that copper supplementation increases COX activity and restores normal steady state levels of COX subunits in COX16 knockout cells, indicating that, even in the absence of a canonical copper binding motif, COX16 could be involved in copper delivery to COX2
High viral load in upper respiratory specimens of persons with SARS-CoV-2 Infection in a Northern Italian Area
No full textBackground: An assessment of viral load in biologic specimens of subjects with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may have important implications for public health planning. The aim of this study was to estimate the prevalence of high viral load in upper respiratory specimens of patients with SARS-CoV-2 infection during the first Italian wave (spring) and at the beginning of the second wave (summer) of the COVID-19 epidemic, through themeasurement of cycle threshold (Ct) values from real-Time reverse transcription-polymerase chain reaction tests conducted at the University Hospital of Udine, Italy, serving 530,000 inhabitants. Methods: We compared the prevalence of high viral load, defined as Ct < 20 at the first positive test result, in 262 subjects from the spring and 453 from the summer period. Logistic regression was used to account for potential confounding due to sex, age, and severity of infection. Results: In the spring, 9.2% of subjects had Ct < 20 versus 21.4% in the summer. After adjusting for confounders, the likelihood of having high viral load was 2.9 times higher in the summer than in the spring (95% confidence interval, 1.7 5.0). Conclusions: In this Italian area,more COVID-19 patients had high viral load in the spring epidemic wave than at the beginning of the second, during the summer. Cycle threshold values may represent useful information to monitor viral load at a population level in subjects with SARSCoV-2 infection
Copper and bezafibrate cooperate to rescue cytochrome c oxidase deficiency in cells of patients with sco2 mutations.
No full textBACKGROUND:
Mutations in SCO2 cause cytochrome c oxidase deficiency (COX) and a fatal infantile cardioencephalomyopathy. SCO2 encodes a protein involved in COX copper metabolism; supplementation with copper salts rescues the defect in patients' cells. Bezafibrate (BZF), an approved hypolipidemic agent, ameliorates the COX deficiency in mice with mutations in COX10, another COX-assembly gene.
METHODS:
We have investigated the effect of BZF and copper in cells with SCO2 mutations using spectrophotometric methods to analyse respiratory chain activities and a luciferase assay to measure ATP production..
RESULTS:
Individual mitochondrial enzymes displayed different responses to BZF. COX activity increased by about 40% above basal levels (both in controls and patients), with SCO2 cells reaching 75-80% COX activity compared to untreated controls. The increase in COX was paralleled by an increase in ATP production. The effect was dose-dependent: it was negligible with 100 uM BZF, and peaked at 400 uM BZF. Higher BZF concentrations were associated with a relative decline of COX activity, indicating that the therapeutic range of this drug is very narrow. Combined treatment with 100 uM CuCl2 and 200 uM BZF (which are only marginally effective when administered individually) achieved complete rescue of COX activity in SCO2 cells.
CONCLUSIONS:
These data are crucial to design therapeutic trials for this otherwise fatal disorder. The additive effect of copper and BZF will allow to employ lower doses of each drug and to reduce their potential toxic effects. The exact mechanism of action of BZF remains to be determined
Pontocerebellar hypoplasia type 6 caused by mutations in RARS2: definition of the clinical spectrum and molecular findings in five patients
No full textRecessive mutations in the mitochondrial arginyltransfer
RNA synthetase (RARS2) gene have been associated
with early onset encephalopathy with signs of oxidative
phosphorylation defects classified as pontocerebellar
hypoplasia 6. We describe clinical, neuroimaging and molecular
features on five patients from three unrelated families
who displayed mutations in RARS2. All patients rapidly developed
a neonatal or early-infantile epileptic encephalopathy
with intractable seizures. The long-term follow-up revealed a
virtual absence of psychomotor development, progressive
microcephaly, and feeding difficulties. Mitochondrial respiratory
chain enzymes in muscle and fibroblasts were normal in
two. Blood and CSF lactate was abnormally elevated in all
five patients at early stages while appearing only occasionally
abnormal with the progression of the disease. Cerebellar vermis
hypoplasia with normal aspect of the cerebral and cerebellar
hemispheres appeared within the first months of life at
brain MRI. In three patients follow-up neuroimaging revealed
a progressive pontocerebellar and cerebral cortical atrophy.
Molecular investigations of RARS2 disclosed the c.25A>G/
p.I9V and the c.1586+3A>T in family A, the c.734G>A/
p.R245Q and the c.1406G>A/p.R469H in family B, and the
c.721T>A/p.W241R and c.35A>G/p.Q12R in family C.
Functional complementation studies in Saccharomyces
cerevisiae showed that mutation MSR1-R531H (equivalent
to human p.R469H) abolished respiration whereas the MSR1-
R306Q strain (corresponding to p.R245Q) displayed a reduced
growth on non-fermentable YPG medium. Although
mutations functionally disrupted yeast we found a relatively
well preserved arginine aminoacylation of mitochondrial
tRNA. Clinical and neuroimaging findings are important clues
to raise suspicion and to reach diagnostic accuracy for RARS2
mutations considering that biochemical abnormalities may be
absent in muscle biopsy
Functional Analysis of Missense Mutations of OAT, Causing Gyrate Atrophy of Choroid and Retina.
No full textWe studied eight kindreds with gyrate atrophy of choroid and retina (GA), a rare autosomal recessive disorder caused by mutations of the OAT gene, encoding the homoexameric enzyme ornithine-delta-aminotransferase. We identified four novel and five previously reported mutations. Missense alleles were expressed in yeast strain carrying a deletion of the orthologous of human OAT. All mutations markedly reduced enzymatic activity. However, the effect on the yeast growth was variable, suggesting that some mutations retain residual activity, below the threshold of the enzymatic assay. Mutant proteins were either highly unstable and rapidly degraded, or failed to assemble to form the active OAT hexamer. Where possible, fibroblast analysis confirmed these data. We found no correlation between the residual enzymatic activity and the age of onset, or the severity of symptoms. Moreover, the response to B6 was apparently not related to the specific mutations carried by patients. Overall these data suggest that other factors besides the specific OAT genotype modulate (GA) phenotype in patients. Finally, we found that 5-aminoimidazole-4-carboxamide ribonucleoside, an AMPK activator known to increase mitochondrial biogenesis, markedly stimulates OAT expression, thus representing a possible treatment for a subset of GA patients with hypomorphic alleles
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