Using a quantitative quadruple immunofluorescent assay to diagnose isolated mitochondrial Complex I deficiency

Isolated Complex I (CI) deficiency is the most commonly observed mitochondrial respiratory chain biochemical defect, affecting the largest OXPHOS component. CI is genetically heterogeneous; pathogenic variants affect one of 38 nuclear-encoded subunits, 7 mitochondrial DNA (mtDNA)-encoded subunits or 14 known CI assembly factors. The laboratory diagnosis relies on the spectrophotometric assay of enzyme activity in mitochondrially-enriched tissue homogenates, requiring at least 50 mg skeletal muscle, as there is no reliable histochemical method for assessing CI activity directly in tissue cryosections. We have assessed a validated quadruple immunofluorescent OXPHOS (IHC) assay to detect CI deficiency in the diagnostic setting, using 10 µm transverse muscle sections from 25 patients with genetically-proven pathogenic CI variants. We observed loss of NDUFB8 immunoreactivity in all patients with mutations affecting nuclear-encoding structural subunits and assembly factors, whilst only 3 of the 10 patients with mutations affecting mtDNA-encoded structural subunits showed loss of NDUFB8, confirmed by BN-PAGE analysis of CI assembly and IHC using an alternative, commercially-available CI (NDUFS3) antibody. The IHC assay has clear diagnostic potential to identify patients with a CI defect of Mendelian origins, whilst highlighting the necessity of complete mitochondrial genome sequencing in the diagnostic work-up of patients with suspected mitochondrial disease

A recessive homozygous p.Asp92Gly SDHD mutation causes prenatal cardiomyopathy and a severe mitochondrial complex II deficiency

Succinate dehydrogenase (SDH) is a crucial metabolic enzyme complex that is involved in ATP production, playing roles in both the tricarboxylic cycle and the mitochondrial respiratory chain (complex II). Isolated complex II deficiency is one of the rarest oxidative phosphorylation disorders with mutations described in three structural subunits and one of the assembly factors; just one case is attributed to recessively inherited SDHD mutations. We report the pathological, biochemical, histochemical and molecular genetic investigations of a male neonate who had left ventricular hypertrophy detected on antenatal scan and died on day one of life. Subsequent postmortem examination confirmed hypertrophic cardiomyopathy with left ventricular non-compaction. Biochemical analysis of his skeletal muscle biopsy revealed evidence of a severe isolated complex II deficiency and candidate gene sequencing revealed a novel homozygous c.275A>G, p.(Asp92Gly) SDHD mutation which was shown to be recessively inherited through segregation studies. The affected amino acid has been reported as a Dutch founder mutation p.(Asp92Tyr) in families with hereditary head and neck paraganglioma. By introducing both mutations into Saccharomyces cerevisiae, we were able to confirm that the p.(Asp92Gly) mutation causes a more severe oxidative growth phenotype than the p.(Asp92Tyr) mutant, and provides functional evidence to support the pathogenicity of the patient’s SDHD mutation. This is only the second case of mitochondrial complex II deficiency due to inherited SDHD mutations and highlights the importance of sequencing all SDH genes in patients with biochemical and histochemical evidence of isolated mitochondrial complex II deficiency

What happens if you single out? An experiment

We present an experiment investigating the effects of singling out an individual on trust and trustworthiness. We find that (a) trustworthiness falls if there is a singled out subject; (b) non-singled out subjects discriminate against the singled out subject when they are not responsible of the distinct status of this person; (c) under a negative frame, the singled out subject returns significantly less; (d) under a positive frame, the singled out subject behaves bimodally, either selecting very low or very high return rates. Overall, singling out induces a negligible effect on trust but is potentially disruptive for trustworthiness

Early‐onset coenzyme Q10 deficiency associated with ataxia and respiratory chain dysfunction due to novel pathogenic COQ8A variants, including a large intragenic deletion

Coenzyme Q10 (CoQ10) deficiency is a clinically and genetically heterogeneous subtype of mitochondrial disease. We report two girls with ataxia and mitochondrial respiratory chain deficiency who were shown to have primary CoQ10 deficiency. Muscle histochemistry displayed signs of mitochondrial dysfunction—ragged red fibers, mitochondrial paracrystalline inclusions, and lipid deposits while biochemical analyses revealed complex II+III respiratory chain deficiencies. MRI brain demonstrated cerebral and cerebellar atrophy. Targeted molecular analysis identified a homozygous c.1015G>A, p.(Ala339Thr) COQ8A variant in subject 1, while subject 2 was found to harbor a single heterozygous c.1029_1030delinsCA variant predicting a p.Gln343_Val344delinsHisMet amino acid substitution. Subsequent investigations identified a large‐scale COQ8A deletion in trans to the c.1029_1030delinsCA allele. A skin biopsy facilitated cDNA studies that confirmed exon skipping in the fibroblast derived COQ8A mRNA transcript. This report expands the molecular genetic spectrum associated with COQ8A ‐related mitochondrial disease and highlights the importance of thorough investigation of candidate pathogenic variants to establish phase. Rapid diagnosis is of the utmost importance as patients may benefit from therapeutic CoQ10 supplementation

Cisplatin-induced emesis: systematic review and meta-analysis of the ferret model and the effects of 5-HT3 receptor antagonists

PURPOSE: The ferret cisplatin emesis model has been used for ~30 years and enabled identification of clinically used anti-emetics. We provide an objective assessment of this model including efficacy of 5-HT(3) receptor antagonists to assess its translational validity. METHODS: A systematic review identified available evidence and was used to perform meta-analyses. RESULTS: Of 182 potentially relevant publications, 115 reported cisplatin-induced emesis in ferrets and 68 were included in the analysis. The majority (n = 53) used a 10 mg kg(−1) dose to induce acute emesis, which peaked after 2 h. More recent studies (n = 11) also used 5 mg kg(−1), which induced a biphasic response peaking at 12 h and 48 h. Overall, 5-HT(3) receptor antagonists reduced cisplatin (5 mg kg(−1)) emesis by 68% (45–91%) during the acute phase (day 1) and by 67% (48–86%) and 53% (38–68%, all P < 0.001), during the delayed phase (days 2, 3). In an analysis focused on the acute phase, the efficacy of ondansetron was dependent on the dosage and observation period but not on the dose of cisplatin. CONCLUSION: Our analysis enabled novel findings to be extracted from the literature including factors which may impact on the applicability of preclinical results to humans. It reveals that the efficacy of ondansetron is similar against low and high doses of cisplatin. Additionally, we showed that 5-HT(3) receptor antagonists have a similar efficacy during acute and delayed emesis, which provides a novel insight into the pharmacology of delayed emesis in the ferret

Internal lipid synthesis and vesicle growth as a step toward self-reproduction of the minimal cell

One of the major properties of the semi-synthetic minimal cell, as a model for early living cells, is the ability to self-reproduce itself, and the reproduction of the boundary layer or vesicle compartment is part of this process. A minimal bio-molecular mechanism based on the activity of one single enzyme, the FAS-B (Fatty Acid Synthase) Type I enzyme from Brevibacterium ammoniagenes, is encapsulated in 1-palmitoyl-2oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes to control lipid synthesis. Consequently molecules of palmitic acid released from the FAS catalysis, within the internal lumen, move toward the membrane compartment and become incorporated into the phospholipid bilayer. As a result the vesicle membranes change in lipid composition and liposome growth can be monitored. Here we report the first experiments showing vesicles growth by catalysis of one enzyme only that produces cell boundary from within. This is the prototype of the simplest autopoietic minimal cell

Home-based voluntary HIV counselling and testing found highly acceptable and to reduce inequalities

<p>Abstract</p> <p>Background</p> <p>Low uptake of voluntary HIV counselling and testing (VCT) in sub-Saharan Africa is raising acceptability concerns which might be associated with ways by which it is offered. We investigated the acceptability of home-based delivery of counselling and HIV testing in urban and rural populations in Zambia where VCT has been offered mostly from local clinics.</p> <p>Methods</p> <p>A population-based HIV survey was conducted in selected communities in 2003 (n = 5035). All participants stating willingness to be HIV tested were offered VCT at home and all counselling was conducted in the participants' homes. In the urban area post-test counselling and giving of results were done the following day whereas in rural areas this could take 1-3 weeks.</p> <p>Results</p> <p>Of those who indicated willingness to be HIV tested, 76.1% (95%CI 74.9-77.2) were counselled and received the test result. Overall, there was an increase in the proportion ever HIV tested from 18% before provision of home-based VCT to 38% after. The highest increase was in rural areas; among young rural men aged 15-24 years up from 14% to 42% vs. for urban men from 17% to 37%. Test rates by educational attainment changed from being positively associated to be evenly distributed after home-based VCT.</p> <p>Conclusions</p> <p>A high uptake was achieved by delivering HIV counselling and testing at home. The highest uptakes were seen in rural areas, in young people and groups with low educational attainment, resulting in substantial reductions in existing inequalities in accessing VCT services.</p