bims-cytox1 2018-07-01 papers

Issue of 2018–07–01
three papers selected by
Gavin McStay, Staffordshire University

J Mol Diagn. 2018 Jun 21. pii: S1525-1578(17)30491-9. [Epub ahead of print]

A Monochrome Multiplex qPCR Assay for the Measurement of Mitochondrial DNA Content.

Anthony Y Y Hsieh, Matthew Budd, David Deng, Izabella Gadawska, Hélène C F Côté.

Mitochondrial DNA copies per cell (mtDNA content) can fluctuate with cellular aging, oxidative stress, and mitochondrial dysfunction, and has been investigated in cancer, diabetes, Human immunodeficiency virus, and metabolic disease. mtDNA content testing in both clinical and basic settings is expected to rise as research uncovers its biological relevance. Here, we present a novel mtDNA content assay developed on monochrome, multiplex qPCR (MMqPCR) principles. This assay offers a >2-fold improvement on time- and cost-effectiveness over conventional (monoplex) qPCR, as well as improved reproducibility given the reduced effects of human pipetting errors. The new MMqPCR method was compared with the gold standard monoplex qPCR assay on DNA from a variety of sources, including human whole blood, skeletal muscle, and commercial cell lines. The MMqPCR assay is reproducible (n=98, r=0.99, P < 0.0001) and highly correlated to the monoplex qPCR assay (n=160, r>0.98, P < 0.0001). Intra- and inter-assay variabilities, as established independently by multiple operators, range between 4.3% to 7.9% and 2.9% to 9.2%, respectively. This robust assay can quantify >82 pg of template DNA per reaction, with a minimum mtDNA:nuclear DNA ratio of 20, and is especially suitable for studies that require high throughput.

DOI: https://doi.org/10.1016/j.jmoldx.2018.05.001

Biochim Biophys Acta. 2018 Jun 20. pii: S1388-1981(18)30139-2. [Epub ahead of print]

Genetic re-engineering of polyunsaturated phospholipid profile of Saccharomyces cerevisiae identifies a novel role for Cld1 in mitigating the effects of cardiolipin peroxidation.

Wenjia Lou, Hsiu-Chi Ting, Christian jA Reynolds, Yulia Y Tyurina, Vladimir A Tyurin, Yiran Li, Jiajia Ji, Wenxi Yu, Zhuqing Liang, Detcho A Stoyanovsky, Tamil S Anthonymuthu, Michael A Frasso, Peter Wipf, Joel S Greenberger, Hülya Bayir, Valerian E Kagan, Miriam L Greenberg.

Cardiolipin (CL) is a unique phospholipid localized almost exclusively within the mitochondrial membranes where it is synthesized. Newly synthesized CL undergoes acyl remodeling to produce CL species enriched with unsaturated acyl groups. Cld1 is the only identified CL specific phospholipase in yeast and is required to initiate the CL remodeling pathway. In higher eukaryotes, peroxidation of CL, yielding CLOX, has been implicated in the cellular signaling events that initiate apoptosis. CLOX can undergo enzymatic hydrolysis, resulting in the release of lipid mediators with signaling properties. Our previous findings suggested that CLD1 expression is upregulated in response to oxidative stress, and that one of the physiological roles of CL remodeling is to remove peroxidized CL. To exploit the powerful yeast model to study functions of CLD1 in CL peroxidation, we expressed the H. brasiliensis Δ12-desaturase gene in yeast, which then synthesized PUFAs that are incorporated into CL species. Using LC-MS based redox phospholipidomics, we identified and quantified the molecular species of CL and other phospholipids in cld1Δ vs. WT cells. Loss of CLD1 led to a dramatic decrease in chronological lifespan, mitochondrial membrane potential, and respiratory capacity; and increased levels of mono-hydroperoxy-CLs, particularly among the highly unsaturated CL species, including tetralinoleoyl-CL. In addition, purified Cld1 exhibited a higher affinity for CLOX, and treatment of cells with H2O2 increased CLD1 expression in the logarithmic growth phase. These data suggest that CLD1 expression is required to mitigate oxidative stress. The findings from this study contribute to our overall understanding of CL remodeling and its role in mitigating oxidative stress.

Keywords: Cld1; cardiolipin; lipid peroxidation; mass spectrometry (MS); polyunsaturated fatty acid (PUFA); remodeling; yeast

DOI: https://doi.org/10.1016/j.bbalip.2018.06.016

Eur Arch Otorhinolaryngol. 2018 Jun 23.

Tinnitus in patients with hearing loss due to mitochondrial DNA pathogenic variants.

Urszula Lechowicz, Agnieszka Pollak, Danuta Raj-Koziak, Beata Dziendziel, Piotr Henryk Skarżyński, Henryk Skarżyński, Monika Ołdak.

PURPOSE: Tinnitus described as individual perception of phantom sound constitutes a significant medical problem and has become an essential subject of many studies conducted worldwide. In the study, we aimed to examine the prevalence of tinnitus among Polish hearing loss (HL) patients with identified mitochondrial DNA (mtDNA) variants.
METHODS: Among the selected group of unrelated HL patients with known mtDNA pathogenic variants, two questionnaires were conducted, i.e. Tinnitus Handicap Inventory translated into Polish (THI-POL) and Visual Analogue Scale (VAS) for measuring subjectively perceived tinnitus loudness, distress, annoyance and possibility of coping with this condition (VASs). Pathogenic mtDNA variants were detected with real-time PCR and sequencing of the whole mtDNA.
RESULTS: This is the first extensive tinnitus characterization using THI-POL and VASs questionnaires in HL patients due to mtDNA variants. We have established the prevalence of tinnitus among the studied group at 23.5%. We found that there are no statistically significant differences in the prevalence of tinnitus and its characteristic features between HL patients with known HL mtDNA variants and the general Polish population. In Polish HL patients with tinnitus, m.7511T>C was significantly more frequent than in patients without tinnitus. We observed that the prevalence of tinnitus is lower in Polish patients with m.1555A>G as compared to other available data.
CONCLUSIONS: Our data suggest that the mtDNA variants causative of HL may affect tinnitus development but this effect seems to be ethnic-specific.

Keywords: Hearing loss; Mitochondrial variants; Tinnitus; m.1555A>G; m.3243A>G; m.7511T>C

DOI: https://doi.org/10.1007/s00405-018-5028-y