Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error

A key to selected rockfishes (Sebastes spp.) based on mitochondrial DNA restriction fragment analysis

Larval and juvenile rockfishes (Sebastes spp.) are difficult to identify using morphological characters. We developed a key based on sizes of restriction endonuclease fragments of the NADH dehydrogenase-3 and -4 (ND3/ND4) and 12S and 16S ribosomal RNA (12S/16S) mitochondrial regions. The key makes use of variation in the ND3/ND4 region. Restriction endonuclease Dde I variation can corroborate identifications, as can 12S/16S variation. The key, based on 71 species, includes most North American taxa, several Asian species, and Sebastolobus alascanus and Helicolenus hilgendorfi that are closely related to rockfishes. Fifty-eight of 71 rockfish species in our database can be distinguished unequivocally, using one to five restriction enzymes; identities of the remaining species are narrowed to small groups: 1) S. polyspinis, S. crameri, and S. ciliatus or variabilis (the two species could not be distinguished and were considered as a single species) ; 2) S. chlorostictus, S. eos, and S. rosenblatti; 3) S. entomelas and S. mystinus; 4)S. emphaeus, S. variegatus, and S. wilsoni; and 5) S. carnatus and S. chrysomelas

Very Rare Complementation between Mitochondria Carrying Different Mitochondrial DNA Mutations Points to Intrinsic Genetic Autonomy of the Organelles in Cultured Human Cells

In the present work, a large scale investigation was done regarding the capacity of cultured human cell lines (carrying in homoplasmic form either the mitochondrial tRNALys A8344G mutation associated with the myoclonic epilepsy and ragged red fiber (MERRF) encephalomyopathy or a frameshift mutation, isolated in vitro, in the gene for the ND4 subunit of NADH dehydrogenase) to undergo transcomplementation of their recessive mitochondrial DNA (mtDNA) mutations after cell fusion. The presence of appropriate nuclear drug resistance markers in the two cell lines allowed measurements of the frequency of cell fusion in glucose-containing medium, non-selective for respiratory capacity, whereas the frequency of transcomplementation of the two mtDNA mutations was determined by growing the same cell fusion mixture in galactose-containing medium, selective for respiratory competence. Transcomplementation of the two mutations was revealed by the re-establishment of normal mitochondrial protein synthesis and respiratory activity and by the relative rates synthesis of two isoforms of the ND3 subunit of NADH dehydrogenase. The results of several experiments showed a cell fusion frequency between 1.4 and 3.4% and an absolute transcomplementation frequency that varied between 1.2 × 10^-5 and 5.5 × 10^-4. Thus, only 0.3-1.6% of the fusion products exhibited transcomplementation of the two mutations. These rare transcomplementing clones were very sluggish in developing, grew very slowly thereafter, and showed a substantial rate of cell death (22-28%). The present results strongly support the conclusion that the capacity of mitochondria to fuse and mix their contents is not a general intrinsic property of these organelles in mammalian cells, although it may become activated in some developmental or physiological situations

Gardner's Minichess Variant is solved

A 5x5 board is the smallest board on which one can set up all kind of chess pieces as a start position. We consider Gardner's minichess variant in which all pieces are set as in a standard chessboard (from Rook to King). This game has roughly 9x10^{18} legal positions and is comparable in this respect with checkers. We weakly solve this game, that is we prove its game-theoretic value and give a strategy to draw against best play for White and Black sides. Our approach requires surprisingly small computing power. We give a human readable proof. The way the result is obtained is generic and could be generalized to bigger chess settings or to other games

Magnetically-induced ferroelectricity in the (ND4)2[FeCl5(D2O)] molecular compound

The number of magnetoelectric multiferroic materials reported to date is scarce, as magnetic structures that break inversion symmetry and induce an improper ferroelectric polarization typically arise through subtle competition between different magnetic interactions. The (NH4)2[FeCl5(H2O)] compound is a rare case where such improper ferroelectricity has been observed in a molecular material. We have used single crystal and powder neutron diffraction to obtain detailed solutions for the crystal and magnetic structures of (NH4)2[FeCl5(H2O)], from which we determined the mechanism of multiferroicity. From the crystal structure analysis, we observed an order-disorder phase transition related to the ordering of the ammonium counterion. We have determined the magnetic structure below TN, at 2K and zero magnetic field, which corresponds to a cycloidal spin arrangement with magnetic moments contained in the ac-plane, propagating parallel to the c-axis. The observed ferroelectricity can be explained, from the obtained magnetic structure, via the inverse Dzyaloshinskii-Moriya mechanism

Reorientational Dynamics and Solid-Phase Transformation of Ammonium Dicyanamide into Dicyandiamide

The reorientational dynamics of ammonium dicyanamide ND4[N(C≡N)2] and the kinetics as well as the mechanism of the solid-state isomerization reaction from ammonium dicyanamide into dicyandiamide (N≡C-N=C(NH2)2) was studied by means of 2H and 14N solid-state NMR spectroscopy in a temperature range between 38 and 390 K. Whereas in previous investigations the mechanism of the solid-state transformation was investigated by means of vibrational and magic angle spinning solid-state NMR spectroscopy as well as neutron diffraction, we here present a comprehensive 2H study of the ammonium ion dynamics prior to and during the course of the reaction, thereby highlighting possible cross correlations between dynamics and reactivity involving the ammonium ion. The ND4+ group was found to undergo thermally activated random jumps in a tetrahedral potential, which is increasingly distorted with increasing temperature, giving rise to an asymmetrically compressed or elongated tetrahedron with deviations from the tetrahedral angle of up to 6°. The correlation time follows an Arrhenius law with an activation energy of Ea = 25.8(2) kJ mol-1 and an attempt frequency of τ0-1 = 440(80) THz. The spin−lattice relaxation times were fitted according to a simple Bloembergen−Purcell−Pound type model with a T1 minimum of 4 ms at 230 K. Temperature-dependent librational amplitudes were extracted by line-shape simulations between 38 and 390 K and contrasted with those obtained by neutron diffraction, their values ranging between 5 and 28°. The onset and progress of the solid-phase transformation were followed in situ at temperatures above 372 K and could be classified as a strongly temperature-dependent, heterogeneous two-step reaction proceeding with rapid evolution of ammonia and comparatively slow subsequent reintegration into the solid. On the microscopic level, this correlates with a rapid proton transferpossibly triggered by a coupling between the ammonium ion dynamics and phonon modes on the terahertz time scaleand an essentially decoupled nucleophilic attack of ammonia at the nitrile carbon, giving rise to significantly differing time constants for the two processes

The identification of mitochondrial DNA variants in glioblastoma multiforme

Background: Mitochondrial DNA (mtDNA) encodes key proteins of the electron transfer chain (ETC), which produces ATP through oxidative phosphorylation (OXPHOS) and is essential for cells to perform specialised functions. Tumor-initiating cells use aerobic glycolysis, a combination of glycolysis and low levels of OXPHOS, to promote rapid cell proliferation and tumor growth. Glioblastoma multiforme (GBM) is an aggressively malignant brain tumor and mitochondria have been proposed to play a vital role in GBM tumorigenesis. Results: Using next generation sequencing and high resolution melt analysis, we identified a large number of mtDNA variants within coding and non-coding regions of GBM cell lines and predicted their disease-causing potential through in silico modeling. The frequency of variants was greatest in the D-loop and origin of light strand replication in non-coding regions. ND6 was the most susceptible coding gene to mutation whilst ND4 had the highest frequency of mutation. Both genes encode subunits of complex I of the ETC. These variants were not detected in unaffected brain samples and many have not been previously reported. Depletion of HSR-GBM1 cells to varying degrees of their mtDNA followed by transplantation into immunedeficient mice resulted in the repopulation of the same variants during tumorigenesis. Likewise, de novo variants identified in other GBM cell lines were also incorporated. Nevertheless, ND4 and ND6 were still the most affected genes. We confirmed the presence of these variants in high grade gliomas. Conclusions: These novel variants contribute to GBM by rendering the ETC. partially dysfunctional. This restricts metabolism to anaerobic glycolysis and promotes cell proliferation

Lack of complex I activity in human cells carrying a mutation in MtDNA-encoded ND4 subunit is corrected by the Saccharomyces cerevisiae NADH-quinone oxidoreductase (NDI1) gene

The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene. Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis. In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive. Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain. Furthermore, the P:O ratio with malate/glutamate-dependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK cells, as expected from the lack of proton pumping activity in the yeast enzyme. Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium. The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency

A black hole instability in five dimensions?

We study the moduli-space scattering of a two-charge supertube in the background of a rotating BPS D1-D5-P black hole in 4+1 dimensions, extending the static analysis of Bena and Kraus (hep-th/0402144). While the magnetic forces associated with this motion change the details considerably, the final conclusion is similar to that of the static analysis: we find that one can bring the supertube to the horizon, so that the BMPV black hole and the supertube merge. However, our analysis shows that this can occur even at significantly larger values of the angular momentum than was indicated by the static analysis. For a range of parameters, conservation laws and the area theorem forbid the result of the merger from being any single known object: neither near-extremal black holes nor non-supersymmetric black rings are allowed. Such results suggest that the merger triggers an instability of the rotating D1-D5-P black hole, perhaps leading to bifurcation into a pair of black objects.Comment: 25 pages, 4 figures; v2: references adde

Developing cardiac and skeletal muscle share fast-skeletal myosin heavy chain and cardiac troponin-I expression

Skeletal muscle derived stem cells (MDSCs) transplanted into injured myocardium can differentiate into fast skeletal muscle specific myosin heavy chain (sk-fMHC) and cardiac specific troponin-I (cTn-I) positive cells sustaining recipient myocardial function. We have recently found that MDSCs differentiate into a cardiomyocyte phenotype within a three-dimensional gel bioreactor. It is generally accepted that terminally differentiated myocardium or skeletal muscle only express cTn-I or sk-fMHC, respectively. Studies have shown the presence of non-cardiac muscle proteins in the developing myocardium or cardiac proteins in pathological skeletal muscle. In the current study, we tested the hypothesis that normal developing myocardium and skeletal muscle transiently share both sk-fMHC and cTn-I proteins. Immunohistochemistry, western blot, and RT-PCR analyses were carried out in embryonic day 13 (ED13) and 20 (ED20), neonatal day 0 (ND0) and 4 (ND4), postnatal day 10 (PND10), and 8 week-old adult female Lewis rat ventricular myocardium and gastrocnemius muscle. Confocal laser microscopy revealed that sk-fMHC was expressed as a typical striated muscle pattern within ED13 ventricular myocardium, and the striated sk-fMHC expression was lost by ND4 and became negative in adult myocardium. cTn-I was not expressed as a typical striated muscle pattern throughout the myocardium until PND10. Western blot and RT-PCR analyses revealed that gene and protein expression patterns of cardiac and skeletal muscle transcription factors and sk-fMHC within ventricular myocardium and skeletal muscle were similar at ED20, and the expression patterns became cardiac or skeletal muscle specific during postnatal development. These findings provide new insight into cardiac muscle development and highlight previously unknown common developmental features of cardiac and skeletal muscle. © 2012 Clause et al