Reduced mitochondrial D-loop methylation levels in sporadic amyotrophic lateral sclerosis

Background: Mitochondrial dysregulation and aberrant epigenetic mechanisms have been frequently reported in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and several researchers suggested that epigenetic dysregulation in mitochondrial DNA (mtDNA) could contribute to the neurodegenerative process. We recently screened families with mutations in the major ALS causative genes, namely C9orf72, SOD1, FUS, and TARDBP, observing reduced methylation levels of the mtDNA regulatory region (D-loop) only in peripheral lymphocytes of SOD1 carriers. However, until now no studies investigated the potential role of mtDNA methylation impairment in the sporadic form of ALS, which accounts for the majority of disease cases. The aim of the current study was to investigate the D-loop methylation levels and the mtDNA copy number in sporadic ALS patients and compare them to those observed in healthy controls and in familial ALS patients. Pyrosequencing analysis of D-loop methylation levels and quantitative analysis of mtDNA copy number were performed in peripheral white blood cells from 36 sporadic ALS patients, 51 age- and sex-matched controls, and 27 familial ALS patients with germinal mutations in SOD1 or C9orf72 that represent the major familial ALS forms. Results: In the total sample, D-loop methylation levels were significantly lower in ALS patients compared to controls, and a significant inverse correlation between D-loop methylation levels and the mtDNA copy number was observed. Stratification of ALS patients into different subtypes revealed that both SOD1-mutant and sporadic ALS patients showed lower D-loop methylation levels compared to controls, while C9orf72-ALS patients showed similar D-loop methylation levels than controls. In healthy controls, but not in ALS patients, D-loop methylation levels decreased with increasing age at sampling and were higher in males compared to females. Conclusions: Present data reveal altered D-loop methylation levels in sporadic ALS and confirm previous evidence of an inverse correlation between D-loop methylation levels and the mtDNA copy number, as well as differences among the major familial ALS subtypes. Overall, present results suggest that D-loop methylation and mitochondrial replication are strictly related to each other and could represent compensatory mechanisms to counteract mitochondrial impairment in sporadic and SOD1-related ALS forms

Preferential X Chromosome Inactivation as a Mechanism to Explain Female Preponderance in Myasthenia Gravis

Myasthenia gravis (MG) is a neuromuscular autoimmune disease characterized by preva- lence in young women (3:1). Several mechanisms proposed as explanations for gender bias, including skewed X chromosome inactivation (XCI) and dosage or sex hormones, are often involved in the development of autoimmunity. The skewed XCI pattern can lead to an unbalanced expression of some X-linked genes, as observed in several autoimmune disorders characterized by female predominance. No data are yet available regarding XCI and MG. We hypothesize that the preferential XCI pattern may contribute to the female bias observed in the onset of MG, especially among younger women. XCI analysis was performed on blood samples of 284 women between the ages of 20 and 82. XCI was tested using the Human Androgen Receptor Assay (HUMARA). XCI patterns were classified as random (XCI < 75%) and preferential (XCI 75%). In 121 informative patients, the frequency of skewed XCI patterns was 47%, significantly higher than in healthy controls (17%; p 0.00001). Interestingly, the phenomenon was observed mainly in younger patients (<45 years; p 0.00001). Furthermore, considering the XCI pattern and the other clinical characteristics of patients, no signif- icant differences were found. In conclusion, we observed preferential XCI in MG female patients, suggesting its potential role in the aetiology of MG, as observed in other autoimmune diseases in women

Comparison Study of MS-HRM and Pyrosequencing Techniques for Quantification of APC and CDKN2A Gene Methylation

There is increasing interest in the development of cost-effective techniques for the quantification of DNA methylation biomarkers. We analyzed 90 samples of surgically resected colorectal cancer tissues for APC and CDKN2A promoter methylation using methylation sensitive-high resolution melting (MS-HRM) and pyrosequencing. MS-HRM is a less expensive technique compared with pyrosequencing but is usually more limited because it gives a range of methylation estimates rather than a single value. Here, we developed a method for deriving single estimates, rather than a range, of methylation using MS-HRM and compared the values obtained in this way with those obtained using the gold standard quantitative method of pyrosequencing. We derived an interpolation curve using standards of known methylated/ unmethylated ratio (0%, 12.5%, 25%, 50%, 75%, and 100% of methylation) to obtain the best estimate of the extent of methylation for each of our samples. We observed similar profiles of methylation and a high correlation coefficient between the two techniques. Overall, our new approach allows MS-HRM to be used as a quantitative assay which provides results which are comparable with those obtained by pyrosequencing

MONO AND DINUCLEAR GOLD(I) COMPLEXES WITH NEUTRAL AND DEPROTONATED 1,4-BENZODIAZEPIN-2-ONES - CRYSTAL AND MOLECULAR-STRUCTURE OF (L-H)AU(PPH3).ET2O, WHERE L=1,3-DIHYDRO-7-NITRO-5-PHENYL-2H-1,4-BENZODIAZEPIN-2-ONE, NITRAZEPAM

The preparation of a series of neutral (L)AuCL (1a-3a) and cationic [(L)Au(PPh3)] [BF4] (1b-5b) gold(I) complexes is described (L = DIAZEPAM, 1; PRAZEPAM, 2; NITRAZEPAM, 3; LORAZEPAM, 4; NIMETAZEPAM, 5). In the presence of alkali, (PPh3)AuCl reacts with 1-unsubstituted 1,4-benzodiazepin-2-ones such as NITRAZEPAM (3) and LORAZEPAM (4) to give neutral gold(I) species (L-H)Au(PPh3) (3c and 4c). The benzodiazepine anion is bonded to Au via the N(1) atom, as shown, in the case of 3c, by X-ray analysis: (L-H)Au(PPh3)\ub7Et2O, C33H25AuN3O3P\ub7 C4H10O, monoclinic, space group P21/c, a= 12.717(9), b=19.270(7), c=14.696(3) \uc5, \u3b2= 107.85(3)\ub0, Z= 4. AuP = 2.238(1), AuN = 2.071(3) \uc5. The crystal structure was determined by standard methods and refined to R = 0.028 and Rw = 0.035 on the basis of 4456 unique reflections. p ]The complex 3c reacts with [(Ph3P)Au(S)][BF4], as obtained from (PPh3)AuCl and AgBF4 in methanol, to give the dinuclear cationic species [(Ph3P)Au{\u3bc(L-H)}Au(PPh3)] [BF4] (3d) where the deprotonated NITRAZEPAM bridges two (Ph3P)Au groups through the N(1) and N(4) atoms. All the new derivatives were characterized by elemental analyses and spectroscopic methods (IR; 1H, 31P NMR)

Dynamic NMR study of ethene exchange in cationic CNN-type platinum(II) complexes

Cationic ethylene platinum(II) complexes of the type [Pt(CNN)(C2H4)]+, containing a methyl fragment and different diimines (NN), or terdentate (\u3baC-\u3ba2NN\u2032) anionic ligands, were synthesized and fully characterized both as solids and in solution [NN = 2,2\u2032-dipyridylamine, 1; 2,2\u2032-dipyridylsulfide, 2; 1,10-phenanthroline, 3; 4,7-diphenyl-1,10-phenanthroline, 4; 3,4,7,8-tetramethyl-1,10-phenanthroline, 5; 2,2\u2032-bipyridine, 6; HC-NN = 6-tert-butyl-2,2\u2032-bipyridine, 7; 6-neo-pentyl-2,2\u2032-bipyridine, 8; 6-phenyl-2,2\u2032-bipyridine, 9; 6-(\u3b1-methyl)benzyl-2,2\u2032-bipyridine, 10; 6-(\u3b1-ethyl)benzyl-2,2\u2032-bipyridine, 11; 6-(\u3b1,\u3b1-dimethyl)benzyl-2,2\u2032-bipyridine, 12]. Crystals suitable for X-ray analysis of complexes 5 and 7 were obtained. Ethene exchange at the cyclometalated platinum(II) complexes 7, 8, and 10 1212 was studied by 1H NMR line-broadening experiments in chloroform-d, as a function of both temperature and olefin concentrations. For the other prepared complexes the process was too fast to be monitored on the NMR time scale even at the lowest temperature. The ethylene exchange rates show a linear dependence on the concentration of the free ligand, with a negligible k1 term indicating that either a solvolytic or a dissociative pathway to the products is absent or negligible. The values of the second-order rate constants kexc, as obtained by linear regression analysis of the experimental data at 298 K, are in a range of ca. 104 12105 s 121 m 121. The activation entropies are negative, ranging between 12129 and 12112 J K 121 mol 121, as expected for associative processes. The activation process is largely entropy controlled: the T\u394S contribution to the free energy of activation is extremely large, amounting to more than 80% for all complexes, with a smaller enthalpy contribution. All the experimental findings evidence that the mechanism takes place via an associative attack by the entering olefin, through a well-ordered, stable pentacoordinated transition state with the two ethene molecules on the trigonal plane. The reactivity of [Pt(CNN)(C2H4)]+ complexes is strongly dependent on the choice of coordinated 6-substituted-2,2\u2032-bipyridines, especially when the terdentate anionic fragment is capable of generating steric crowding and congestion on the coordination plane