Genome-Wide DNA Methylation Profiles Indicate CD8+ T Cell Hypermethylation in Multiple Sclerosis

Objective

Determine whether MS-specific DNA methylation profiles can be identified in whole blood or purified immune cells from untreated MS patients.

Methods

Whole blood, CD4+ and CD8+ T cell DNA from 16 female, treatment naïve MS patients and 14 matched controls was profiled using the HumanMethylation450K BeadChip. Genotype data were used to assess genetic homogeneity of our sample and to exclude potential SNP-induced DNA methylation measurement errors.

Results

As expected, significant differences between CD4+ T cells, CD8+ T cells and whole blood DNA methylation profiles were observed, regardless of disease status. Strong evidence for hypermethylation of CD8+ T cell, but not CD4+ T cell or whole blood DNA in MS patients compared to controls was observed. Genome-wide significant individual CpG-site DNA methylation differences were not identified. Furthermore, significant differences in gene DNA methylation of 148 established MS-associated risk genes were not observed.

Conclusion

While genome-wide significant DNA methylation differences were not detected for individual CpG-sites, strong evidence for DNA hypermethylation of CD8+ T cells for MS patients was observed, indicating a role for DNA methylation in MS. Further, our results suggest that large DNA methylation differences for CpG-sites tested here do not contribute to MS susceptibility. In particular, large DNA methylation differences for CpG-sites within 148 established MS candidate genes tested in our study cannot explain missing heritability. Larger studies of homogenous MS patients and matched controls are warranted to further elucidate the impact of CD8+ T cell and more subtle DNA methylation changes in MS development and pathogenesis.     

Determination of total chromium in tanned leather samples used in car industry

Despite the high competition of synthetic fibers leather is nowadays still widely used for many applications. In order to ensure a sufficient stability of the skin matrix against many factors, such as microbial degradation, heat and sweat, a tanning process is indispensable. Using chromium (III) for this purpose offers a multitude of advantages, thus this way of tanning is widely applied. During the use of chromium tanned leather as clothing material as well as for decoration/covering purposes, chromium is extracted from the leather and may then cause nocuous effects to human skin, e.g. allergic reactions. Thus the knowledge of the total chromium content of leather samples expected to come into prolonged touch with human skin is very important. In car industry leather is used as cover for seats, steering wheel and gearshift lever The chromium contents often chromium tanned leather samples used in car industry were determined. First all samples were dried at 65 degrees C overnight and then cut in small pieces using a ceramic knife, weighed and analyzed by inductively coupled plasma--optical emission spectrometry (ICP-OES) after acidic microwave assisted digestion. The total chromium amounts found were in the range from 19 mg/g up to 32 mg/g. The extraction yield of chromium from leather samples in sweat is approximately 2-7%. Thus especially during long journeys in summer chromium can be extracted in amounts which may cause nocuous effects for example on the palm of the hands or on the back.     

The American cranberry mitochondrial genome reveals the presence of selenocysteine (tRNA-Sec and SECIS) insertion machinery in land plants

This is the first de novo assembly and annotation of a complete mitochondrial genome in the Ericales order from the American cranberry (Vaccinium macrocarpon Ait.). Moreover, only four complete Asterid mitochondrial genomes have been made publicly available. The cranberry mitochondrial genome was assembled and reconstructed from whole genome 454 Roche GS-FLX and Illumina shotgun sequences. Compared with other Asterids, the reconstruction of the genome revealed an average size mitochondrion (459,678 nt) with relatively little repetitive sequences and DNA of plastid origin. The complete mitochondrial genome of cranberry was annotated obtaining a total of 34 genes classified based on their putative function, plus three ribosomal RNAs, and 17 transfer RNAs. Maternal organellar cranberry inheritance was inferred by analyzing gene variation in the cranberry mitochondria and plastid genomes. The annotation of cranberry mitochondrial genome revealed the presence of two copies of tRNA-Sec and a selenocysteine insertion sequence (SECIS) element which were lost in plants during evolution. This is the first report of a land plant possessing selenocysteine insertion machinery at the sequence level.     

Discrimination of American Cranberry Cultivars and Assessment of Clonal Heterogeneity Using Microsatellite Markers

Cranberries (Vaccinium macrocarpon Ait.) are an economically important fruit crop derived from a North American native species. We report the application of 12 simple sequence repeats (SSR) or microsatellite markers to assess the genetic diversity of cranberry cultivars. We studied 164 samples of 21 different cranberry cultivars, 11 experimental hybrids, and 6 representative accessions of wild species. Genetic cluster analysis, based on 117 SSR alleles, differentiated the major cranberry cultivars. However, some cranberry cultivar subclone variants and mislabeled samples were observed. Consensus genetic profiles identified the most likely clonal representatives of several important cranberry cultivars (e.g., “Ben Lear,” “Howes,” and “Stevens”). The markers were further used to confirm putative parents of several hybrid progenies. The long-term goal of our studies is to identify, preserve, and utilize unique genetic materials to breed improved cranberries. Attaining this goal will help growers maintain sustainability under changing economic and environmental conditions.     

Threonine 201 in the diiron enzyme toluene 4-monooxygenase is not required for catalysis

The diiron enzyme toluene 4-monooxygenase from Pseudomonas mendocina KR1 catalyzes the NADH- and O(2)-dependent hydroxylation of toluene. A combination of sequence alignments and spectroscopic studies indicate that T4MO has an active site structure closely related to the crystallographically characterized methane monooxygenase hydroxylase. In the methane monooxygenase hydroxylase, active site residue T213 has been proposed to participate in O(2) activation by analogy to certain proposals made for cytochrome P450. In this work, mutagenesis of the comparable residue in the toluene 4-monooxygenase hydroxylase, T201, has been used to investigate the role of an active site hydroxyl group in catalysis. Five isoforms (T201S, T201A, T201G, T201F, and T201K) that retain catalytic activity based on an in vivo indigo formation assay were identified, and detailed characterizations of the purified T201S, T201A, and T201G variants are reported. These isoforms have k(cat) values of 1.2, 1.0, and 0.6 s(-)(1), respectively, and k(cat)/K(M) values that vary by only approximately 4-fold relative to that of the native isoform. Moreover, these isoforms exhibit 80-90% coupling efficiency, which also compares favorably to the >94% coupling efficiency determined for the native isoform. For the T201S, T201A, and T201G isoforms, the regiospecificity of toluene hydroxylation was nearly identical to that of the natural isoform, with p-cresol representing 90-95% of the total product distribution. In contrast, the T201F isoform caused a substantial shift in the product distribution, and gave o- and p-cresol in a 1:1 ratio. In addition, the amount of benzyl alcohol was increased approximately 10-fold with the T201F isoform. For reaction with p-xylene, previous studies have shown that the native isoform reacted to give 4-methybenzyl alcohol and 2, 5-dimethylphenol in a 4:1 ratio [Pikus, J. D., Studts, J. M., McClay, K., Steffan, R. J., and Fox, B. G. (1997) Biochemistry 36, 9283-9289]. For comparison, the T201S, T201A, and T201F isoforms gave a slightly relaxed 3:1 ratio of these products, while the T201G isoform gave a dramatically relaxed 1:1 ratio. On the basis of these studies, we conclude that the hydroxyl group of T201 is not essential to maintaining the turnover rate or the coupling of the toluene 4-monooxygenase complex. However, changing the volume occupied by the side chain at the position of T201 can lead to alterations in the regiospecificity of the hydroxylation, presumably by producing different orientations for substrate binding during catalysis.     

Membrane lipids of hepatocytes,Kupffer cells and endothelial cells

The membrane lipid composition of isolated hepatocytes, Kupffer cells and endothelial cells was determined. The hepatocytes are characterized by a lower quantity of gangliosides, cholesterol, sphingomyelin and a reduced cholesterol/phospholipid molar ratio when compared to the two other liver cell types. The main gangliosides of Kupffer and endothelial cells are the GM3 species, and those of hepatocytes are of the polysialogangliosides species.     

Drain the lysosome: Development of the novel orally available autophagy inhibitor ROC-325

Although macroautophagy/autophagy is a key contributor to malignant pathogenesis and therapeutic resistance, there are few FDA-approved agents that significantly affect this pathway. We used medicinal chemistry strategies to develop ROC-325, an orally available novel inhibitor of lysosomal-mediated autophagy. Detailed in vitro and in vivo studies in preclinical models of renal cell carcinoma demonstrated that ROC-325 triggered the hallmark features of lysosomal autophagy inhibition, was very well tolerated, and exhibited significant superiority with respect to autophagy inhibition and anticancer activity over hydroxychloroquine. Our findings support the clinical investigation of the safety and preliminary efficacy of ROC-325 in patients with autophagy-dependent malignancies and other disorders where aberrant autophagy contributes to disease pathogenesis.     

Reference concentrations of trace elements in urine of the budapestian population

Reference values in biological specimens are crucial to estimate the type and magnitude of environmental and occupational exposure: Because of its importance in the excretion of noxious substances and to the noninvasive mode of its collection, urine is a useful specimen for monitoring studies. Thus, the concentrations of six trace elements (Al, Co, Mo, Nb, Ni, and Ti) were determined in 100 urine samples of the Budapestian population by inductively coupled plasma-mass spectrometry. The obtained creatinine adjusted concentrations (medians) are (in μg/g) 9.9, 0.6, 53.5, 0.4, 1.5, and 8.5 for Al, Co, Mo, Nb, Ni, and Ti, respectively.