Effect of adding B-vitamins to vitamin D and calcium supplementation on CpG methylation of epigenetic aging markers.

BACKGROUND AND AIM: B-vitamins may influence DNA methylation. We studied the effects of vitamin D + Ca + B versus D + Ca on epigenetic age markers and biological age. METHODS AND RESULTS: Participants (mean ± SD of age = 68.4 ± 10.1 years) were randomized to receive 1200 IE vitamin D3 plus 800 mg Ca-carbonate alone (n = 31) or with 0.5 mg B9, 50 mg B6, and 0.5 mg B12 (n = 32). The CpG methylation of 3 genes (ASPA, ITGA2B, and PDE4C) and the changes in methylation were compared between the groups after 1 year. The changes of ASPA methylation from baseline were higher in the D + Ca + B than in the D + Ca group (1.40 ± 4.02 vs. -0.96 ± 5.12, respectively; p = 0.046, adjusted for age, sex, and baseline methylation). The changes in PDE4C from baseline were slightly higher in the D + Ca + B group (1.95 ± 3.57 vs. 0.22 ± 3.57; adjusted p = 0.062). Methylation of ITGA2B and its changes from baseline were not different between the intervention groups. Sex-adjusted odds ratio of accelerated aging (chronological age < biological age at 1 year) was 5.26 (95% confidence interval 1.51-18.28) in the D + Ca + B compared with the D + Ca group. Accelerated aging in both groups was associated with younger age. In the D + Ca + B group, it was additionally associated with lower baseline homocysteine. CONCLUSIONS: Vitamin D + Ca + B and D + Ca differentially affected epigenetic age markers, although the effect size appeared to be small after 1 year. B-vitamins effect in young subjects with low homocysteine requires further investigation. ClinicalTrials.gov ID: NCT02586181.

Candidate gene analysis of anthocyanin pigmentation loci in the Solanaceae.

Crop species in the Solanaceae, which includes tomato ( Lycopersicon esculentum), potato ( Solanum tuberosum), pepper ( Capsicum spp.), and eggplant ( S. melongena), exhibit natural variation in the types, levels, and tissue-specific expression patterns of anthocyanin pigments. While the identities of the genes underpinning natural variation in anthocyanin traits in these crops are largely unknown, many structural genes and regulators of anthocyanin biosynthesis have been isolated from the solanaceous ornamental species Petunia. To identify candidate genes that may correspond to loci controlling natural variation in the four crops, 13 anthocyanin-related genes were localized on a tomato F(2) genetic map. Gene map positions were then compared to mapped mutants in tomato and through comparative genetic maps to natural variants in potato, eggplant, and pepper. Similar map positions suggest that the tomato mutants anthocyaninless, entirely anthocyaninless, and anthocyanin gainer correspond to flavonoid 3'5'-hydroxylase ( f3'5'h), anthocyanidin synthase, and the Petunia Myb domain trancriptional regulatory gene an2, respectively. Similarly potato R, required for the production of red pelargonidin-based pigments, P, required for production of purple delphinidin-based pigments, and I, required for tissue-specific expression in tuber skin, appear to correspond to dihydroflavonol 4-reductase, f3'5'h and an2, respectively. The map location of an2 also overlaps pepper A and eggplant fap10.1, lla10.1, lra10.1, sa10.1, pa10.1 and ca10.1, suggesting that a homologous regulatory locus has been subjected to parallel selection in the domestication of many solanaceous crops. To test the hypothesis that tomato anthocyaninless corresponds to f3'5'h, a portion of the gene was sequenced. A premature stop codon was observed in an anthocyaninless mutant, but not in wild-type.

A fluorogenic 5' nuclease (TaqMan) assay to assess dosage of a marker tightly linked to red skin color in autotetraploid potato.

We have recently identified an allele of dihydroflavonol 4-reductase ( dfr) that cosegregates with the ability of potato ( Solanum tuberosum L) to produce red pelargonidin-based anthocyanin pigments. A rapid assay to assess dosage of this allele in cultivated potato, an autotetraploid, would be useful for breeding programs that develop red-skinned cultivars. To identify regions of dfr that are conserved between alleles, as well as regions that are variable, a portion of the gene was sequenced from several cultivated and wild potato clones. In one region the sequence of the 'red' dfr allele differed at two nucleotide positions from the three other sequence classes observed. A fluorogenic oligonucleotide probe labeled with 6-FAM was designed to anneal specifically to the red allele in this region, while a second probe labeled with VIC was designed to anneal to the 'not-red' dfr alleles. PCR primers that annealed to conserved sequences flanking the variable region were also developed. When subjected to a fluorogenic 5' nuclease (TaqMan) allelic discrimination assay all diploid clones tested clustered into three distinct groups based on the relative amounts of FAM and VIC released. These three groups represented clones homozygous for the red allele, heterozygous for the red allele, and homozygous for the not-red allele(s). When tetraploid clones were tested they separated into five distinct clusters, three of which were shared with diploid clones. The five clusters were interpreted to represent clones quadruplex, triplex, duplex, simplex and nulliplex for the red dfr allele. This interpretation was supported by monitoring the segregation of red allele dosage in several tetraploid crosses. To the best of our knowledge this is the first report of a fluorogenic 5' nuclease assay being used for allelic discrimination in an autopolyploid.

An allele of dihydroflavonol 4-reductase associated with the ability to produce red anthocyanin pigments in potato (Solanum tuberosum L.).

The potato R locus is necessary for the production of red pelargonidin-based anthocyanin pigments in any tissue of the plant, including tuber skin and flower petals. The production of pelargonidins in plants requires the activity of dihydroflavonol 4-reductase (DFR) to catalyze the reduction of dihydrokaempferol into leucopelargonidin. To test the hypothesis that potato R encodes DFR, portions of both dfr alleles were sequenced from a diploid potato clone known to be heterozygous Rr. Sequence comparison revealed a polymorphic BamHI restriction site. The presence or absence of this site was monitored in three diploid populations that segregated for R, as well as in a wide range of tetraploid breeding clones and cultivars, by amplifying a fragment of dfr and digesting the products with BamHI. An identically sized dfr restriction fragment lacking the BamHI site was present in all potato clones that produced red anthocyanin pigments, while the same fragment was absent in many potato clones with white tuber skin and flowers. An independent RFLP test using DraI to detect sequence polymorphism was performed on a subset of the potato clones. This test also revealed dfr-derived bands that were present in all red-colored potatoes and absent in several white clones. The presence of shared restriction fragments in all red-colored potatoes provides strong evidence that R does indeed code for DFR. The data are also consistent with a 48 year-old hypothesis by Dodds and Long, that R was selected just once during the domestication of potato. A cDNA clone corresponding to the red allele of dfr was sequenced and compared to two other alleles. The red allele is predicted to encode a 382 amino acid protein that differs at ten amino acid positions from the gene products of the two alternative alleles. Several of these differences map in a region known to influence DFR substrate specificity in Gerbera.

Genetic defects as important factors for moderate hyperhomocysteinemia.

The genes for the enzymes methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MS), methionine synthase reductase (MSR) and cytathionine-beta-synthase (CBS) play an important role in homocysteine metabolism. Rare mutations in these genes cause severe hyperhomocysteinemia and clinical symptoms. Growing interest has focused on common mutations with moderate effects on homocysteine levels. We studied 280 subjects of different age groups for the following mutations: MTHFR677C-->T and 1298A-->C, MS2756A-->G, MSR66A-->G and the 68 bp insertion in the CBS gene. The median value for homocysteine increased significantly with age (median homocysteine levels: 7.5, 12.4 and 16.5 micromol/l in the age groups 20-43, 65-75 and 85-96 years, respectively). The genotypes of the MTHFR677C-->T mutation were associated with differences in plasma homocysteine levels, but without reaching significance. Individuals homozygous for the MTHFR677C-->T mutation had a 2.3 micromol/l higher median homocysteine level compared to individuals with the wild-type allele. This effect was pronounced in combination with low folate levels and abolished with higher folate in plasma. For the other three mutations no association with homocysteine values could be determined. The analysis of homocysteine metabolite cystathionine by backward regression analysis revealed a significant correlation of the MS2756A-->G mutation with cystathionine level. This increase could indicate a disturbed remethylation. In summary, larger and homogeneous study populations are necessary to quantify the small effects of common mutations on homocysteine levels. This may also be the reason that no effects of genetic interactions between two genotypes were observed.

Molecular epidemiologic features and antimicrobial susceptibility profiles of various ribotypes of Pseudomonas aeruginosa isolated from humans and ruminants.

OBJECTIVES: To assess automated ribotyping for characterization of Pseudomonas aeruginosa isolates and to identify their type prevalence and geographic distribution. SAMPLE POPULATION: 39 human and 56 ruminant P aeruginosa isolates. PROCEDURES: Isolates were identified by use of bacteriologic techniques and automated Pvull-based ribotyping. Susceptibility to antimicrobials was tested in vitro. Data were analyzed for index of discrimination; prevalence ratio; geographic distribution of ribotypes found only in humans, only in cows, or only in goats (single-host ribotypes); and geographic distribution of ribotypes found in humans and ruminants (multihost ribotypes). RESULTS: All isolates were typeable (45 ribotypes, 35 single-host ribotypes). Ribotyping index of discrimination was 0.976. More isolates (45.3%) than expected yielded multihost ribotypes (22% of all ribotypes). Although 8.6% of single-host ribotypes were found in 4 or more isolates, 60% of multihost ribotypes were found in 4 or more isolates. Ninety percent of multihost ribotypes were isolated from different geographic areas, whereas 3.0% of single-host ribotypes were isolated from different geographic areas. All ruminant isolates were susceptible to gentamicin and polymyxin B. In contrast, antibiogram profiles differed for human isolates from different geographic areas. Susceptibility to antimicrobials differentiated 6 isolates not distinguished by ribotyping. CONCLUSIONS AND CLINICAL RELEVANCE: Automated ribotyping with Pvull discriminated more isolates than in vitro antimicrobial susceptibility. In combination, both tests provided more information than either test alone. Given the greater prevalence and geographic distribution of multihost ribotypes, immunocompromised humans and lactating ruminants may have a greater risk for disease if exposed to multihost P aeruginosa ribotypes, compared with single-host ribotypes.

Role of homocysteine, cystathionine and methylmalonic acid measurement for diagnosis of vitamin deficiency in high-aged subjects.

BACKGROUND: Intracellular B-vitamin and folate deficiency indicated by hyperhomocysteinemia is very frequent in the elderly population. Hyperhomocysteinemia increases the risk of atherothrombotic diseases and neuropsychiatric complications. Our aim was to evaluate the prevalence of increased serum metabolite concentrations in subjects of a higher age, and whether the measurement of metabolite concentrations is more effective in diagnosing B-vitamin deficiency than mere homocysteine. MATERIALS AND METHODS: Homocysteine (HCY), cystathionine (CYS) and methylmalonic acid (MMA) were investigated in serum together with vitamin B-12, B-6 and folate in 90 high-aged subjects (85-102 years), 92 seniors (65-75 years), and in 50 younger subjects (19-50 years). RESULTS: Elderly subjects (high-aged and senior) had elevated serum concentrations of metabolites. High-aged subjects had a higher frequency of pathological increases than seniors: HCY 62% vs. 24%; MMA 62% vs. 23%; CYS 81% vs. 36%. Folate and vitamin B-6 concentrations were significantly decreased in both elderly groups; vitamin B-12 was only decreased in high-aged subjects. Utilising vitamin B-6, B-12 and folate for diagnosis of intracellular vitamin deficiency, the rate was 30% in seniors and 55% in high aged subjects. However, utilising the metabolites (HCY, MMA and CYS) for the diagnosis of intracellular vitamin deficiency, there was a distinctly increased rate of 55% in seniors respective to 90% in high-aged subjects. Backward multiple regression analysis revealed that only folate, MMA, creatinine and age were independent variables influencing the HCY concentration. Furthermore, the MMA concentration was significantly and independently influenced by folate, vitamin B-12, HCY and creatinine, and the serum concentration of CYS by vitamin B-12, creatinine and age. CONCLUSION: The metabolites HCY, MMA and CYS are sensitive indicators diagnosing impaired remethylation of homocysteine to methionine with parallel activation of catabolic pathway. Compared to mere HCY or B-vitamins in serum, the efficiency of diagnosing a disturbed HCY metabolism increases very much in utilising the metabolites HCY, MMA and CYS. For differential diagnosis, parallel measurement of folate and creatinine is recommended. The early and correct diagnosis of B-vitamin deficiency in elderly subjects is of high clinical relevance.

Fluorescence-based single-strand conformation polymorphism analysis of mutations by capillary electrophoresis.

Capillary electrophoresis in combination with fluorescence-based single-strand conformation polymorphism (SSCP) analysis was used to screen for known mutations as well as for unknown mutations. The mutations causing hemochromatosis and thrombogenetic diseases (factor V Leiden mutation and prothrombin mutation) are well defined. Familial hypercholesterolemia is caused by mutations in the low density lipoprotein (LDL) receptor gene. Because the mutations are heterogeneously localized in all 18 exons of the LDL receptor gene, effective screening procedures are necessary. The three well known mutations and 59 of 61 previously characterized mutations in the LDL receptor gene were detected by a distinct abnormal fragment pattern in capillary electrophoresis. The remaining two mutations in the LDL receptor gene showed only slight abnormalities under standard electrophoresis conditions (13 kV, 30 degrees C, 30 min). However, the abnormal pattern could be amplified by increasing the electrophoresis temperature. In all cases, heterozygous and homozygous mutations could clearly be differentiated from wild-type alleles. Because of the high efficiency of mutation detection, capillary electrophoresis in combination with fluorescence-based SSCP analysis would be attractive for the detection of well-defined mutations as well as for the screening of unknown mutations. The accuracy and the degree of automation make this technique well suited for routine genetic diagnosis.

Hyperhomocysteinemia in high-aged subjects: relation of B-vitamins, folic acid, renal function and the methylenetetrahydrofolate reductase mutation.

Moderate hyperhomocysteinemia is an atherogenic risk factor and plays an important role in geriatrics. Here, we have investigated the role of hyperhomocysteinemia in two elderly groups: 104 longeval subjects of 85-102 years, 100 seniors aged 65-75 years, and 75 controls of 19-60 years. Elevated homocysteine levels were found in 58% of longeval subjects in comparison with 32% in seniors. The homocysteine level in serum correlated positively with age as well as serum creatinine, and inversely with serum folate, but there was no correlation with serum B-vitamins. The frequency of vitamin B deficiency in serum of longeval subjects compared to seniors was as follows: vitamin B6 43% vs. 22%, vitamin B12 20% vs. 8%, and folic acid 1% in both groups. Increased serum creatinine levels (> 1.1 mg/dl) were found in 63% of the longeval subjects and in 32% of seniors. The 677-missense mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, responsible for moderate homocysteine elevation, was found in 35, 37 and 27% of alleles in longeval persons, senior subjects and younger controls, respectively, showing no significant difference in frequency distributions of the MTHFR gene mutation. It can be concluded that hyperhomocysteinemia is very common with increased age. Its importance as an atherogenic risk factor with advanced age has to be clarified.

Fluorescence-based single-strand conformation polymorphism analysis of the low density lipoprotein receptor gene by capillary electrophoresis.

We describe here a new method to screen for unknown mutations in the low density lipoprotein (LDL) receptor gene by the use of capillary electrophoresis in single-strand conformation polymorphism (SSCP) analysis. To analyze the promoter and all 18 exons, 20 different amplification reactions were necessary. For each polymerase chain reaction (PCR), the forward and reverse primers were 5' fluorescent-labelled with FAM and HEX, respectively. To test the accuracy of the newly developed method, 61 genetic variants distributed in 16 exons were analyzed. Under identical electrophoresis conditions (13 kV, 30 degrees C, 30 min), 59 mutations were detected by a distinct abnormal SSCP pattern. The two remaining mutations showed only slight abnormalities, which could be amplified by increasing the electrophoresis temperature. The high accuracy, the degree of automation and the speed of analysis make fluorescence-based SSCP analysis with capillary electrophoresis ideal for rapid mutation screening and the technique is well-suited for clinical applications.

Ribotype diversity of Listeria monocytogenes strains associated with outbreaks of listeriosis in ruminants.

Ribotyping is a molecular method for the characterization, identification, and typing of bacterial isolates that has value in epidemiological studies. To demonstrate the utility of this technique for typing of Listeria monocytogenes, four outbreaks of epizootic listeriosis in ruminants were investigated through coordinated detection and characterization methods utilizing classical microbiology and nucleic acid-based techniques. L. monocytogenes strains isolated from clinical samples and the silage consumed by the affected animals were ribotyped to establish the causal relationship between feed and the disease outbreak. For all but one outbreak, we were able to isolate L. monocytogenes strains represented by the same ribotype from both clinical and silage samples. Additional L. monocytogenes strains with ribotypes different from those of the respective clinical samples were isolated from all silage samples. This indicates that a diverse population of L. monocytogenes strains exists in farm environments, of which some may be more likely than others to cause disease.

Diagnosis and epidemiological association of Listeria monocytogenes strains in two outbreaks of listerial encephalitis in small ruminants.

Two outbreaks of epizootic listerial encephalitis, one in sheep and one in goats, were investigated through pathology, microbiology, and DNA amplification-based techniques. Efforts were made to survey the diversity of Listeria monocytogenes strains in the silage consumed by affected animals and to verify the causal relationship between silage and disease outbreak. In both outbreaks, L. monocytogenes was isolated from silage and brain tissue samples. Random amplified polymorphic DNA patterns revealed two distinct L. monocytogenes strains, one of which was identical to the sheep brain isolate, in the silage associated with the outbreak in sheep. Three brain isolates and one silage isolate, all of which had different random amplified polymorphic DNA patterns, were found in the outbreak involving goats. All isolates from both outbreaks were indistinguishable in an in vitro assay for cell-to-cell spread and growth in macrophages. All brain isolates from the goat outbreak had identical intracellular ActA patterns, which were different from the pattern for the silage isolate. While the sheep brain isolate had an ActA pattern different from that of the corresponding silage isolate, the patterns for the brain isolates from the two outbreaks were not identical. This survey demonstrates the diversity of L. monocytogenes in silage and suggests the existence of one or more selective processes by which certain strains are more prone to give rise to disease.

Nucleotide sequence and fine structural analysis of the Corynebacterium glutamicum hom-thrB operon.

The complete nucleotide sequence of the Corynebacterium glutamicum hom-thrB operon has been determined and the structural genes and promoter region mapped. A polypeptide of Mr 46,136 is encoded by hom and a polypeptide of Mr 32,618 is encoded by thrB. Both predicted protein sequences show amino acid sequence homology to their counterparts in Escherichia coli and Bacillus subtilis. The promoter region has been mapped by S1-nuclease and deletion analysis. Located between -88, RNA start site and -219 (smallest deletion clone with complete activity) are sequence elements similar to those found in E. coli and B. subtilis promoters. Although there are no obvious attenuator-like structures in the 5'-untranslated region, there is a dyad-symmetry element, which may act as an operator.