Low-pressure chromatographic separation and UV/Vis spectrophotometric characterization of the native and desialylated human apo-transferrin.

Low-pressure pH gradient ion exchange separation provides a fast, simple and cost-effective method for preparative purification of native and desialylated apo-transferrin. The method enables easy monitoring of the extent of the desialylation reaction and also the efficient separation and purification of protein fractions after desialylation. The N-glycan analysis shows that the modified desialylation protocol successfully reduces the content of the sialylated fractions relative to the native apo-transferrin. In the optimized protocol, the desialylation capacity is increased by 150 %, compared to the original protocol provided by the manufacturer. The molar absorption coefficients in the near-UV region for the native and desialylated apo-transferrin differ by several percent, suggesting a subtle dependence of the glycoprotein absorbance on the variable sialic acid content. The method can easily be modified for other glycoproteins and is particularly appropriate for quick testing of sialic acid content in the protein glycosylation patterns prior to further verification by mass spectrometry.

Epigenetic Differentiation of Natural Populations of Lilium bosniacum Associated with Contrasting Habitat Conditions.

Epigenetic variation in natural populations with contrasting habitats might be an important element, in addition to the genetic variation, in plant adaptation to environmental stress. Here, we assessed genetic, epigenetic, and cytogenetic structure of the three Lilium bosniacum populations growing on distinct habitats. One population was growing under habitual ecological conditions for this species and the other two were growing under stress associated with high altitude and serpentine soil. Amplified fragment length polymorphism and methylation-sensitive amplification polymorphism analyses revealed that the three populations did not differentiate genetically, but were clearly separated in three distinct clusters according to DNA methylation profiles. Principal coordinate analysis showed that overall epigenetic variation was closely related to habitat conditions. A new methylation-sensitive amplification polymorphism scoring approach allowed identification of mainly unmethylated (φST = 0.190) and fully CpG methylated (φST = 0.118) subepiloci playing a role in overall population differentiation, in comparison with hemimethylated sites (φST = 0.073). In addition, unusual rDNA repatterning and the presence of B chromosomes bearing 5S rDNA loci were recorded in the population growing on serpentine soil, suggesting dynamic chromosome rearrangements probably linked to global genome demethylation, which might have reactivated some mobile elements. We discuss our results considering our earlier data on morphology and leaf anatomy of several L. bosniacum populations, and suggest a possible role of epigenetics as a key element in population differentiation associated with environmental stress in these particular lily populations.

Promoter methylation of the MGAT3 and BACH2 genes correlates with the composition of the immunoglobulin G glycome in inflammatory bowel disease.

Background: Many genome- and epigenome-wide association studies (GWAS and EWAS) and studies of promoter methylation of candidate genes for inflammatory bowel disease (IBD) have demonstrated significant associations between genetic and epigenetic changes and IBD. Independent GWA studies have identified genetic variants in the BACH2, IL6ST, LAMB1, IKZF1, and MGAT3 loci to be associated with both IBD and immunoglobulin G (IgG) glycosylation. Methods: Using bisulfite pyrosequencing, we analyzed CpG methylation in promoter regions of these five genes from peripheral blood of several hundred IBD patients and healthy controls (HCs) from two independent cohorts, respectively. Results: We found significant differences in the methylation levels in the MGAT3 and BACH2 genes between both Crohn's disease and ulcerative colitis when compared to HC. The same pattern of methylation changes was identified for both genes in CD19+ B cells isolated from the whole blood of a subset of the IBD patients. A correlation analysis was performed between the MGAT3 and BACH2 promoter methylation and individual IgG glycans, measured in the same individuals of the two large cohorts. MGAT3 promoter methylation correlated significantly with galactosylation, sialylation, and bisecting GlcNAc on IgG of the same patients, suggesting that activity of the GnT-III enzyme, encoded by this gene, might be altered in IBD. The correlations between the BACH2 promoter methylation and IgG glycans were less obvious, since BACH2 is not a glycosyltransferase and therefore may affect IgG glycosylation only indirectly. Conclusions: Our results suggest that epigenetic deregulation of key glycosylation genes might lead to an increase in pro-inflammatory properties of IgG in IBD through a decrease in galactosylation and sialylation and an increase of bisecting GlcNAc on digalactosylated glycan structures. Finally, we showed that CpG methylation in the promoter of the MGAT3 gene is altered in CD3+ T cells isolated from inflamed mucosa of patients with ulcerative colitis from a third smaller cohort, for which biopsies were available, suggesting a functional role of this glyco-gene in IBD pathogenesis.

Population substructure can significantly affect reliability of a DNA-led process of identification of mass fatality victims.

Aiming to evaluate the effects of population substructure on the reliability of a DNA correspondence in the process of human identification, we used the model of "in silico" constructed populations with and without substructure. Effects of population substructure were evaluated at the level of locus heterozygosity, Hardy-Weinberg equilibrium and mini-haplotype distribution. Inbreeding in a subpopulation of 100 individuals through 10 generations did not significantly alter the level of heterozygosity and Hardy-Weinberg equilibrium. However, analysis of mini-haplotype distribution revealed a significant homogenization in separated subpopulations. Average observed mini-haplotype frequency (f(o)) increased to threefold from expected values (f(e)), and the number of mini-haplotypes with f(o)/f(e) above 10 increased over sixfold, suggesting that the effects of population substructure on calculated likelihood ratios (LR) might be larger than previously estimated. In most criminal cases, this would not represent a problem, whereas for identifications in large-scale mass fatality events, population substructure might considerably increase the risk of false identification.

Nucleotide sequence, structural organization and length heterogeneity of ribosomal DNA intergenic spacer in Quercus petraea (Matt.) Liebl. and Q. robur L.

18S-5.8S-26S rDNA family comprises tandemly arranged, repeating units separated by an intergenic spacer (IGS) that contains transcription initiation/termination signals and usually repeating elements. In this study, we performed for the first time thorough sequence analysis of rDNA IGS region in two dominant European oaks, Quercus petraea and Q. robur, in order to investigate (1) if IGS sequence composition allows discrimination between these two species, and (2) if there is an rDNA length heterogeneity arising from IGS sequence. Two spacer length variants (slvs), 2 and 4 kb in length, were found in the genomes of both species. Inter-comparison of both slvs revealed no species-specificity in sequence or structural organization. Both slvs could be divided into four subregions; (1) the subrepeat region containing three repeated elements, (2) the AT-rich region containing matrix attachment sites and putative origin of replication, (3) the promoter region containing putative transcription initiation site and (4) the 5'ETS region. In the 4-kb slvs all four subregions are extended, and the subrepeat, AT-rich and promoter regions are duplicated. This is unique compared to other known IGS sequences where the variation in number of subrepeats is responsible for slvs creation. We also propose a possible evolutionary scenario to explain the formation of the subrepeat region in oak IGS. Results obtained in this work add to the previous picture of low-genetic differentiation of the two oaks and provide important data for further analyses of the function of IGS in control of rRNA gene expression.

How high should paternity index be for reliable identification of war victims by DNA typing?

AIM: To analyze statistically and logically the significance of genetic matches between skeletal remains and relatives of missing persons in the process of identification of war victims by DNA typing. METHODS: DNA was isolated from bone and blood samples and short tandem repeat (STR) loci were typed by using AmpFLSTR Profiler, Profiler Plus, and Identifiler kits. Novel mini-haplotype analysis that compares matches in all three-locus combinations of alleles was developed and used in the analysis of inbreeding in the group of 295 unrelated individuals. RESULTS: While comparing 98 skeletal remains exhumed in the process of identification of war victims in Croatia with over 3,000 genotypes of relatives of missing persons, we revealed 20 cases of 14-locus matches and 4 cases of 15-locus matches between unrelated people. We hypothesized that this unexpectedly high number of false matches might be a consequence of local inbreeding and supported this hypothesis with very low correlation between the probability of a genotype and the number of matching genotypes in the database (R(2) = 0.36). Further support for the hypothesis was obtained by the analysis of mini-haplotypes, which revealed up to 90% overrepresentation of some mini-haplotypes. CONCLUSIONS: STR DNA typing is the "golden standard" of human identification, but evidential value of a genetic match can be easily misinterpreted. Therefore, careful use of statistical methods is essential for the proper evaluation of laboratory results. Whenever possible, multiple relatives should be analyzed and other evidence based on the information about time, place, and other conditions of disappearance, as well as anthropological and other "classical" forensic data should always be put together and compared before any final decision about the identity is made.