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    • First the genotype analysis was done under four inheritance

    2018-11-07

    First, the genotype analysis was done under four inheritance models: general genotype model (2 degrees of freedom), additive model (each copy of the minor allele alters the risk in an additive form), dominant model (a single copy of the minor allele is sufficient to modify the risk) and recessive model (two copies of the minor allele are needed to modify the risk) (Table 1). The data show no association of the studied SNPs under any of the model with completed suicide in our sample (p>0.05) (Table 1). Haplotype analysis was carried out using 2-, 3- and 4-adjacent SNPs sliding window approach implemented in PLINK v1.07 [2]. We also carried out the haplotype analysis for all 5 SNPs (rs7124442, rs10767664, rs962369, rs12273363 and rs908867) which are in strong linkage disequilibrium (also see in Ref. [1, Fig. 1]). Haplotypes with frequencies lower than 1% were excluded from the analysis and correction for multiple testing was performed by 10,000 permutations test. As shown in Table 2, none of the haplotypes showed significant association with completed suicide after 10,000 permutations correction (EMP p>0.05). However, the T/C base change in the first position (rs7124442) in a specific context of common order Asiatic acid (/T–/C–/C–/T) of at least two of the four SNPs rs10767664, rs962369, rs12273363 and rs908867, always resulted in at least a nominally significant p-value (Table 2).
    Acknowledgemets Funding for the study was provided by the Slovenian Research Agency, Programme Grant no. P1-0390, and by a Junior Researchers Grant to SR. We would like to thank the Institute of Forensic Medicine in the Faculty of Medicine of the University of Ljubljana, Slovenia, for long term cooperation.
    Experimental design and data Fig. 1 illustrates the general workflow of the AP-MS approach (adapted from [1]). In order to discriminate centromere specific proteins from proteins abundantly found in chromatin, we quantitatively compared proteomes isolated from different chromatin regions. To biochemically isolate these different chromatin regions, we made use Drosophila cell lines expressing GFP-tagged histone H3 variants as baits. These were either the replacement variant H3.3, which is enriched in euchromatin, or the centromere specific H3 variant CENP-ACID. Chromatin from these cells and the parental cell line was isolated and solubilized by Micrococcal Nuclease digestion. This soluble extract served as input material for anti GFP-affinity purification, thereby enriching chromatin fragments together with the associated proteins. Repeated washes were performed to remove unspecifically bound contaminants. The associated proteins were eluted under denaturating conditions and fractionated via SDS-PAGE. After in gel-tryptic digestion, peptides were extracted, concentrated and analyzed by LC–MS/MS. Three independent AP-MS experiments were performed per cell line used. Intensity-based absolute quantification (iBAQ) values from the output of the MaxQuant software package were used as a measure for the abundance of identified proteins. Average iBAQ values were calculated for the different samples and in case the protein was not detected, the iBAQ values were imputed from a random distribution (see Section 2.5). Centromere enrichment was calculated by dividing the average iBAQ value for each protein in the CENP-ACID–GFP purification by the corresponding iBAQ value in the chromatin purification from untransfected parental or H3.3-GFP expressing cell lines, respectively. A factor was considered centromere specific if its log2-fold enrichment over both controls was more than four. Using these criteria, we identified 86 proteins that were specifically enriched in CENP-ACID–GFP containing chromatin (Table 1 and [1]). Known CENP-ACID-binding proteins such as Cal1, the centromeric proteins CENP-C, or CAF-1 were also found enriched in CENP-ACID chromatin, demonstrating the general applicability of the technique to detect proteins enriched in centromeric chromatin [2]. While centromere association of most of the 86 identified proteins has not been reported so far, several associations among the proteins are predicted using the “Search Tool for the Retrieval of Interacting Genes/Proteins” (STRING) (Fig. 2) [3]. This indicates that a complex network of interactions contributes to centromere function or maintenance.