„By wspomnienia o nich poznały miliony”. O nagrobkach księży na wirtualnych cmentarzach

Virtual Cemetery is today a place of memory. Every an Internet user has the opportunity to visit and-most importantly-to found among others virtual tombstone for spiritual person and anybody can use with these services as: www. wirtualnycmentarz.pl, www.virtualheaven.pl, www.nekropolia.pl, www.zaduszki. pl or www.miejscapamieci.pl. One of the elements that creators of virtual cemeteries must take into account in their design, there are religious or not religious character. Religious or nonreligious character of the virtual cemetery, just like a traditional cemetery, located in the real world, involves symbolic functioning in two types of codes: verbal and nonverbal. Among the frequent visitors are virtual cemeteries and those who opted for „founds” tombstones for spiritual person. Why we can encounter tombstones: ordinary priests, bishops, priests-poets and the highest dignitaries of the church, like Pope John Paul II. Virtual Cemetery definitely is not a holy place or even the devoted. The formation of such sites on the Internet is a manifestation of the secularization of the virtual cemeteries. The more famous and well deserved ecclesiastical dignitary got the more extended inscriptions. The clergy, no matter what they had views on modern methods of cultivation of the memory-unintentionally become part of a virtual cemetery, which by its nature becoming more and more followers

Optimization of Bifunctional Antisense Oligonucleotides for Regulation of Mutually Exclusive Alternative Splicing of PKM Gene

Oligonucleotide tools, as modulators of alternative splicing, have been extensively studied, giving a rise to new therapeutic approaches. In this article, we report detailed research on the optimization of bifunctional antisense oligonucleotides (BASOs), which are targeted towards interactions with hnRNP A1 protein. We performed a binding screening assay, Kd determination, and UV melting experiments to select sequences that can be used as a high potency binding platform for hnRNP A1. Newly designed BASOs were applied to regulate the mutually exclusive alternative splicing of the PKM gene. Our studies demonstrate that at least three repetitions of regulatory sequence are necessary to increase expression of the PKM1 isoform. On the other hand, PKM2 expression can be inhibited by a lower number of regulatory sequences. Importantly, a novel branched type of BASOs was developed, which significantly increased the efficiency of splicing modulation. Herein, we provide new insights into BASOs design and show, for the first time, the possibility to regulate mutually exclusive alternative splicing via BASOs

Correction: A Tandem Oligonucleotide Approach for SNP-Selective RNA Degradation Using Modified Antisense Oligonucleotides.

[This corrects the article DOI: 10.1371/journal.pone.0142139.]

Thermodynamic, Anticoagulant, and Antiproliferative Properties of Thrombin Binding Aptamer Containing Novel UNA Derivative

Thrombin is a serine protease that plays a crucial role in hemostasis, fibrinolysis, cell proliferation, and migration. Thrombin binding aptamer (TBA) is able to inhibit the activity of thrombin molecule via binding to its exosite I. This 15-nt DNA oligonucleotide forms an intramolecular, antiparallel G-quadruplex structure with a chair-like conformation. In this paper, we report on our investigations on the influence of certain modified nucleotide residues on thermodynamic stability, folding topology, and biological properties of TBA variants. In particular, the effect of single incorporation of a novel 4-thiouracil derivative of unlocked nucleic acid (UNA), as well as single incorporation of 4-thiouridine and all four canonical UNAs, was evaluated. The studies presented herein have shown that 4-thiouridine in RNA and UNA series, as well as all four canonical UNAs, can efficiently modulate G-quadruplex thermodynamic and biological stability, and that the effect is strongly position dependent. Interestingly, TBA variants containing the modified nucleotide residues are characterized by unchanged folding topology. Thrombin time assay revealed that incorporation of certain UNA residues may improve G-quadruplex anticoagulant properties. Noteworthy, some TBA variants, characterized by decreased ability to inhibit thrombin activity, possess significant antiproliferative properties reducing the viability of the HeLa cell line even by 95% at 10 μM concentration

Structural determinants for alternative splicing regulation of the MAPT pre-mRNA

<p>Alternative splicing at the MAPT gene exon 10 yields similar levels of the 3R and 4R tau protein isoforms.<a href="#cit0001" target="_blank">¹</a> The presence of mutations, particularly in exon 10 and intron 10–11, changes the quantity of tau isoforms. Domination each of the isoform yields tau protein aggregation and frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Here, we report for the first time the secondary structure of the 194/195 nucleotide region for the wild type (WT) and 10 mutants of the MAPT gene pre-mRNA determined using both chemical and microarray mapping. Thermodynamic analyses indicate that single nucleotide mutations in the splicing regulatory element (SRE) that form a hairpin affect its stability by up to 4 and 7 kcal/mol. Moreover, binding the regulatory hairpin of small molecule ligands (neomycin, kanamycin, tobramycin and mitoxantrone) enhance its stability depending on the nature of the ligands and the RNA mutations. Experiments using the cos-7 cell line indicate that the presence of ligands and modified antisense oligonucleotides affect the quantity of 3R and 4R isoforms. This finding correlates with the thermodynamic stability of the regulatory hairpin. An alternative splicing regulation mechanism for exon 10 is postulated based on our experimental data and on published data.</p

A Tandem Oligonucleotide Approach for SNP-Selective RNA Degradation Using Modified Antisense Oligonucleotides

<div><p>Antisense oligonucleotides have been studied for many years as a tool for gene silencing. One of the most difficult cases of selective RNA silencing involves the alleles of single nucleotide polymorphisms, in which the allele sequence is differentiated by a single nucleotide. A new approach to improve the performance of allele selectivity for antisense oligonucleotides is proposed. It is based on the simultaneous application of two oligonucleotides. One is complementary to the mutated form of the targeted RNA and is able to activate RNase H to cleave the RNA. The other oligonucleotide, which is complementary to the wild type allele of the targeted RNA, is able to inhibit RNase H cleavage. Five types of SNPs, C/G, G/C, G/A, A/G, and C/U, were analyzed within the sequence context of genes associated with neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, ALS (Amyotrophic Lateral Sclerosis), and Machado-Joseph disease. For most analyzed cases, the application of the tandem approach increased allele-selective RNA degradation 1.5–15 fold relative to the use of a single antisense oligonucleotide. The presented study proves that differentiation between single substitution is highly dependent on the nature of the SNP and surrounding nucleotides. These variables are crucial for determining the proper length of the inhibitor antisense oligonucleotide. In the tandem approach, the comparison of thermodynamic stability of the favorable duplexes WT RNA-inhibitor and Mut RNA-gapmer with the other possible duplexes allows for the evaluation of chances for the allele-selective degradation of RNA. A larger difference in thermodynamic stability between favorable duplexes and those that could possibly form, usually results in the better allele selectivity of RNA degradation.</p></div

The RNase H assay results for G46A SNP within <i>SNCA</i> mRNA.

<p><b>(A)</b> kinetics of the WT and Mut RNAs cleavage in the presence of the gapmer (kKM) and the shorter (Ik1) or longer (Ik2) inhibitor, where black squares indicate degradation of WT RNA in the presence of the Ik1/kKM/Mut RNA mixture, red dots—degradation of WT RNA in the presence of the Ik2/kKM/ Mut RNA mixture, blue triangles—degradation of Mut RNA in the presence of the Ik1/kKM/WT RNA mixture and green triangles—degradation of Mut RNA in the presence of the Ik2/kKM/WT RNA mixture, <b>(B)</b> stability of the WT RNA (green bar) and Mut RNA (red bar) in the presence of: gapmer kKM only (first pair of bars from the left), gapmer kKM and short inhibitor Ik1 (second) or gapmer kKM and longer inhibitor Ik2 (third), and in the WT/Mut RNA/kKM/Ik1 mixture (fourth) and WT/Mut RNA/kKM/Ik2 mixture (fifth). Statistically significant differences between the mean hydrolysis efficiency of the RNA variants (P<0.05) are marked with asterisk, <b>(C,D)</b> Results of <i>HeLa</i> cells cotransfection with WT/Mut G46A -pEGFP constructs and different amounts of inhibitor and gapmer antisense oligonucleotides. qPCR results of tandem approach with <b>(C)</b> shorter inhibitor Ik1, <b>(D)</b> Longer inhibitor Ik2. Statistically significant differences between the mean of the RNA variants expression (P<0.05) are marked with asterisk.</p