BACKGROUND AND OBJECTIVES: Utilisation of the one-step nucleic acid amplification (OSNA) molecular biology method for the detection of the metastatic involvement of sentinel lymph nodes (SLNs) in endometrial cancer (EC) patients. A comparison with histopathological ultrastaging and a description of the clinical consequences. METHODS: Surgically treated EC patients underwent detection of SLNs. Nodes greater than 5 mm were cut into sections 2-mm thick parallel to the short axis of the node. Odd sections were examined according to the OSNA method, while even ones according to an appropriate ultrastaging protocol. Nodes less than or equal to 5 mm were cut into halves along the longitudinal axis with one half examined according to the OSNA method and the other half by ultrastaging. RESULTS: Fifty-eight patients were included and 135 SLNs were acquired. Both ultrastaging and OSNA agreed on 116 results. According to the OSNA method, 20.69% more patients were classified into International Federation of Gynecology and Obstetrics (FIGO) stage III. When comparing the results of the OSNA method to the conclusions of ultrastaging as a reference method, sensitivity of 90.9%, specificity of 85.5% and concordance of 85.9% were attained. CONCLUSIONS: The results of the OSNA method showed a higher frequency of detection of micrometastases and included 20.69% more patients into FIGO stage III.

• Klíčová slova
• cytokeratin 19, micrometastasis, one-step nucleic acid amplification assay,
• MeSH
• adenokarcinom z jasných buněk genetika   sekundární   chirurgie   MeSH
• biopsie sentinelové lymfatické uzliny MeSH
• dospělí MeSH
• keratin-19 genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lymfatické metastázy MeSH
• mikrometastázy MeSH
• míra přežití MeSH
• nádorové biomarkery genetika   MeSH
• nádory endometria genetika   patologie   chirurgie   MeSH
• následné studie MeSH
• nukleové kyseliny analýza   genetika   MeSH
• peroperační doba MeSH
• prognóza MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• sentinelová uzlina patologie   chirurgie   MeSH
• serózní cystadenokarcinom genetika   sekundární   chirurgie   MeSH
• techniky amplifikace nukleových kyselin metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• keratin-19 MeSH
• nádorové biomarkery MeSH
• nukleové kyseliny MeSH

We have carried out extended set of μs-scale explicit solvent MD simulations of all possible G-triplexes which can participate in folding pathways of the human telomeric quadruplex. Our study accumulates almost 60 μs of simulation data, which is by about three orders of magnitude larger sampling compared to the earlier simulations of human telomeric G-DNA triplexes. Starting structures were obtained from experimental quadruplex structures by deleting either the first or the last strand. The life-times of antiparallel triplexes with lateral and diagonal loops are at least on μs-scale, which should be sufficient to contribute to the folding pathways. However, the triplex states may involve structures with various local deviations from the ideal triplexes, such as strand tilting and various alternative and incomplete triads. The simulations reveal easy rearrangements between lateral and diagonal loop triplex topologies. Propeller loops of antiparallel triplexes may to certain extent interfere with the G-triplexes but these structures are still viable candidates to participate in the folding. In contrast, all-parallel all-anti triplexes are very unstable and are unlikely to contribute to the folding. Although our simulations demonstrate that antiparallel G-triplexes, if folded, would have life-times sufficient to participate in the quadruplex folding, the results do not rule out the possibility that the G-triplexes are out-competed by other structures not included in our study. Among them, numerous possible misfolded structures containing guanine quartets can act as off-path intermediates with longer life-times than the triplexes. Besides analyzing the structural dynamics of a diverse set of G-DNA triplexes, we also provide a brief discussion of the limitations of the simulation methodology, which is necessary for proper understanding of the simulation data.

• Klíčová slova
• G-DNA folding, Molecular dynamics, Quadruplex, Telomere, Triplex,
• MeSH
• DNA chemie   MeSH
• G-kvadruplexy * MeSH
• konformace nukleové kyseliny * MeSH
• lidé MeSH
• nukleové kyseliny chemie   genetika   MeSH
• sekvence nukleotidů MeSH
• simulace molekulární dynamiky MeSH
• telomery chemie   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• nukleové kyseliny MeSH

Based on the matrix-addressing sequence of mitochondrial ribosomal 5S-rRNA (termed MAM), which is naturally imported into mitochondria, we have constructed an import system for in vivo targeting of mitochondrial DNA (mtDNA) or mt-mRNA, in order to provide fluorescence hybridization of the desired sequences. Thus DNA oligonucleotides were constructed, containing the 5'-flanked T7 RNA polymerase promoter. After in vitro transcription and fluorescent labeling with Alexa Fluor(®) 488 or 647 dye, we obtained the fluorescent "L-ND5 probe" containing MAM and exemplar cargo, i.e., annealing sequence to a short portion of ND5 mRNA and to the light-strand mtDNA complementary to the heavy strand nd5 mt gene (5'-end 21 base pair sequence). For mitochondrial in vivo fluorescent hybridization, HepG2 cells were treated with dequalinium micelles, containing the fluorescent probes, bringing the probes proximally to the mitochondrial outer membrane and to the natural import system. A verification of import into the mitochondrial matrix of cultured HepG2 cells was provided by confocal microscopy colocalizations. Transfections using lipofectamine or probes without 5S-rRNA addressing MAM sequence or with MAM only were ineffective. Alternatively, the same DNA oligonucleotides with 5'-CACC overhang (substituting T7 promoter) were transcribed from the tetracycline-inducible pENTRH1/TO vector in human embryonic kidney T-REx®-293 cells, while mitochondrial matrix localization after import of the resulting unlabeled RNA was detected by PCR. The MAM-containing probe was then enriched by three-order of magnitude over the natural ND5 mRNA in the mitochondrial matrix. In conclusion, we present a proof-of-principle for mitochondrial in vivo hybridization and mitochondrial nucleic acid import.

• MeSH
• genetická transkripce MeSH
• lidé MeSH
• mitochondriální DNA chemie   genetika   MeSH
• nukleové kyseliny chemie   genetika   MeSH
• oligonukleotidy chemie   genetika   MeSH
• RNA ribozomální chemie   genetika   MeSH
• RNA chemie   genetika   MeSH
• sekvenční homologie nukleových kyselin MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální DNA MeSH
• nukleové kyseliny MeSH
• oligonukleotidy MeSH
• RNA ribozomální MeSH
• RNA MeSH

Förster resonance energy transfer (FRET) microscopy is a powerful technique routinely used to monitor interactions between biomolecules. Here, we focus on the techniques that are used for investigating the structure and interactions of nucleic acids (NAs). We present a brief overview of the most commonly used FRET microscopy techniques, their advantages and drawbacks. We list experimental approaches recently used for either in vitro or in vivo studies. Next, we summarize how FRET contributed to the understanding of pre-mRNA splicing and spliceosome assembly.

• MeSH
• alternativní sestřih MeSH
• fluorescenční mikroskopie * MeSH
• molekulární konformace MeSH
• nukleoproteiny metabolismus   MeSH
• nukleové kyseliny chemie   genetika   metabolismus   MeSH
• prekurzory RNA chemie   genetika   metabolismus   MeSH
• rezonanční přenos fluorescenční energie * MeSH
• sestřih RNA * MeSH
• spliceozomy genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• nukleoproteiny MeSH
• nukleové kyseliny MeSH
• prekurzory RNA MeSH

If we look at the history of our knowledge of nucleic acids, we would see in the distant past of 140 years Friedrich Miescher who had identified the acidic substance within the cell nucleus, which he called nuclein. About 70 years after his initial observation, this substance was connected with genetic information. This very substantial finding happened during the World War II. This was the impulse that research of nucleic acids received to speed up continuously growing mountain of information, which is more and more difficult to understand. Another eruption of new information about our genome was the result of ten years of intensive cooperation of many manufacturers divided into two competitive blocks which offered us knowledge of nucleotide sequence of all 46 DNA molecules. The year 2000 became the landmark marking the start of the postgenomic era. It did not mean that human genome was totally explored, but the cornerstone has been settled. Since then, we could concentrate our efforts on variability; use of the project of 1,000 genomes brought many important findings, eg. copy number variability (CNV) exceeds the single nucleotide polymophisms (SNP). Also intergenomic relationships, studies on function and pathways began to be much more understandable by elucidation of the genome primary structure. NGS as a tool also accelerated the epigenetic research. All this improved molecular diagnostics by discovering many new markers playing their role in disease and treatment and allowed us to enter the field of multifactorial illnesses including cancer. The progress in diagnostic technologies which has happened during the last decade forced our research teams to include other professions - eg. bioinformatics.

• MeSH
• dějiny 20. století MeSH
• dějiny 21. století MeSH
• diagnostické techniky molekulární * MeSH
• lékařská genetika MeSH
• lidé MeSH
• nukleové kyseliny * genetika   dějiny   MeSH
• Check Tag
• dějiny 20. století MeSH
• dějiny 21. století MeSH
• lidé MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• historické články MeSH
• přehledy MeSH
• Názvy látek
• nukleové kyseliny * MeSH

Three- and four-body nonadditivities in the uracil tetramer (in DNA-like geometry) and the GC step (in crystal geometry) were investigated at various levels of the wave-function theory: HF, MP2, MP3, L-CCD, CCSD and CCSD(T). All of the calculations were performed using the 6-31G**(0.25,0.15) basis set, whereas the HF, MP2 and the MP3 nonadditivities were, for the sake of comparison, also determined with the much larger aug-cc-pVDZ basis set. The HF and MP2 levels do not provide reliable values for many-body terms, making it necessary to go beyond the MP2 level. The benchmark CCSD(T) three- and four-body nonadditivities are reasonably well reproduced at the MP3 level, and almost quantitative agreement is obtained (fortuitously) either on the L-CCD level or as an average of the MP3 and the CCSD results. Reliable values of many-body terms (especially their higher-order correlation contributions) are obtained already when the rather small 6-31G**(0.25,0.15) basis set is used. The four-body term is much smaller when compared to the three-body terms, but it is definitely not negligible, e.g. in the case of the GC step it represents about 16% of all of the three- and four-body terms. While investigating the geometry dependence of many-body terms for the GG step at the MP3/6-31G**(0.25,0.15) level, we found that it is necessary to include at least three-body terms in the determination of optimal geometry parameters.

• MeSH
• cytosin chemie   MeSH
• guanin chemie   MeSH
• kvantová teorie * MeSH
• molekulární modely MeSH
• nukleové kyseliny chemie   genetika   MeSH
• párování bází MeSH
• polymery chemie   MeSH
• sekvence nukleotidů MeSH
• studie proveditelnosti MeSH
• termodynamika MeSH
• uracil chemie   MeSH
• vodíková vazba MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytosin MeSH
• guanin MeSH
• nukleové kyseliny MeSH
• polymery MeSH
• uracil MeSH

We analyzed occurrences of bases in 20,352 introns, exons of 25,574 protein-coding genes, and among the three codon positions in the protein-coding sequences. The nucleotide sequences originated from the whole spectrum of organisms from bacteria to primates. The analysis revealed the following: (1) In most exons, adenine dominates over thymine. In other words, adenine and thymine are distributed in an asymmetric way between the exon and the complementary strand, and the coding sequence is mostly located in the adenine-rich strand. (2) Thymine dominates over adenine not only in the strand complementary to the exon but also in introns. (3) A general bias is further revealed in the distribution of adenine and thymine among the three codon positions in the exons, where adenine dominates over thymine in the second and mainly the first codon position while the reverse holds in the third codon position. The product (A1/T1)x(A2/T2)x(T3/A3) is smaller than one in only a few analyzed genes.

• MeSH
• adenin analýza   MeSH
• DNA chemie   genetika   MeSH
• eukaryotické buňky MeSH
• exony genetika   MeSH
• genetický kód MeSH
• genom MeSH
• geny genetika   MeSH
• introny genetika   MeSH
• nukleové kyseliny chemie   genetika   MeSH
• prokaryotické buňky MeSH
• RNA chemie   genetika   MeSH
• thymin analýza   MeSH
• zastoupení bazí * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• adenin MeSH
• DNA MeSH
• nukleové kyseliny MeSH
• RNA MeSH
• thymin MeSH