Laserová záchytná mikrodisekce (Laser capture microdissection, LCM) je rychlá a spolehlivá metoda, která umožňuje izolaci cílových buněk ze specifického komplexu tkáně pro jejich následnou molekulární nebo proteinovou analýzu. Základem LCM je inverzní mikroskop se zabudovaným nízkovýkonnostním infračerveným laserem. Nařezané tkáně jsou upevněny na standardní podložní sklo a termoplastická membrána (TM) je umístuna nad dehydratovaný preparát. V ohnisku laserového mikroskopu je umístněna TM, kterou laser roztaví v požadovaném místě a naváže tak cílovou buňku či strukturu k membráně. V současné době máme k dispozici několik laserových mikrodisekčních systémů, které se liší způsobem zachycení disekovaných buněk, v konfiguraci systému i v jednotlivých aplikacích. Laserovou mikrodisekci lze použít pro izolaci buněk u řady typů buněčných i tkáňových preparátů, včetně zamražených vzorků, formalínem fixovaných parafinizovaných tkání či cytologických preparátů. V závislosti na použitém materiálu je možno z mikrodisekovaných buněk extrahovat DNA, RNA či proteiny v dobré kvalitě. Kombinací s dalšími technikami, jako je například cDNA microarray, LCM pomáhá identifikovat nové diagnostické a prognostické znaky vedoucí ke zlepšení diagnosticko terapeutických metod v léčbě onkologických onemocnění. Na našem pracovišti jsme laserovou mikrodisekcí izolovali buňky z cytologických preparátů adenokarcinomu plic získaných punkční cytologií zmražených či parafinizovaných nádorů a také buněčné linie, např. myeloidní leukemii K562. V této práci popisujeme naše zkušenosti se zavedením LCM s následnou mikroizolací DNA/RNA a lineární amplifikací DNA/RNA pro účely dalších genetických analýz jako je např. komparativní genomická hybridizace, expresní studie, či přímé sekvenování vyšetřovaných genů z biologického materiálu s minimálním obsahem nádorových buněk, či pro studium nádorové heterogenity na jednobuněčné úrovni.

Laser capture microdissection (LCM) is a rapid, reliable method to obtain pure populations of targeted cells from specific microscopic regions of tissue sections for subsequent analysis. LCM is based on the adherence of visually selected cells to a thermoplastic membrane, which overlies the dehydrated tissue section and is focally melted by triggering of a low energy infrared laser pulse. Tissue sections are mounted on standard glass slides, and transparent thermoplastic membrane is then placed over the dry section. The laser provides enough energy to transiently melt this thermoplastic film in to the target cells. Several systems are available for LCM, and vary in cell-capture method, system configuration and applications. LCM was applied to a wide range of cell and tissue preparations including frozen samples, formalin-fixed paraffin-embedded tissues or cytology smears. Depending on the starting material, DNA, good quality mRNA, and proteins can by extracted successfully from captured tissue fragments, down to the single cell level. In combination with another techniques like expression library construction and cDNA array hybridisation, LCM will allow the establishment of new diagnostic and prognostic markers, in order to indicate therapy individually tailored to the molecular profile of a given tumour. In this paper we refer our experiences with the LCM isolation of single cells from cytology smears of lung carcinomas, frozen and paraffin embedded tumour tissues as well as cell line cytospin preparation. Our ultimate goal was to introduce LCM technology in combination with DNA/RNA isolation and linear amplification for subsequent genomic analyses such as comparative genomic hybridisation, RNA expression studies and specific amplifications of investigated genes from tissue specimens with minority of tumour cells and/or for tumour heterogeneity studies based on one the single cell level.

• MeSH
• erbB receptory genetika   izolace a purifikace   MeSH
• financování organizované MeSH
• geny erbB-1 genetika   MeSH
• geny ras genetika   MeSH
• hybridizace genetická MeSH
• lasery využití   MeSH
• lékařská onkologie metody   trendy   MeSH
• lidé MeSH
• mikrodisekce metody   přístrojové vybavení   využití   MeSH
• odběr biologického vzorku metody   využití   MeSH
• srovnávací genomová hybridizace MeSH
• techniky amplifikace nukleových kyselin metody   přístrojové vybavení   využití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

The application of rapid, specific, and sensitive methods for pathogen detection and quantification is very advantageous in diagnosis of human pathogens in several applications, including food analysis. The aim of this study was the evaluation of a method for the multiplexed detection and quantification of three significant foodborne pathogenic species (Escherichia coli O157, Salmonella spp., and Listeria monocytogenes). The assay combines specific DNA extraction by multiplex magnetic capture hybridization (mMCH) with multiplex real-time PCR. The amplification assay showed linearity in the range 106-10 genomic units (GU)/PCR for each co-amplified species. The sensitivity corresponded to 1 GU/PCR for E. coli O157 and L. monocytogenes, and 10 GU/PCR for Salmonella spp. The immobilization process and the hybrid capture of the MCH showed good efficiency and reproducibility for all targets, allowing the combination in equal amounts of the different nanoparticle types in mMCH. MCH and mMCH efficiencies were similar. The detection limit of the method was 10 CFU in samples with individual pathogens and 102 CFU in samples with combination of the three pathogens in unequal amounts (amount's differences of 2 or 3 log). In conclusion, this multiplex molecular platform can be applied to determine the presence of target species in food samples after culture enrichment. In this way, this method could be a time-saving and sensitive tool to be used in routine diagnosis.

• MeSH
• Escherichia coli O157 klasifikace   genetika   MeSH
• hybridizace nukleových kyselin MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• Listeria monocytogenes klasifikace   genetika   MeSH
• multiplexová polymerázová řetězová reakce MeSH
• Salmonella klasifikace   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a single-stranded positive-sense RNA virus. Like other coronaviruses, SARS-CoV-2 has an unusually large genome that encodes four structural proteins and sixteen nonstructural proteins. The structural nucleocapsid phosphoprotein N is essential for linking the viral genome to the viral membrane. Both N-terminal RNA binding (N-NTD) and C-terminal dimerization domains are involved in capturing the RNA genome and, the intrinsically disordered region between these domains anchors the ribonucleoprotein complex to the viral membrane. Here, we characterized the structure of the N-NTD and its interaction with RNA using NMR spectroscopy. We observed a positively charged canyon on the surface of the N-NTD that might serve as a putative RNA binding site similarly to other coronaviruses. The subsequent NMR titrations using single-stranded and double-stranded RNA revealed a much more extensive U-shaped RNA-binding cleft lined with regularly distributed arginines and lysines. The NMR data supported by mutational analysis allowed us to construct hybrid atomic models of the N-NTD/RNA complex that provided detailed insight into RNA recognition.

• MeSH
• COVID-19 * MeSH
• fosfoproteiny chemie   genetika   MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• nukleokapsida - proteiny chemie   genetika   MeSH
• RNA virová chemie   genetika   MeSH
• SARS-CoV-2 chemie   genetika   MeSH
• simulace molekulového dockingu * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

OBJECTIVE: In this work we aim to investigate the origins and genetic affinities of Bronze Age populations (2,400-1,100 BC) from the region of southern Poland and to trace maternal kinship patterns present in the burials of those populations by the use of complete mitochondrial genomes. MATERIALS AND METHODS: We performed ancient DNA analyses for Bronze Age individuals from present-day Poland associated with the Strzyżow culture, the Mierzanowice culture, and the Trzciniec Cultural circle. To obtain complete mitochondrial genomes, we sequenced genomic libraries using Illumina platform. Additionally, hybridization capture was used to enrich some of the samples for mitochondrial DNA. AMS 14 C-dating was conducted for 51 individuals to verify chronological and cultural attribution of the analyzed samples. RESULTS: Complete ancient mitochondrial genomes were generated for 80 of the Bronze Age individuals from present-day Poland. The results of the population genetic analyses indicate close maternal genetic affinity between Mierzanowice, Trzciniec, and Corded Ware culture-associated populations. This is in contrast to the genetically more distant Strzyżów people that displayed closer maternal genetic relation to steppe populations associated with the preceding Yamnaya culture and Catacomb culture, and with later Scythians. Potential maternal kinship relations were identified in burials of Mierzanowice and Trzciniec populations analyzed in this study. DISCUSSION: Results revealed genetic continuity from the Late Neolithic Corded Ware groups to Bronze Age Mierzanowice and Trzciniec-associated populations, and possible additional genetic contribution from the steppe to the formation of the Strzyżów-associated group at the end of 3rd millennium BC. Mitochondrial patterns indicated several pairs of potentially maternally related individuals mostly in Trzciniec-associated group.

• MeSH
• antropologie fyzická MeSH
• běloši genetika   MeSH
• dějiny starověku MeSH
• dítě MeSH
• dospělí MeSH
• genom mitochondriální genetika   MeSH
• haplotypy genetika   MeSH
• hřbitovy MeSH
• lidé MeSH
• migrace lidstva MeSH
• populační genetika * MeSH
• starobylá DNA analýza   MeSH
• Check Tag
• dějiny starověku MeSH
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• historické články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Polsko MeSH

• Klíčová slova
• Gardasil 9, anoskopie, genom Hybrid Capture 2,
• MeSH
• cytologické techniky MeSH
• diagnostické techniky a postupy MeSH
• dítě MeSH
• DNA testy na papilomavirus MeSH
• dospělí MeSH
• gynekologické vyšetření * metody   ošetřování   přístrojové vybavení   využití   MeSH
• gynekologie * MeSH
• hodnocení léčiv MeSH
• infekce papilomavirem * diagnóza   epidemiologie   prevence a kontrola   MeSH
• klinické zkoušky jako téma MeSH
• kongresy jako téma * MeSH
• lékařská genetika metody   normy   pracovní síly   zákonodárství a právo   MeSH
• lidé MeSH
• mladiství MeSH
• molekulární biologie MeSH
• multicentrické studie jako téma MeSH
• nádorové biomarkery MeSH
• nádory anu diagnóza   etiologie   mortalita   prevence a kontrola   MeSH
• nádory děložního čípku diagnóza   prevence a kontrola   MeSH
• palpační vyšetření konečníku * metody   ošetřování   přístrojové vybavení   využití   MeSH
• plošný screening * metody   metody   normy   organizace a řízení   využití   MeSH
• prekancerózy MeSH
• prenatální péče * metody   MeSH
• randomizované kontrolované studie jako téma MeSH
• registrace * MeSH
• senzitivita a specificita MeSH
• směrnice pro lékařskou praxi jako téma MeSH
• těhotenství * MeSH
• vakcíny proti papilomavirům * farmakologie   terapeutické užití   MeSH
• výsledek terapie MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• těhotenství * MeSH
• ženské pohlaví MeSH

A taxonomic division of the family Bovidae (Artiodactyla) is difficult and the evolutionary relationships among most bovid subfamilies remain uncertain. In this study, we isolated the cattle satellite I clone BTREP15 (1.715 satellite DNA family) and autosomal centromeric DNAs of members of ten bovid tribes. We wished to determine whether the analysis of fluorescence in situ hybridization patterns of the cattle satellite I clone (BTREP15) and tribe-specific centromeric repeats isolated by laser microdissection would help to reveal some of the ambiguities occurring in the systematic classification of the family Bovidae. The FISH study of the presence and distribution of the cattle satellite I clone BTREP15 (1.715 satellite DNA family) within members of ten bovid tribes was not informative. FISH analysis of autosomal centromeric DNA probes in several species within one tribe revealed similar hybridization patterns in autosomes confirming tribal homogeneity of these probes. Sex chromosomes showed considerable variation in sequence composition and arrangement not only between tribes but also between species of one tribe. According to our findings it seems that Oreotragus oreotragus developed its own specific satellite DNA which does not hybridize to any other bovid species analysed. Our results suggest O. oreotragus as well as Aepyceros melampus may be unique species not particularly closely related to any of the recognized bovid tribes. This study indicates the isolation of tribe-specific centromeric DNAs by laser microdissection and cloning the sequence representing the main motif of these repetitive DNAs could offer the perspectives for comparative phylogenetic studies.

• MeSH
• biologická evoluce MeSH
• centromera chemie   genetika   MeSH
• DNA sondy MeSH
• fylogeneze MeSH
• hybridizace in situ fluorescenční MeSH
• laserová záchytná mikrodisekce MeSH
• přežvýkavci klasifikace   genetika   MeSH
• satelitní DNA genetika   MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Salivary gland carcinomas represent a heterogeneous group of poorly characterized head and neck tumors. The purpose of this study was to evaluate ALK gene and protein aberrations in a large, well-characterized cohort of these tumors. A total of 182 salivary gland carcinomas were tested for anaplastic lymphoma kinase (ALK) positivity by immunohistochemistry (IHC) using the cut-off of 10% positive cells. ALK positive tumors were subjected to FISH analysis and followed by hybrid capture-based next generation sequencing (NGS). Of the 182 tumors, 8 were ALK positive by IHC. Further analysis using hybrid capture NGS analysis revealed a novel MYO18A (Exon1-40)-ALK (exon 20-29) gene fusion in one case of intraductal carcinoma. Additional genomic analyses resulted in the detection of inactivating mutations in BRAF and TP53, as well as amplifications of ERBB2 and ALK. ALK rearrangements are a rare entity in salivary gland carcinomas. We identified a potentially targetable novel ALK fusion in an intraductal carcinoma of minor salivary glands.

• MeSH
• amplifikace genu MeSH
• anaplastická lymfomová kináza genetika   MeSH
• dítě MeSH
• dospělí MeSH
• fúze genů MeSH
• hybridizace in situ fluorescenční MeSH
• imunohistochemie MeSH
• intraduktální neinfiltrující karcinom enzymologie   genetika   patologie   MeSH
• karcinom enzymologie   genetika   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• nádorové biomarkery genetika   MeSH
• nádory slinných žláz enzymologie   genetika   patologie   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Peptide nucleic acids (PNAs) are synthetic DNA mimics in which the sugar phosphate backbone is replaced by repeating N-(2-aminoethyl) glycine units linked by an amine bond and to which the nucleobases are fixed. Peptide nucleic acids hybridize with complementary nucleic acids with remarkably high affinity and specificity, essentially because of their uncharged and flexible polyamide backbone. The unique physicochemical properties of PNAs have led to the development of a large variety of biological research assays, and, over the last few years, PNAs have proved their powerful usefulness in genetic and cytogenetic diagnostic procedures. Several sensitive and robust PNA-dependent methods have been designed for modulating polymerase chain reactions, detecting genomic mutation or capturing nucleic acids. The more recent applications of PNA involve their use as molecular hybridization probes. Thus, the in situ detection of several human chromosomes has been reported in various types of tissues.

• MeSH
• hybridizace in situ fluorescenční MeSH
• lidé MeSH
• lidské chromozomy * MeSH
• peptidové nukleové kyseliny * genetika   chemie   MeSH
• regulace genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

There is a demand for efficient tools for the monitoring of RNase H activity. We report on a new assay which allows for simultaneous (1) real-time monitoring of RNase H activity and (2) detection of cleavage reaction products. The dual assay is implemented using a multichannel surface plasmon resonance (SPR) biosensor with two independently functionalized sensing areas in a single fluidic path. In the first sensing area the RNA cleavage by RNase H is monitored, while the products of the cleavage reaction are captured in the second sensing area with specific DNA probes. The assay was optimized with respect to AON concentration and temperature. A significant improvement was obtained with special chimeric probes, which contain RNA substrate for RNase H and a longer deoxyribonucleotide tail, which enhances the SPR signal. It has been shown that RNase H stabilizes the RNA:DNA hybrid duplex before the cleavage. The potential of the assay is demonstrated in the study in which the ability of natural and modified oligonucleotides to activate RNase H is examined.

• MeSH
• DNA sondy MeSH
• heteroduplexy nukleové kyseliny MeSH
• povrchová plasmonová rezonance přístrojové vybavení   metody   MeSH
• proteiny z Escherichia coli analýza   metabolismus   MeSH
• ribonukleasa H analýza   metabolismus   MeSH
• RNA sondy MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

Triticale (X Triticosecale Wittmack), a wheat-rye small grain crop hybrid, combines wheat and rye attributes in one hexaploid genome. It is characterized by high adaptability to adverse environmental conditions: drought, soil acidity, salinity and heavy metal ions, poorer soil quality, and waterlogging. So that its cultivation is prospective in a changing climate. Here, we describe RGB on-ground phenotyping of field-grown eighteen triticale market-available cultivars, made in naturally changing light conditions, in two consecutive winter cereals growing seasons: 2018-2019 and 2019-2020. The number of ears was counted on top-down images with an accuracy of 95% and mean average precision (mAP) of 0.71 using advanced object detection algorithm YOLOv4, with ensemble modeling of field imaging captured in two different illumination conditions. A correlation between the number of ears and yield was achieved at the statistical importance of 0.16 for data from 2019. Results are discussed from the perspective of modern breeding and phenotyping bottleneck.

• MeSH
• jedlá semena genetika   MeSH
• prospektivní studie MeSH
• půda MeSH
• šlechtění rostlin MeSH
• triticale * MeSH
• Publikační typ
• časopisecké články MeSH