Quantitative genomic mapping of DNA damage may provide insights into the underlying mechanisms of damage and repair. Sequencing based approaches are bound to the limitations of PCR amplification bias and read length which hamper both the accurate quantitation of damage events and the ability to map them to structurally complex genomic regions. Optical Genome mapping in arrays of parallel nanochannels allows physical extension and genetic profiling of millions of long genomic DNA fragments, and has matured to clinical utility for characterization of complex structural aberrations in cancer genomes. Here we present a new mapping modality, Repair-Assisted Damage Detection - Optical Genome Mapping (RADD-OGM), a method for single-molecule level mapping of DNA damage on a genome-wide scale. Leveraging ultra-long reads to assemble the complex structure of a sarcoma cell-line genome, we mapped the genomic distribution of oxidative DNA damage, identifying regions more susceptible to DNA oxidation. We also investigated DNA repair by allowing cells to repair chemically induced DNA damage, pinpointing locations of concentrated repair activity, and highlighting variations in repair efficiency. Our results showcase the potential of the method for toxicogenomic studies, mapping the effect of DNA damaging agents such as drugs and radiation, as well as following specific DNA repair pathways by selective induction of DNA damage. The facile integration with optical genome mapping enables performing such analyses even in highly rearranged genomes such as those common in many cancers, a challenging task for sequencing-based approaches.

• MeSH
• bromičnany toxicita   MeSH
• lidé MeSH
• mapování chromozomů * přístrojové vybavení   metody   MeSH
• mikrofluidní analytické techniky * přístrojové vybavení   metody   MeSH
• nádorové buněčné linie MeSH
• nanotechnologie * přístrojové vybavení   metody   MeSH
• oprava DNA genetika   MeSH
• oxidační stres účinky léků   genetika   MeSH
• poškození DNA * genetika   MeSH
• regulace genové exprese MeSH
• stanovení celkové genové exprese MeSH
• toxikogenetika * přístrojové vybavení   metody   MeSH
• variabilita počtu kopií segmentů DNA MeSH
• zobrazení jednotlivé molekuly * přístrojové vybavení   metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Classical models of gene expression were built using genetics and biochemistry. Although these approaches are powerful, they have very limited consideration of the spatial and temporal organization of gene expression. Although the spatial organization and dynamics of RNA polymerase II (RNAPII) transcription machinery have fundamental functional consequences for gene expression, its detailed studies have been abrogated by the limits of classical light microscopy for a long time. The advent of super-resolution microscopy (SRM) techniques allowed for the visualization of the RNAPII transcription machinery with nanometer resolution and millisecond precision. In this review, we summarize the recent methodological advances in SRM, focus on its application for studies of the nanoscale organization in space and time of RNAPII transcription, and discuss its consequences for the mechanistic understanding of gene expression.

• MeSH
• fluorescenční mikroskopie * metody   MeSH
• genetická transkripce * MeSH
• lidé MeSH
• regulace genové exprese * MeSH
• RNA-polymerasa II metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• vazba proteinů MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

• MeSH
• DNA vazebné proteiny analýza   chemie   ultrastruktura   MeSH
• DNA chemie   MeSH
• fluorescenční mikroskopie MeSH
• konfokální mikroskopie MeSH
• nukleotidy chemie   MeSH
• oprava DNA MeSH

DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET 15N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10⁻20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90°) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10° relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site.

• MeSH
• 53BP1 metabolismus   MeSH
• konfokální mikroskopie metody   MeSH
• kultivované buňky MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• rekombinační oprava DNA * MeSH
• transport proteinů MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: The association between increases in cardiac troponin and adverse cardiac outcomes is well established. There is a growing interest in exploring routine cardiac troponin monitoring as a potential early indicator of adverse heart health trends. Prognostic use of cardiac troponin measurements requires an assay with very high sensitivity and outstanding analytical performance. We report development and preliminary validation of an investigational assay meeting these requirements and demonstrate its applicability to cohorts of healthy individuals and patients with heart failure. METHODS: On the basis of single molecule array technology, we developed a 45-min immunoassay for cardiac troponin I (cTnI) for use on a novel, fully automated digital analyzer. We characterized its analytical performance and measured cTnI in healthy individuals and heart failure patients in a preliminary study of assay analytical efficacy. RESULTS: The assay exhibited a limit of detection of 0.01 ng/L, a limit of quantification of 0.08 ng/L, and a total CV of 10% at 2.0 ng/L. cTnI concentrations were well above the assay limit of detection for all samples tested, including samples from healthy individuals. cTnI was significantly higher in heart failure patients, and exhibited increasing median and interquartile concentrations with increasing New York Heart Association classification of heart failure severity. CONCLUSIONS: The robust 2-log increase in sensitivity relative to contemporary high-sensitivity cardiac troponin immunoassays, combined with full automation, make this assay suitable for exploring cTnI concentrations in cohorts of healthy individuals and for the potential prognostic application of serial cardiac troponin measurements in both apparently healthy and diseased individuals.

• MeSH
• čipová analýza proteinů ekonomika   metody   MeSH
• dospělí MeSH
• imunoanalýza ekonomika   metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• limita detekce MeSH
• mladý dospělý MeSH
• srdeční selhání krev   diagnóza   MeSH
• troponin I analýza   krev   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

• MeSH
• chromozomy rostlin chemie   genetika   metabolismus   MeSH
• DNA-topoisomerasy typu II metabolismus   MeSH
• fluorescenční barviva chemie   MeSH
• fluorescenční mikroskopie metody   MeSH
• indoly chemie   MeSH
• ječmen (rod) cytologie   genetika   MeSH
• konfokální mikroskopie metody   MeSH
• metafáze genetika   MeSH
• reprodukovatelnost výsledků MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

RNA-binding proteins (RBPs) are critical to posttranscriptional gene regulation. Therefore, characterization of the RNA molecules bound by RBPs in vivo represent a key step in elucidating their function. The recently developed iCLIP technique allows single nucleotide resolution of the RNA binding footprints of RBPs. We present the iCLIP technique modified for its application to Trypanosoma brucei and most likely other kinetoplastid flagellates. By using the immuno- or affinity purification approach, it was successfully applied to the analysis of several RBPs. Furthermore, we also provide a detailed description of the iCLIP/iCLAP protocol that shall be particularly suitable for the studies of trypanosome RBPs.

• MeSH
• imunoprecipitace metody   MeSH
• nukleotidy genetika   metabolismus   MeSH
• parazitologie metody   MeSH
• proteiny vázající RNA analýza   genetika   metabolismus   MeSH
• protozoální proteiny analýza   genetika   metabolismus   MeSH
• RNA protozoální genetika   metabolismus   MeSH
• RNA genetika   metabolismus   MeSH
• Trypanosoma brucei brucei genetika   MeSH
• ultrafialové záření MeSH
• vazba proteinů genetika   účinky záření   MeSH
• vazebná místa genetika   MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We report a powerful method for capturing the time-resolved concentration profiles, liquid swelling and surface phenomena during the absorption of methane (CH4) in still liquid ethanol (C2D6O) and n-decane (n-C10D22) and at high spatial resolution (pixel size 21.07 μm) using neutron imaging. Absorption of supercritical methane was followed at two temperatures and two pressures of methane, namely 7.0, 37.8 °C and 80, 120 bar. Fick's second law, which was used in the liquid-fixed coordinates, enabled for an adequate parameterization of the observed concentration profiles and liquid levels using simple analytical expressions. For both studied liquids, anomalously slow diffusion was observed in the initial stage of the absorption experiment. This was ascribed to the slow formation of the surface excess on the interface, time constant ranged 130-275 s. The axial symmetry of the cell allowed for the tomographic reconstructions of the profiles of the menisci. Based on these profiles, contact angle and surface tension were evaluated using the Young-Laplace equation. Overall, neutron imaging made it possible to capture time- and space-resolved information from which the methane concentration, liquid level and meniscus shape under high-pressure conditions inside a cylindrical titanium vessel were quantitatively derived. Multiple characteristics of ethanol, a methane hydrate inhibitor, and n-decane, a model constituent of crude oil, were thus measured for the first time under industrially relevant conditions in a one-pot experiment.

• MeSH
• alkany chemie   MeSH
• difuze MeSH
• ethanol chemie   MeSH
• fyzikální jevy MeSH
• methan chemie   MeSH
• neutrony MeSH
• ropa MeSH
• statistické modely MeSH
• teplota MeSH
• voda MeSH
• zobrazení jednotlivé molekuly přístrojové vybavení   metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

PURPOSE: To demonstrate the utility of an amplification-free long-read sequencing method to characterize the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (CTG18.1). METHODS: We applied an amplification-free method, utilizing the CRISPR/Cas9 system, in combination with PacBio single-molecule real-time (SMRT) long-read sequencing, to study CTG18.1. FECD patient samples displaying a diverse range of CTG18.1 allele lengths and zygosity status (n = 11) were analyzed. A robust data analysis pipeline was developed to effectively filter, align, and interrogate CTG18.1-specific reads. All results were compared with conventional polymerase chain reaction (PCR)-based fragment analysis. RESULTS: CRISPR-guided SMRT sequencing of CTG18.1 provided accurate genotyping information for all samples and phasing was possible for 18/22 alleles sequenced. Repeat length instability was observed for all expanded (≥50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length ≥91 repeats) indicating that expanded alleles behave dynamically. CONCLUSION: CRISPR-guided SMRT sequencing of CTG18.1 has revealed novel insights into CTG18.1 length instability. Furthermore, this study provides a framework to improve the molecular diagnostic accuracy for CTG18.1-mediated FECD, which we anticipate will become increasingly important as gene-directed therapies are developed for this common age-related and sight threatening disease.

• MeSH
• alely MeSH
• CRISPR-Cas systémy genetika   MeSH
• dospělí MeSH
• expanze trinukleotidových repetic genetika   MeSH
• Fuchsova endoteliální dystrofie genetika   patologie   MeSH
• genetická predispozice k nemoci * MeSH
• genotyp MeSH
• introny genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• sekvenční analýza DNA MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• transkripční faktor 4 genetika   MeSH
• trinukleotidové repetice genetika   MeSH
• zobrazení jednotlivé molekuly MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cryo-electron microscopy has established as a mature structural biology technique to elucidate the three-dimensional structure of biological macromolecules. The Coulomb potential of the sample is imaged by an electron beam, and fast semi-conductor detectors produce movies of the sample under study. These movies have to be further processed by a whole pipeline of image-processing algorithms that produce the final structure of the macromolecule. In this chapter, we illustrate this whole processing pipeline putting in value the strength of "meta algorithms," which are the combination of several algorithms, each one with different mathematical rationale, in order to distinguish correctly from incorrectly estimated parameters. We show how this strategy leads to superior performance of the whole pipeline as well as more confident assessments about the reconstructed structures. The "meta algorithms" strategy is common to many fields and, in particular, it has provided excellent results in bioinformatics. We illustrate this combination using the workflow engine, Scipion.

• MeSH
• algoritmy * MeSH
• elektronová kryomikroskopie metody   MeSH
• makromolekulární látky ultrastruktura   MeSH
• molekulární biologie metody   MeSH
• počítačové zpracování obrazu metody   MeSH
• průběh práce MeSH
• výpočetní biologie MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• zobrazování trojrozměrné metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH