• MeSH
• genom * MeSH
• oprava DNA genetika   MeSH

• Konspekt
• Obecná genetika. Obecná cytogenetika. Evoluce
• NLK Obory
• genetika, lékařská genetika
• NLK Publikační typ
• elektronické časopisy

The crucial requirement of molecular genetic methods is high-quality input material. The key question is "how to preserve DNA during long-term storage." Biobanks are recommended to aliquot isolated DNA into provided volumes. The aim of this study was to analyse the effect of repeated freezing and thawing on the genomic DNA integrity, quality and concentration. The aliquoted DNA isolated from blood cells using the automatic MagNA system and manual salting out method underwent freeze/thaw cycles at different storage conditions (-20 °C, -80 °C and liquid nitrogen). The average initial concentrations were 270.6 ng/μl (salting out method) and 125.0 ng/μl (MagNA). All concentration deviations relative to the concentration after the first freeze/ thaw cycle were less than 5 % for -20 °C and -80 °C cycling with both isolation methods. The average percentage differences of liquid nitrogen samples were higher, and the MagNA isolation method showed significant differences. There were no significant changes in the DNA purity or quality. The repeating freeze/ thaw up to 100 cycles (through -20 °C and -80 °C, respectively) did not significantly influence the integrity, concentration, or purity of genomic DNA, suggesting that storage of samples in high-volume pools without multiple aliquoting is possible. Storage in a freezer seems to be the most suitable way of long-term DNA preservation, because liquid nitrogen storage leads to formation of DNA clumps.

• MeSH
• DNA * MeSH
• genomika * MeSH
• zmrazování MeSH
• Publikační typ
• časopisecké články MeSH

Sequencing enabled the world to rapidly identify SARS-CoV-2 and develop diagnostic tests and other tools for outbreak management. Continued genome sequencing supports the monitoring of the disease’s spread and evolution of the virus. Accelerated integration of genome sequencing into the practices of the global health community is required if we want to be better prepared for the future threats. This document provides guidance for laboratories on maximizing the impact of SARS-CoV-2 sequencing now and other emerging pathogens in the future.

• MeSH
• Betacoronavirus MeSH
• COVID-19 MeSH
• epidemický výskyt choroby MeSH
• genom virový MeSH
• ochrana veřejného zdraví MeSH
• sekvenování celého genomu MeSH

• Konspekt
• Veřejné zdraví a hygiena
• NLK Obory
• veřejné zdravotnictví
• virologie
• NLK Publikační typ
• publikace WHO

Following the discovery in 1998 of -H2AX, the first histone modification induced by DNA damage, interest in the changes to chromatin induced by DNA damage has exploded, and a vast amount of information has been generated. However, there has been a discrepancy between our rapidly advancing knowledge of how chromatin responds to DNA damage and the understanding of why cells mobilize large segments of chromatin to protect the genome against destabilizing effects posed by tiny DNA lesions. Recent research has provided insights into these issues and suggests that chromatin responses induced by DNA damage are not simply the accumulation of 'nuclear foci' but are mechanisms required to guard genome integrity.

• MeSH
• buněčný cyklus * genetika   MeSH
• histony * metabolismus   MeSH
• lidé MeSH
• nestabilita genomu * MeSH
• oprava DNA MeSH
• poškození DNA * MeSH
• posttranslační úpravy proteinů MeSH
• restrukturace chromatinu * genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH

The sturgeon is a highly endangered fish species mostly due to over-fishing, habitat destruction, and water pollution. Duroquinone (derivative of 1,4-benzoquinone) is a xenobiotic compound widespread in the environment. The effect of duroquinone on motility, DNA integrity, and oxidative stress indices in sterlet, Acispenser ruthenus, spermatozoa was investigated. Sterlet sperm was exposed for 2h to duroquinone at concentrations of 25, 50, 100, and 150 μM. Spermatozoa motility, velocity, and ATP content were significantly decreased with exposure to duroquinone. The level of DNA damage significantly increased at concentrations of 50 μM and above. Oxidative stress indices (lipid peroxidation and content of carbonyl proteins) and superoxide dismutase (SOD) activity increased significantly with increasing concentrations of duroquinone. Oxidative stress in sterlet spermatozoa induced by duroquinone was shown to impair spermatozoa DNA integrity, motility parameters, and the antioxidant defense system. Spermatozoa motility, content of carbonyl proteins, and SOD activity were shown to be sensitive biomarkers, exhibiting strong responses to low concentrations of the xenobiotic. Results also suggested that fish spermatozoa in vitro assays may provide a simple and efficient means of monitoring residual pollutants in the aquatic environment.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• benzochinony toxicita   MeSH
• chemické látky znečišťující vodu toxicita   MeSH
• motilita spermií účinky léků   MeSH
• oxidační stres účinky léků   MeSH
• poškození DNA MeSH
• ryby fyziologie   MeSH
• spermie účinky léků   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Telomeres are indispensable for genome stability maintenance. They are maintained by the telomere-associated protein complex, which include Ku proteins and a telomerase among others. Here, we investigated a role of Ku80 in Leishmania mexicana. Leishmania is a genus of parasitic protists of the family Trypanosomatidae causing a vector-born disease called leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: We used the previously established CRISPR/Cas9 system to mediate ablation of Ku80- and Ku70-encoding genes in L. mexicana. Complete knock-outs of both genes were confirmed by Southern blotting, whole-genome Illumina sequencing, and RT-qPCR. Resulting telomeric phenotypes were subsequently investigated using Southern blotting detection of terminal restriction fragments. The genome integrity in the Ku80- deficient cells was further investigated by whole-genome sequencing. Our work revealed that telomeres in the ΔKu80 L. mexicana are elongated compared to those of the wild type. This is a surprising finding considering that in another model trypanosomatid, Trypanosoma brucei, they are shortened upon ablation of the same gene. A telomere elongation phenotype has been documented in other species and associated with a presence of telomerase-independent alternative telomere lengthening pathway. Our results also showed that Ku80 appears to be not involved in genome stability maintenance in L. mexicana. CONCLUSION/SIGNIFICANCE: Ablation of the Ku proteins in L. mexicana triggers telomere elongation, but does not have an adverse impact on genome integrity.

• MeSH
• antigen Ku genetika   metabolismus   MeSH
• genom protozoální MeSH
• Leishmania mexicana genetika   metabolismus   MeSH
• leishmanióza kožní parazitologie   MeSH
• lidé MeSH
• nestabilita genomu * MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• telomery genetika   metabolismus   MeSH
• Trypanosoma brucei brucei genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

R-loops are common non-B nucleic acid structures formed by a three-stranded nucleic acid composed of an RNA-DNA hybrid and a displaced single-stranded DNA (ssDNA) loop. Because the aberrant R-loop formation leads to increased mutagenesis, hyper-recombination, rearrangements, and transcription-replication collisions, it is regarded as important in human diseases. Therefore, its prevalence and distribution in genomes are studied intensively. However, in silico tools for R-loop prediction are limited, and therefore, we have developed the R-loop tracker tool, which was implemented as a part of the DNA Analyser web server. This new tool is focused upon (1) prediction of R-loops in genomic DNA without length and sequence limitations; (2) integration of R-loop tracker results with other tools for nucleic acids analyses, including Genome Browser; (3) internal cross-evaluation of in silico results with experimental data, where available; (4) easy export and correlation analyses with other genome features and markers; and (5) enhanced visualization outputs. Our new R-loop tracker tool is freely accessible on the web pages of DNA Analyser tools, and its implementation on the web-based server allows effective analyses not only for DNA segments but also for full chromosomes and genomes.

• MeSH
• algoritmy * MeSH
• DNA chemie   genetika   MeSH
• genomika metody   MeSH
• internet statistika a číselné údaje   MeSH
• lidé MeSH
• nestabilita genomu * MeSH
• R-smyčka * MeSH
• software MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Genomic integrity and instability mechanisms in the pathogenesis and potential personalized targeted treatment of prostate cancer. PI: J. Bártek The key topic of this project is analysis of cell cycle checkpoints and genome integrity maintenance mechanisms (e.g. in radio/chemotherapy) in prostate cancer, to better understand molecular pathogenesis, treatment responsiveness or resistance, and the role of cancer stem cells in these processes. We will use cell culture and gene transfer models, clinical material and a wide range of immunohistochemical, cell and molecular biology methods. The project will elucidate resistance to standard treatment, predictive assessment and individualized management, especially in androgen-independent prostate tumours, to provide new insights into genetic instability and proposals for combining standard radio/chemotherapy and targeted modulation of DNA damage signaling and repair pathways. This project should therefore address one of the major concerns in current oncology.

Mechanismy integrity a nestability genomu v patogeneze a potenciální individualizovaná molekulárně-cílená léčba karcinomu prostaty Hl. řešitel J. Bártek Předmětem projektu je studium mechanismů regulace buněčného cyklu a integrity genomu (např. u radio-a chemoterapie) u rakoviny prostaty, s cílem lépe pochopit molekulární patogenezi, citlivost či rezistenci na léčbu a úlohu nádorových kmenových buněk v těchto procesech. K řešení bude využito modelů buněčných kultur a genového přenosu, klinického materiálu a imunohistochemie a metod buněčné a molekulární biologie. Projekt přispěje k objasnění rezistence na klasickou léčbu, k odhadu odpovědi na terapii a k přiblížení individualizace léčby, zejména u obtížně léčitelných, na androgenech nezávislych nadorůprostaty, včetně lepšího pochopení genetické nestability a návrhu strategie kombinace klasické radio-/chemoterapie a molekulárně-cílené modulace signalizace a opravy poškozené DNA. Projekt tedy přispěje k řešení závažného problému současné onkologie.

• MeSH
• buněčný cyklus MeSH
• cílená molekulární terapie MeSH
• DNA vazebné proteiny MeSH
• genom lidský MeSH
• nádorové kmenové buňky MeSH
• nádory prostaty MeSH
• oprava DNA MeSH
• poškození DNA MeSH
• sekvenční analýza RNA MeSH
• western blotting MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• andrologie
• onkologie
• biologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

53BP1 is a mediator of DNA damage response (DDR) and a tumor suppressor whose accumulation on damaged chromatin promotes DNA repair and enhances DDR signaling. Using foci formation of 53BP1 as a readout in two human cell lines, we performed an siRNA-based functional high-content microscopy screen for modulators of cellular response to ionizing radiation (IR). Here, we provide the complete results of this screen as an information resource, and validate and functionally characterize one of the identified 'hits': a nuclear pore component NUP153 as a novel factor specifically required for 53BP1 nuclear import. Using a range of cell and molecular biology approaches including live-cell imaging, we show that knockdown of NUP153 prevents 53BP1, but not several other DDR factors, from entering the nuclei in the newly forming daughter cells. This translates into decreased IR-induced 53BP1 focus formation, delayed DNA repair and impaired cell survival after IR. In addition, NUP153 depletion exacerbates DNA damage caused by replication stress. Finally, we show that the C-terminal part of NUP153 is required for effective 53BP1 nuclear import, and that 53BP1 is imported to the nucleus through the NUP153-importin-β interplay. Our data define the structure-function relationships within this emerging 53BP1-NUP153/importin-β pathway and implicate this mechanism in the maintenance of genome integrity.

• MeSH
• buněčné jádro metabolismus   MeSH
• genom lidský genetika   MeSH
• HeLa buňky MeSH
• imunoblotting MeSH
• imunoprecipitace MeSH
• intracelulární signální peptidy a proteiny genetika   metabolismus   MeSH
• komplex proteinů jaderného póru genetika   metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• RNA interference fyziologie   MeSH
• vazba proteinů genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Keratoconus is characterised by reduced rigidity of the cornea with distortion and focal thinning that causes blurred vision, however, the pathogenetic mechanisms are unknown. It can lead to severe visual morbidity in children and young adults and is a common indication for corneal transplantation worldwide. Here we report the first large scale genome-wide association study of keratoconus including 4,669 cases and 116,547 controls. We have identified significant association with 36 genomic loci that, for the first time, implicate both dysregulation of corneal collagen matrix integrity and cell differentiation pathways as primary disease-causing mechanisms. The results also suggest pleiotropy, with some disease mechanisms shared with other corneal diseases, such as Fuchs endothelial corneal dystrophy. The common variants associated with keratoconus explain 12.5% of the genetic variance, which shows potential for the future development of a diagnostic test to detect susceptibility to disease.

• MeSH
• buněčná diferenciace genetika   MeSH
• celogenomová asociační studie MeSH
• extracelulární matrix metabolismus   patologie   MeSH
• fenotyp MeSH
• genetická predispozice k nemoci MeSH
• genetické lokusy * MeSH
• hodnocení rizik MeSH
• jednonukleotidový polymorfismus * MeSH
• keratokonus diagnóza   etnologie   genetika   metabolismus   MeSH
• kolagen metabolismus   MeSH
• lidé MeSH
• rizikové faktory MeSH
• studie případů a kontrol MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• metaanalýza MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Geografické názvy
• Austrálie MeSH
• Evropa MeSH