• MeSH
• cytogenetika MeSH
• diagnostické techniky molekulární MeSH
• polymerázová řetězová reakce MeSH
• preimplantační diagnóza MeSH
• ženská infertilita terapie   MeSH
• Publikační typ
• monografie MeSH

• Konspekt
• Biotechnologie. Genetické inženýrství
• NLK Obory
• biologie
• genetika, lékařská genetika

Correct determination of the instantaneous level and changes of relevant proteins inside individual cells is essential for correct interpretation and understanding of physiological, diagnostic, and therapeutic events. Thus, single-cell analyses are important for quantification of natural cellular heterogeneity, which cannot be evaluated from averaged data of a cell population measurements. Here, we developed an original highly sensitive and selective instrumentation and methodology based on homogeneous single-step bioluminescence assay to quantify caspases and evaluate their heterogeneity in individual cells. Individual suspended cells are selected under microscope and reliably transferred into the 7 μl detection vials by a micromanipulator. The sensitivity of the method is given by implementation of photomultiplying tube with a cooled photocathode working in the photon counting mode. By optimization of our device and methodology, the limits of detection and quantitation were decreased down to 2.1 and 7.0 fg of recombinant caspase-3, respectively. These masses are lower than average amounts of caspase-3/7 in individual apoptotic and even non-apoptotic cells. As a proof of concept, the content of caspase-3/7 in single treated and untreated HeLa cells was determined to be 154 and 25 fg, respectively. Based on these results, we aim to use the technology for investigations of non-apoptotic functions of caspases.

• MeSH
• apoptóza * MeSH
• HeLa buňky MeSH
• kaspasa 3 MeSH
• kaspasy * MeSH
• lidé MeSH
• technologie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Analysing the chemical content of individual cells has already been proven to reveal unique information on various biological processes. Single-cell analysis provides more accurate and reliable results for biology and medicine than analyses of extracts from cell populations, where a natural heterogeneity is averaged. To meet the requirements in the research of important biologically active molecules, such as caspases, we have developed a miniaturized device for simultaneous analyses of individual cells. A stainless steel body with a carousel holder enables high-sensitivity parallel detections in eight microvials. The holder is mounted in front of a photomultiplier tube with cooled photocathode working in photon counting mode. The detection of active caspase-3/7, central effector caspases in apoptosis, in single cells is based on the bioluminescence chemistry commercially available as Caspase-Glo®3/7 reagent developed by Promega. Individual cells were captured from a culture medium under microscope and transferred by micromanipulator into detection microvial filled with the reagent. As a result of testing, the limits of detection and quantification were determined to be 0.27/0.86 of active caspase-3/7 content in an average apoptotic cell and 0.46/2.92 for non-apoptotic cells. Application potential of this technology in laboratory diagnostics and related medical research is discussed. Graphical abstract Miniaturized device for simultaneous analyses of individual cells.

• MeSH
• analýza jednotlivých buněk přístrojové vybavení   metody   MeSH
• apoptóza * MeSH
• design vybavení MeSH
• enzymatické testy přístrojové vybavení   metody   MeSH
• kaspasa 3 analýza   metabolismus   MeSH
• kaspasa 7 analýza   metabolismus   MeSH
• kultivované buňky MeSH
• luminiscenční měření přístrojové vybavení   metody   MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Caspases are key enzymes activated during the apoptotic machinery. Apoptosis as a way of programmed cell death becomes deregulated in some pathologies including cancer transformations, neurodegenerative, or autoimmune diseases. Most of the methods available for the detection of apoptosis and caspases provide qualitative information only or quantification data as an average from cell populations or cell lysates. Several reports point to the importance of more accurate single-cell analyses in biomedical studies due to heterogeneity at tissue as well as cell level. To meet these requirements, we developed a miniaturized device enabling detection and quantification of active caspase-3/7 in individual cells at a femtogram level (10(-15) g). The active caspase-3/7 detection protocol is based on the bioluminescence chemistry commercially available as a Caspase-Glo™ 3/7 reagent developed by Promega. As a model, we used human stem cells treated by camptothecin to induce apoptosis. Individual apoptotic cells were captured from a culture medium under a microscope and transferred by a micromanipulation system into a detection capillary containing 2 μl of the reagent. Cells without activation by camptothecin served as negative controls. The detection limit of active caspase-3/7 achieved in the miniaturized system was determined as 0.20 and limit of quantification as 0.65 of the amount found in a single apoptotic human stem cell. Such a sensitive method could have a wide application potential in laboratory medicine and related clinically oriented research.

• MeSH
• analýza jednotlivých buněk přístrojové vybavení   MeSH
• apoptóza * MeSH
• buněčná diferenciace MeSH
• crista neuralis cytologie   MeSH
• design vybavení MeSH
• kamptothecin chemie   MeSH
• kaspasa 3 metabolismus   MeSH
• kaspasa 7 metabolismus   MeSH
• kmenové buňky účinky léků   patologie   MeSH
• lidé MeSH
• luminiscence MeSH
• mikromanipulace MeSH
• miniaturizace přístrojové vybavení   MeSH
• reprodukovatelnost výsledků MeSH
• zánět MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We describe here a new reversed-phase high-performance liquid chromatography with mass spectrometry detection method for quantifying intact cytokinin nucleotides in human K-562 leukemia cells. Tandem mass spectrometry was used to identify the intracellular metabolites (cytokinin monophosphorylated, diphosphorylated, and triphosphorylated nucleotides) in riboside-treated cells. For the protein precipitation and sample preparation, a trichloroacetic acid extraction method is used. Samples are then back-extracted with diethyl ether, lyophilized, reconstituted, and injected into the LC system. Analytes were quantified in negative selected ion monitoring mode using a single quadrupole mass spectrometer. The method was validated in terms of retention time stabilities, limits of detection, linearity, recovery, and analytical accuracy. The developed method was linear in the range of 1-1,000 pmol for all studied compounds. The limits of detection for the analytes vary from 0.2 to 0.6 pmol.

• MeSH
• chromatografie kapalinová MeSH
• cytokininy analýza   chemie   MeSH
• hmotnostní spektrometrie MeSH
• lidé MeSH
• molekulární struktura MeSH
• nádorové buněčné linie MeSH
• nukleotidy analýza   chemie   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The human p73 protein is essential for normal morphogenesis and maintenance of neural tissue. Recently, several TP73 transcripts have been revealed in medulloblastoma (MB), the most common malignant brain tumor in children. Here, we performed immunohistochemical analysis on 29 MB specimens using anti-p73alpha and anti-DeltaNp73 antibodies. Real-time PCR quantification was performed to assess TAp73 and DeltaNp73 transcripts in a subset of 13 MB samples. Normal cerebellar tissues and RNA were used for comparison. Pilot clinical-pathological correlations were also provided. We report significant differences for TAp73 and DeltaNp73 mRNA expression between tumor tissues and reference (P = 0.013, P = 0.028). Immunohistochemically, 52 and 29% MB samples were positive for p73alpha and DeltaNp73, respectively. p73alpha expression was found to be in both the nucleus and cytoplasm, whereas DeltaNp73 was localized predominantly in the cytoplasm. In normal cerebellum, positive staining for p73alpha and DeltaNp73 was observed in the Purkinje cells of newborns, not adult samples, which supports the developmental role of TP73 during organogenesis of the human cerebellum. Survival analysis has shown negative relationship of DeltaNp73-immunoreactivity with overall survival (OS) and event free survival (EFS) (P = 0.026 and P = 0.127, respectively). For p73alpha-positive cases, the negative trend in OS (P = 0.149) and EFS (P = 0.216) was also apparent. Our results indicate the involvement of p73 protein in MB tumorigenesis and define TP73 as a potential prognostic and therapeutic target for medulloblastom.

• MeSH
• dítě MeSH
• DNA vazebné proteiny genetika   imunologie   metabolismus   MeSH
• dospělí MeSH
• financování organizované MeSH
• imunohistochemie MeSH
• lidé MeSH
• meduloblastom genetika   imunologie   metabolismus   MeSH
• messenger RNA analýza   metabolismus   MeSH
• míra přežití MeSH
• mladiství MeSH
• mozek metabolismus   patofyziologie   patologie   MeSH
• nádorová transformace buněk genetika   metabolismus   MeSH
• nádorové biomarkery analýza   genetika   metabolismus   MeSH
• nádorové supresorové proteiny genetika   imunologie   metabolismus   MeSH
• nádory mozku genetika   metabolismus   patofyziologie   MeSH
• předškolní dítě MeSH
• prognóza MeSH
• protein - isoformy genetika   izolace a purifikace   metabolismus   MeSH
• regulace genové exprese u nádorů genetika   MeSH
• retrospektivní studie MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH

Reverse transcription quantitative PCR (RT-qPCR) is already an established tool for mRNA detection and quantification. Since recently, this technique has been successfully employed for gene expression analyses, and also in individual cells (single cell RT-qPCR). Although the advantages of single cell measurements have been proven several times, a study correlating the expression measured on single cells, and in bulk samples consisting of a large number of cells, has been missing. Here, we collected a large data set to explore the relation between gene expression measured in single cells and in bulk samples, reflected by qPCR Cq values. We measured the expression of 95 genes in 12 bulk samples, each containing thousands of astrocytes, and also in 693 individual astrocytes. Combining the data, we described the relation between Cq values measured in bulk samples with either the percentage of the single cells that express the given genes, or the average expression of the genes across the single cells. We show that data obtained with single cell RT-qPCR are fully consistent with measurements in bulk samples. Our results further provide a base for quality control in single cell expression profiling, and bring new insights into the biological process of cellular expression.

• MeSH
• analýza jednotlivých buněk * MeSH
• gliový fibrilární kyselý protein genetika   MeSH
• messenger RNA genetika   MeSH
• myši transgenní MeSH
• myši MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• stanovení celkové genové exprese * MeSH
• zelené fluorescenční proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Prognosis of solid cancers is generally more favorable if the disease is treated early and efficiently. A key to long cancer survival is in radical surgical therapy directed at the primary tumor followed by early detection of possible progression, with swift application of subsequent therapeutic intervention reducing the risk of disease generalization. The conventional follow-up care is based on regular observation of tumor markers in combination with computed tomography/endoscopic ultrasound/magnetic resonance/positron emission tomography imaging to monitor potential tumor progression. A recent development in methodologies allowing screening for a presence of cell-free DNA (cfDNA) brings a new viable tool in early detection and management of major cancers. It is believed that cfDNA is released from tumors primarily due to necrotization, whereas the origin of nontumorous cfDNA is mostly apoptotic. The process of cfDNA detection starts with proper collection and treatment of blood and isolation and storage of blood plasma. The next important steps include cfDNA extraction from plasma and its detection and/or quantification. To distinguish tumor cfDNA from nontumorous cfDNA, specific somatic DNA mutations, previously localized in the primary tumor tissue, are identified in the extracted cfDNA. Apart from conventional mutation detection approaches, several dedicated techniques have been presented to detect low levels of cfDNA in an excess of nontumorous (nonmutated) DNA, including real-time polymerase chain reaction (PCR), "BEAMing" (beads, emulsion, amplification, and magnetics), and denaturing capillary electrophoresis. Techniques to facilitate the mutant detection, such as mutant-enriched PCR and COLD-PCR (coamplification at lower denaturation temperature PCR), are also applicable. Finally, a number of newly developed miniaturized approaches, such as single-molecule sequencing, are promising for the future.

• MeSH
• apoptóza * MeSH
• DNA nádorová krev   genetika   MeSH
• lidé MeSH
• mutace * MeSH
• mutační analýza DNA přístrojové vybavení   metody   MeSH
• nádory krev   genetika   patologie   MeSH
• nekróza MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

The increase of mixed chimerism (MC) after allogeneic hematopoietic stem cell transplantation has been associated with a high risk of relapse. A variety of techniques that use polymorphic markers have been established to survey hematopoietic chimerism status. The highest sensitivity is achieved using real-time quantitative polymerase chain reaction (RQ-PCR) analysis of insertion/deletion polymorphism, which allows the detection of disease recurrence and subsequently the earlier initiation of therapeutic intervention. The purpose of this study is the evaluation of multiplex RQ-PCR for MC assessment (six biallelic genetic systems and Y-specific locus), allowing the amplification and detection of target gene of interest and glyceraldehyde-3-phosphate dehydrogenase reference housekeeping gene in a single microtube. With optimized amounts of primers and probe, the quantification of target DNA was shown to be linear throughout the tested range (100%-0.05%). The efficiencies of multiplex RQ-PCR were in a range of 0.89 to 1.07. The sensitivity of individual systems ranged 0.02% to 0.04% with an average of 0.034%. A high degree of linear correlation between the chimerism results obtained by multiplex RQ-PCR vs singleplex RQ-PCR was observed (P < 0.0001, Spearman's coefficient = 0.9927), while correlation between multiplex RQ-PCR vs short tandem repeat analysis was also statistically significant (P < 0.0001, Spearman's coefficient = 0.9769). This new multiplex RQ-PCR assay is a quick, sensitive, reproducible, and cost-effective method for accurate MC assessment.

• MeSH
• alely MeSH
• chimérismus * MeSH
• DNA primery MeSH
• leukemie terapie   MeSH
• lidé MeSH
• lokální recidiva nádoru diagnóza   etiologie   prevence a kontrola   MeSH
• multiplexová polymerázová řetězová reakce metody   MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   MeSH
• polymorfismus genetický * MeSH
• transplantace hematopoetických kmenových buněk škodlivé účinky   MeSH
• transplantační chiméra krev   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The urokinase-type plasminogen activator (uPA) and PA inhibitor 1 (PAI-1) play important roles in breast cancer metastasis through cell migration and invasion. They are clinically applicable prognostic and predictive markers. High levels of uPA and PAI-1 are associated with high risk of recurrence and adjuvant chemotherapy provides substantial benefit for this breast cancer population. The current sole validated method for quantifying uPA level in breast tumour tissue is ELISA assay. It requires 50–300 mg of fresh or frozen tissue, which is the main limitation for routine use. In this study, we evaluated the performances of customized antibody microarray to quantify uPA concentration from reduced extraction solution of breast tumour tissue and compared it with standard ELISA kit. We firstly optimized the elaboration of customized antibody microarray in order to sensitively detect and quantify uPA standard solutions. In the best conditions, we analysed uPA concentration in 16 cytosolic extracts from breast tumour tissue. Results showed that our customized antibody microarray could correctly quantify uPA concentration while consuming 100 times less volume of tumour tissue extraction solution than ELISA. Our antibody microarray is a powerful and promising tool for the miniaturization of the immunoassay quantification of uPA from breast tumour tissue extracts.

• MeSH
• aktivátor plazminogenu urokinázového typu * analýza   imunologie   škodlivé účinky   MeSH
• biologické markery MeSH
• čipová analýza tkání * metody   přístrojové vybavení   MeSH
• ELISA MeSH
• imunoanalýza metody   MeSH
• lidé MeSH
• metastázy nádorů MeSH
• nádory prsu * diagnóza   imunologie   MeSH
• prognóza MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• srovnávací studie MeSH