single molecule detection
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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
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
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.
Early-stage diagnosis of prostatic carcinoma is essential for successful treatment and, thus, significant prognosis improvement. In laboratory practice, the standard non-invasive diagnostic approach is the immunochemical detection of the associated biomarker, prostate-specific antigen (PSA). Ultrasensitive detection of PSA is essential for both diagnostic and recurrence monitoring purposes. To achieve exceptional sensitivity, we have developed a microfluidic device with a flow-through cell for single-molecule analysis using photon-upconversion nanoparticles (UCNPs) as a detection label. For this purpose, magnetic microparticles (MBs) were first optimized for the capture and preconcentration of PSA and then used to implement a bead-based upconversion-linked immunoassay (ULISA) in the microfluidic device. The digital readout based on counting single nanoparticle-labeled PSA molecules on MBs enabled a detection limit of 1.04 pg mL-1 (36 fM) in 50% fetal bovine serum, which is an 11-fold improvement over the respective analog MB-based ULISA. The microfluidic technique conferred several other advantages, such as easy implementation and the potential for achieving high-throughput analysis. Finally, it was proven that the microfluidic setup is suitable for clinical sample analysis, showing a good correlation with a reference electrochemiluminescence assay (recovery rates between 97% and 105%).
- MeSH
- imunoanalýza přístrojové vybavení metody MeSH
- lidé MeSH
- limita detekce MeSH
- mikrofluidní analytické techniky přístrojové vybavení MeSH
- nádory prostaty diagnóza krev MeSH
- nanočástice chemie MeSH
- prostatický specifický antigen * analýza krev MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Chromosome architecture needs to be investigated in relation with the chemical function of DNA. The kinetics of gene expression, DNA replication, and repair are driven by the mechanisms by which a functional nuclear protein finds its substrate in the nucleus. Single-particle tracking (SPT) is a method to quantify fluorescent molecules dynamics from the tracks of the single molecules recorded by high-resolution microscopes. SPT offers direct observation of the movement and single-molecule resolution. Usually SPT is performed on membranes because of higher contrast. Here, we introduce a novel method to record the trajectories of weakly fluorescent molecules in the nucleus of living cells. I-SPT uses some specific detection and analysis tools to enable the computation of reliable statistics on nuclear particle movement.
AIMS: To compare serum levels of 17 cytokines and 5 adhesion molecules in patients with newly diagnosed acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) using biochip array technology. METHODS: A total of 15 AML and 15 ALL patients were studied. Serum samples were taken prior to anticancer therapy and were analyzed by biochip based immunoassays on the Evidence Investigator analyzer. This approach allows simultaneous detection of multiple analytes from a single sample. T-tests were used for statistical analysis. RESULTS: Comparing cytokine and adhesion molecules levels in newly diagnosed AML and ALL patients, we found significant increase in AML in serum IL-4 (P < 0.0001), IL-2 (P < 0.01), IL-3 (P < 0.05), and significant decrease (P < 0.05) in serum VEGF and VCAM-1. DISCUSSION: Our results indicate that serum profile of cytokines and adhesion molecules differs in newly diagnosed AML and ALL patients. Further studies are needed to establish if these alterations could be used as a clinically relevant biomarker for acute leukemias.
- MeSH
- akutní lymfatická leukemie krev diagnóza MeSH
- akutní myeloidní leukemie krev diagnóza MeSH
- čipová analýza proteinů metody MeSH
- cytokiny metabolismus MeSH
- diferenciální diagnóza MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- molekuly buněčné adheze metabolismus MeSH
- nádorové biomarkery metabolismus 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
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie 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
BACKGROUND: Inactivating mutations of the hypothalamic transcription factor singleminded1 (SIM1) have been shown as a cause of early-onset severe obesity. However, to date, the contribution of SIM1 mutations to the obesity phenotype has only been studied in a few populations. In this study, we screened the functional regions of SIM1 in severely obese children of Slovak and Moravian descent to determine if genetic variants within SIM1 may influence the development of obesity in these populations. METHODS: The SIM1 promoter region, exons and exon-intron boundaries were sequenced in 126 unrelated obese children and adolescents (2-18 years of age) and 41 adult lean controls of Slovak and Moravian origin. Inclusion criteria for the children and adolescents were a body mass index standard deviation score higher than 2 SD for an appropriate age and sex, and obesity onset at less than 5 years of age. The clinical phenotypes of the SIM1 variant carriers were compared with clinical phenotypes of 4 MC4R variant carriers and with 27 unrelated SIM1 and MC4R mutation negative obese controls that were matched for age and gender. RESULTS: Seven previously described SIM1 variants and one novel heterozygous variant p.D134N were identified. The novel variant was predicted to be pathogenic by 7 in silico software analyses and is located at a highly conserved position of the SIM1 protein. The p.D134N variant was found in an 18 year old female proband (BMI 44.2kg/m2; +7.5 SD), and in 3 obese family members. Regardless of early onset severe obesity, the proband and her brother (age 16 years) did not fulfill the criteria of metabolic syndrome. Moreover, the variant carriers had significantly lower preferences for high sugar (p = 0.02) and low fat, low carbohydrate, high protein (p = 0.02) foods compared to the obese controls. CONCLUSIONS: We have identified a novel SIM1 variant, p.D134N, in 4 obese individuals from a single pedigree which is also associated with lower preference for certain foods.
- MeSH
- detekce genetických nosičů MeSH
- dítě MeSH
- fenotyp MeSH
- lidé MeSH
- mladiství MeSH
- mutace MeSH
- nepřímá kalorimetrie MeSH
- obezita etnologie genetika MeSH
- předškolní dítě MeSH
- preference v jídle MeSH
- receptor melanokortinový typ 4 genetika MeSH
- represorové proteiny genetika MeSH
- rodokmen MeSH
- studie případů a kontrol MeSH
- stupeň závažnosti nemoci MeSH
- transkripční faktory bHLH genetika MeSH
- věk při počátku nemoci MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika etnologie MeSH
- Slovenská republika etnologie MeSH
Metody pro studium jednotlivých biomolekul pomáhají objasnit jejich vlastnosti jinak skryté při měřeních v celém objemu vzorku. Velmi významnou metodou pro výzkum chování jednotlivých biomolekul je luminiscenční mikroskopie. Luminiscenční značky, které vykazují fotonovou up‑konverzi, přináší nové možnosti v této oblasti. Předpokládá se, že zjednoduší a přinesou větší rozšíření metod pro vizualizaci jednotlivých biomolekul, např. membránových receptorů, studium jejich interakcí v reálném čase a rovněž nové možnosti pro detekci jednotlivých biomolekul.
Single molecule methods allow us to explain properties of biomolecules on single molecule level, which are not apparent from bulk experiments. Luminescence microscopy is a very important method for single molecule experiments. Novel luminescent labels, which exhibit photon up‑conversion, revealed new possibilities in this field. It is considered that photon up‑conversion nanoparticles will allow for next development of single molecule methods and facilitate for visualization of individual biomolecules, e.g., membrane receptors, studies of their interactions in real time and also provide new possibilities for the detection of individual biomolecules.
