Přestože děláme velké pokroky v objasňování molekulární podstaty mnohých onemocnění, stále je zde mnoho nezodpovězených otázek týkajících se patogeneze nádorových onemocnění, případně vývoje efektivních metod pro včasnou diagnostiku a léčbu. Současný zájem o proteomiku vzrůstá částečně i proto, že proteomika nabízí metody, které dokážou překonat nedostatky jiných metod, a zároveň nabízí možnost sledovat procesy, které vedou k patologickým změnám v organizmu v průběhu onemocnění. Tato práce přináší přehled o vývoji a významu proteomiky pro studium onkohematologických onemocnění. Dále pak uvádí přehled nejnovějších metod používaných v proteomice, zabývá se problémy souvisejícími s používáním komplexního biologického materiálu, popisuje využití proteomiky na Ústavu hematologie a krevní transfuze v charakterizaci myelodysplastického syndromu (MDS) a chronické myeloidní leukemie. Na závěr uvádíme naše zkušenosti s vývojem proteinového čipu s on-line detekcí optickou metodou rezonance povrchového plazmonu pro studium patogeneze MDS onemocnění.
Despite great advances in our understanding of the molecular basis of many diseases, there are still substantial gaps in our understanding of oncohematological diseases as well as in the development of effective strategies for early diagnosis and for treatment. The current interest in proteomics is growing partly due to the prospects that proteomic methods offer and hopefully overcome limitations of other approaches. At the Institute of Hematology and Blood Transfusion the proteomics of oncohematological diseases, especially myelodysplastic syndrome and chronic myeloid leukaemia, has been studied. This work gives an overview of development and importance of proteomics for studying the oncohematological diseases. Furthermore, it introduces several new methods applied in proteomics, deals with problems attached to working with complex biological samples, and at the end describes our latest development of a protein chip with on-line detection using an optical method – the surface plasmon resonance – for characterizing MDS pathogenesis.
- Keywords
- rezonance povrchového plazmonu, myelodysplastický syndrom,
- MeSH
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive diagnosis blood MeSH
- Protein Array Analysis * methods MeSH
- Hematologic Neoplasms * diagnosis blood MeSH
- Plasma chemistry MeSH
- Blood Proteins analysis MeSH
- Humans MeSH
- Myelodysplastic Syndromes diagnosis blood MeSH
- Biomarkers, Tumor blood MeSH
- Surface Plasmon Resonance methods MeSH
- Proteome analysis MeSH
- Proteomics * history methods instrumentation MeSH
- Vascular Endothelial Growth Factor Receptor-1 blood metabolism MeSH
- High-Throughput Screening Assays MeSH
- Vascular Endothelial Growth Factor A blood metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
A novel microfluidic label-free bead-based metallothionein immunosensors was designed. To the surface of superparamagnetic agarose beads coated with protein A, polyclonal chicken IgY specifically recognizing metallothionein (MT) were immobilized via rabbit IgG. The Brdicka reaction was used for metallothionein detection in a microfluidic printed 3D chip. The assembled chip consisted of a single copper wire coated with a thin layer of amalgam as working electrode. Optimization of MT detection using designed microfluidic chip was performed in stationary system as well as in the flow arrangement at various flow rates (0-1800 μL/min). In stationary arrangement it is possible to detect MT concentrations up to 30 ng/mL level, flow arrangement allows reliable detection of even lower concentration (12.5 ng/mL). The assembled miniature flow chip was subsequently tested for the detection of MT elevated levels (at approx. level 100 μg/mL) in samples of patients with cancer. The stability of constructed device for metallothionein detection in flow arrangement was found to be several days without any maintenance needed.
- MeSH
- Equipment Design MeSH
- Electrochemical Techniques instrumentation methods MeSH
- Electrodes MeSH
- Antibodies, Immobilized chemistry metabolism MeSH
- Immunoglobulin G chemistry metabolism MeSH
- Immunoglobulins chemistry metabolism MeSH
- Immunomagnetic Separation instrumentation methods MeSH
- Rabbits MeSH
- Chickens MeSH
- Middle Aged MeSH
- Humans MeSH
- Metallothionein blood MeSH
- Head and Neck Neoplasms blood MeSH
- Animals MeSH
- Check Tag
- Rabbits MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
This study presents a simple, label-free electrochemical technique for the monitoring of DNA ligase activity. DNA ligases are enzymes that catalyze joining of breaks in the backbone of DNA and are of significant scientific interest due to their essential nature in DNA metabolism and their importance to a range of molecular biological methodologies. The electrochemical behavior of DNA at mercury and some amalgam electrodes is strongly influenced by its backbone structure, allowing a perfect discrimination between DNA molecules containing or lacking free ends. This variation in electrochemical behavior has been utilized previously for a sensitive detection of DNA damage involving the sugar-phosphate backbone breakage. Here we show that the same principle can be utilized for monitoring of a reverse process, i.e., the repair of strand breaks by action of the DNA ligases. We demonstrate applications of the electrochemical technique for a distinction between ligatable and unligatable breaks in plasmid DNA using T4 DNA ligase, as well as for studies of the DNA backbone-joining activity in recombinant fragments of E. coli DNA ligase.
A piezoelectric sensor with immobilized polyclonal antibody was developed as a label-free assay for the model bacterium, Escherichia coli. The polyclonal antibody was prepared from mice BALB/c and covalently immobilized on the sensing gold electrode of the piezoelectric quartz crystal. The biosensor was able to detect E. coli in the range of 10(6)-10(9) CFU/mL; signal of the negative control was not statistically relevant in the selected range. Samples could be analyzed in four minutes and one measuring cycle including regeneration was completed within ten minutes. Repeatability of the developed method is discussed; the signal obtained from three different biosensors was 12.9+/-0.4 Hz for the sample containing 10(8) CFU/mL.
- MeSH
- Biosensing Techniques methods MeSH
- Electrodes MeSH
- Escherichia coli immunology isolation & purification MeSH
- Quartz chemistry MeSH
- Mice, Inbred BALB C MeSH
- Mice MeSH
- Antibodies, Bacterial chemistry immunology immunology MeSH
- Gold chemistry MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Evaluation Study MeSH
MicroRNAs (miRNAs) are becoming a very important group of molecules especially since their connection to numerous diseases has been revealed. The potential in gene therapy as well as in diagnostics is being widely investigated leading to the demand of sensitive, selective and simple methods of isolation and detection. The combined advantages of magnetic particle-based separation with sensitive electrochemical detection may offer a very valuable tool for these purposes. In this study, the miR‑124 was targeted as an example analyte for development and optimization of the isolation procedure coupled to the electrochemical detection. The sensitivity of the method was demonstrated by the limit of detection at the level of nanomolar concentration (4 nM). To verify the applicability of the procedure to the real samples, miR‑124 was isolated from the human embryonic kidney cells naturally expressing this miRNA molecule and the results were compared to the amount of miR‑124 isolated from the cells transfected by the pENTR-miR‑124 plasmid leading to the overexpression of miR‑124.
- MeSH
- Biosensing Techniques * MeSH
- Humans MeSH
- Limit of Detection MeSH
- RNA, Messenger biosynthesis genetics MeSH
- MicroRNAs genetics isolation & purification MeSH
- Cell Line, Tumor MeSH
- Gene Expression Regulation, Neoplastic MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: Because of its non-destructive nature, label-free imaging is an important strategy for studying biological processes. However, routine microscopic techniques like phase contrast or DIC suffer from shadow-cast artifacts making automatic segmentation challenging. The aim of this study was to compare the segmentation efficacy of published steps of segmentation work-flow (image reconstruction, foreground segmentation, cell detection (seed-point extraction) and cell (instance) segmentation) on a dataset of the same cells from multiple contrast microscopic modalities. RESULTS: We built a collection of routines aimed at image segmentation of viable adherent cells grown on the culture dish acquired by phase contrast, differential interference contrast, Hoffman modulation contrast and quantitative phase imaging, and we performed a comprehensive comparison of available segmentation methods applicable for label-free data. We demonstrated that it is crucial to perform the image reconstruction step, enabling the use of segmentation methods originally not applicable on label-free images. Further we compared foreground segmentation methods (thresholding, feature-extraction, level-set, graph-cut, learning-based), seed-point extraction methods (Laplacian of Gaussians, radial symmetry and distance transform, iterative radial voting, maximally stable extremal region and learning-based) and single cell segmentation methods. We validated suitable set of methods for each microscopy modality and published them online. CONCLUSIONS: We demonstrate that image reconstruction step allows the use of segmentation methods not originally intended for label-free imaging. In addition to the comprehensive comparison of methods, raw and reconstructed annotated data and Matlab codes are provided.
- MeSH
- Algorithms MeSH
- Cell Fractionation methods MeSH
- Humans MeSH
- Microscopy methods MeSH
- Image Processing, Computer-Assisted MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
The inherent diversity of approaches in proteomics research has led to a wide range of software solutions for data analysis. These software solutions encompass multiple tools, each employing different algorithms for various tasks such as peptide-spectrum matching, protein inference, quantification, statistical analysis, and visualization. To enable an unbiased comparison of commonly used bottom-up label-free proteomics workflows, we introduce WOMBAT-P, a versatile platform designed for automated benchmarking and comparison. WOMBAT-P simplifies the processing of public data by utilizing the sample and data relationship format for proteomics (SDRF-Proteomics) as input. This feature streamlines the analysis of annotated local or public ProteomeXchange data sets, promoting efficient comparisons among diverse outputs. Through an evaluation using experimental ground truth data and a realistic biological data set, we uncover significant disparities and a limited overlap in the quantified proteins. WOMBAT-P not only enables rapid execution and seamless comparison of workflows but also provides valuable insights into the capabilities of different software solutions. These benchmarking metrics are a valuable resource for researchers in selecting the most suitable workflow for their specific data sets. The modular architecture of WOMBAT-P promotes extensibility and customization. The software is available at https://github.com/wombat-p/WOMBAT-Pipelines.
- MeSH
- Data Analysis MeSH
- Benchmarking * MeSH
- Proteins MeSH
- Proteomics * MeSH
- Workflow MeSH
- Software MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Surface plasmon resonance sensors have made vast advancements in the sensing technology and the number of applications achievable. New developments in surface plasmon resonance sensors have gained considerable momentum promoted by the urgent needs of fast, reliable and label-free methods for detection and quantification of analytes in molecular biology, medicine and other life sciences. However, even if enormous improvements in the limits of detections have been achieved, this technology still faces important challenges to be translated to clinical practice or in-field measurements. This paper reviews the important recent advances of this technology for the label-free detection in real biological samples and we discussed the key challenges to be overcome to transit from prototypes to commercial biosensors.
- MeSH
- Biology instrumentation methods MeSH
- Diagnosis MeSH
- Humans MeSH
- Surface Plasmon Resonance instrumentation methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
With the increased demand for beef in emerging markets, the development of quality-control diagnostics that are fast, cheap and easy to handle is essential. Especially where beef must be free from pork residues, due to religious, cultural or allergic reasons, the availability of such diagnostic tools is crucial. In this work, we report a label-free impedimetric genosensor for the sensitive detection of pork residues in meat, by leveraging the biosensing capabilities of graphene acid - a densely and selectively functionalized graphene derivative. A single stranded DNA probe, specific for the pork mitochondrial genome, was immobilized onto carbon screen-printed electrodes modified with graphene acid. It was demonstrated that graphene acid improved the charge transport properties of the electrode, following a simple and rapid electrode modification and detection protocol. Using non-faradaic electrochemical impedance spectroscopy, which does not require any electrochemical indicators or redox pairs, the detection of pork residues in beef was achieved in less than 45 min (including sample preparation), with a limit of detection of 9% w/w pork content in beef samples. Importantly, the sample did not need to be purified or amplified, and the biosensor retained its performance properties unchanged for at least 4 weeks. This set of features places the present pork DNA sensor among the most attractive for further development and commercialization. Furthermore, it paves the way for the development of sensitive and selective point-of-need sensing devices for label-free, fast, simple and reliable monitoring of meat purity.
- MeSH
- Biosensing Techniques * MeSH
- DNA MeSH
- Electrochemical Techniques MeSH
- Electrodes MeSH
- Graphite * MeSH
- Meat MeSH
- Cattle MeSH
- Animals MeSH
- Check Tag
- Cattle MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
This review focuses on recent advances in the development of functionalizable antifouling coatings and their applications in label-free optical biosensors. Approaches to the development of antifouling coatings, ranging from self-assembled monolayers and PEG derivatives to ultra-low-fouling polymer brushes, are reviewed. Methods of preparation and characterization of antifouling coatings and the functionalization of antifouling coatings with bioreceptors are reviewed, and the effect of functionalization on the fouling properties of biofunctional coating is discussed. Special attention is given to biofunctional coatings for label-free bioanalysis of blood plasma and serum for medical diagnostics.
