Q89845452
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This review defines limits of currently used techniques to assess developmental capacity of human embryos in assisted reproduction and provides an overview of techniques assessing embryo's physiology on levels of genomics, transcriptomics, proteomics and metabolomics. Basic principles of respective techniques are included. Discovered biomarkers are discussed with respect to biochemical functions and their prognostic values of embryonal development.
- Klíčová slova
- vývojový potenciál embrya,
- MeSH
- 2D gelová elektroforéza MeSH
- aneuploidie MeSH
- asistovaná reprodukce MeSH
- biologické markery analýza metabolismus MeSH
- biopsie MeSH
- embryo savčí * MeSH
- embryonální vývoj * genetika MeSH
- fertilizace in vitro MeSH
- hybridizace in situ fluorescenční metody MeSH
- lidé MeSH
- messenger RNA analýza MeSH
- metabolom MeSH
- metabolomika klasifikace metody MeSH
- polymerázová řetězová reakce metody MeSH
- preimplantační diagnóza metody MeSH
- proteom analýza MeSH
- sekvenční analýza DNA metody MeSH
- srovnávací genomová hybridizace metody MeSH
- transkriptom MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Cíl studie: Uvést stručný přehled současných metod pro analýzu metabolomu embrya, vlastních zkušeností v této oblasti a perspektivy dalšího vývoje. Typ studie: Literární přehled. Název a sídlo pracoviště: Gynekologicko-porodnická klinika, Lékařská fakulta Masarykovy univerzity a Fakultní nemocnice Brno; Ústav biochemie, Přírodovědecká fakulta a CEITEC, Masarykova univerzita, Brno. Předmět a metoda studie: V současné době jsou k dispozici vysoce citlivé analytické metody umožňující přesné stanovení řady molekul, které souvisí s látkovou přeměnou – metabolismem – embrya v prvních dnech jeho vývoje. Jednou z perspektivních metod je kapilární elektroforéza. V uplynulých letech byly analyzovány parametry metabolismu embryí kultivovaných in vitro a byl hodnocen jejich vztah k morfologii a dosažení těhotenství. Pozornost se soustředila nejčastěji na metabolismus aminokyselin, glukózy a pyruvátu. Výsledky studií však mají zatím nejednoznačné výsledky. Závěr: Analýza kultivačního média kapilární elektroforézou patří ke slibným metodám stanovení metabolomu. Rozsáhlý výzkum přináší nové poznatky, které by ve svém výsledku měly vést k vytvoření klinicky použitelné metody výběru embrya a zvýšení efektivity transferu jednoho embrya. Klíčová slova: metabolomika, analýza metabolomu, kapilární elektroforéza, embryo, asistovaná reprodukce.
Objective: To review current technologies for the analytical examination of the embryonic metabolome and its perspectives. Design: Review article. Setting: Department of Gynecology and Obstetrics, Faculty of Medicine, Masaryk University, and University Hospital, Brno, Department of Biochemistry, Faculty of Science and CEITEC, Masaryk University, Brno. Methods and results: Nowadays, very sensitive analytical technologies are available. They enable exact measurement of various molecules – products of embryo metabolism during first days of cultivation. The capillary electrophoresis is one of promising method. Recent studies analysed metabolic differences between embryos that result in a pregnancy and those that do not. Amino acid levels, glucose or pyruvate in the embryo culture media were analysed most frequently. However, results of these studies are ambiguous. Conclusions: The capillary electrophoresis with contactless conductivity detection may provide a useful data of the embryonic metabolome. A comprehensive analysis of the used culture medium may represent a valuable adjunct to morphological criteria for enhanced rates of implantation and delivery. Key words: metabolomics, metabolome analysis, capillary electrophoresis, embryo, assisted reproductive techniques.
- MeSH
- asistovaná reprodukce MeSH
- chemické techniky analytické * metody MeSH
- elektroforéza kapilární MeSH
- kultivace embrya * MeSH
- kultivační média MeSH
- lidé MeSH
- metabolom * MeSH
- metabolomika metody MeSH
- terminologie jako téma MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
This review defines metabolomics and clarifies its history and significance. Various strategies of metabolomics research are described together with their main application fields. The review focuses on the methods of sample preparation and on the analytical methodologies that are most frequently used in metabolomic studies NMR, MS, GC/MS, HPLC/MS and CE/MS. Practical importance of metabolomics and its role in system biology are mentioned as well.
An original method based on capillary zone electrophoresis with fluorimetric detection has been developed for the determination of terpenic compounds. The method is based on the separation of a terpenes dynamically labeled by the non-ionogenic tenside poly(ethylene glycol) pyrenebutanoate, which was used previously for the labeling of biopolymers. The background electrolytes were composed of taurine-Tris buffer (pH 8.4). In addition to the non-ionogenic tenside aceton and poly(ethylene glycol) were used as the additives. The capillary zone electrophoresis with fluorometric detection at the excitation wavelength 335 nm and the emission wavelength 463 nm was successfully applied to the analysis of tonalid, cholesterol, vitamin A, ergosterol, estrone and farnesol at level of 10(-17) mol L(-1). Farnesol, is produced by Candida albicans as an extracellular quorum-sensing molecule that influences expression of a number of virulence factors, especially morphogenesis and biofilm formation. It enables this yeast to cause serious nosocomial infections. The sensitivity of this method was demonstrated on the separation of farnesol directly from the cultivation medium.
- MeSH
- biofilmy MeSH
- butyráty chemie MeSH
- Candida albicans chemie metabolismus MeSH
- cholesterol chemie MeSH
- elektroforéza kapilární metody MeSH
- ergosterol chemie MeSH
- estron chemie MeSH
- farnesol chemie izolace a purifikace metabolismus MeSH
- fluorometrie metody MeSH
- polymery chemie MeSH
- quorum sensing MeSH
- senzitivita a specificita MeSH
- terpeny chemie izolace a purifikace MeSH
- tetrahydronaftaleny chemie MeSH
- vitamin A chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In view of the fact that several studies have shown that diclofenac hydroxylation by cytochrome P450 2C9 deviated from Michaelis-Menten kinetics at low substrate concentrations, sweeping combined with MEKC was applied for the kinetic study of this pharmacologically important reaction. A 50 μm fused silica capillary (56 cm effective length) was used to carry out all separations. 70 mM SDS in 20 mM phosphate 20 mM tetraborate buffer, pH 8.6, was used as the BGE. Injection was accomplished by the application of 50 mbar (5 kPa) pressure to the sample vial for 52 s. Separation was performed at 22 kV (positive polarity), with a capillary temperature of 25°C and detection at 200 nm. The higher sensitivity of the sweeping-MEKC combination compared with the simple MEKC method enabled this reaction to be fitted to a Hill kinetic model and confirmed the findings of other authors. A Michaelis constant of 2.91±0.10 μM, maximum reaction velocity of 9.16±0.16 nmol/min/nmol and Hill coefficient of 1.66±0.08 were determined. This value of Hill coefficient confirms the presence of a positive cooperativity at low diclofenac concentrations and supports the hypothesis of two substrates binding at or near the active site.
- MeSH
- aromatické hydroxylasy chemie metabolismus MeSH
- chromatografie micelární elektrokinetická kapilární metody MeSH
- diklofenak metabolismus farmakologie MeSH
- inhibitory cyklooxygenasy farmakologie MeSH
- katalytická doména MeSH
- kinetika MeSH
- lidé MeSH
- molekulární struktura MeSH
- protein - isoformy MeSH
- vazba proteinů účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This study presents the in-capillary enzymatic biotransformation of dextromethorphan, an antitusive drug and opioid receptor antagonist, and subsequent electrophoretic separation of its products. The study includes the optimization of separation parameters to fulfill the requirements of an online microreaction. The analyses were performed in a bare fused-silica capillary using 100 mM sodium tetraborate (pH 10.0) mixed with linear polyacrylamide (20%, v/v) and 2-propanol (10%, v/v). This BGE was suitable for monitoring both off-line and in-capillary incubations. The partial filling technique enabled the enzymatic reaction to be carried out in its optimal environment (20 mM sodium phosphate, pH 7.4). Finally, in-capillary microreaction in the presence of cytochrome P450 3A4 gave satisfactory outcomes.
- MeSH
- biosenzitivní techniky MeSH
- dextromethorfan analýza chemie metabolismus MeSH
- elektroforéza kapilární přístrojové vybavení metody MeSH
- jaterní mikrozomy metabolismus MeSH
- lidé MeSH
- spektrofotometrie ultrafialová MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this work, electrophoretically mediated microanalysis (EMMA) was applied to the in-capillary tryptic digestion of proteins for proteomic purposes. Compared with classical in-solution tryptic digestion or the trypsin reactor commonly used for this purpose, the EMMA-based method is rapid, can be automated and requires only a small amount of trypsin preparation. Moreover, the protein digestion and the analysis of the resulting peptides are integrated into one procedure. A combination of the EMMA methodology with a partial filling technique was used in this study, since the pH optimum of the trypsin reaction differs strongly from the best pH for the CZE separation of peptides. In this set-up, a part of the capillary is filled with the best buffer for the tryptic digestion (50 mM Tris-HCl buffer, pH 8.5) whereas the rest of the capillary is filled with the BGE optimal for peptide separation (0.1 M phosphate buffer, pH 2.5). As the proteins differ in their isoelectric points, a sandwich type of injection was used. The analysed protein is thus injected between two trypsin zones, which ensures their mixing and digestion. The analysis of one protein comprising both the digestion and the peptide separation is then completed in 1 h using a commercial instrument for CE with no modifications.
The main aim of this work was to demonstrate the applicability of capillary zone electrophoresis in combination with field enhanced sample stacking in targeted metabolome analyses of adenine nucleotides--AMP, ADP, ATP, coenzymes NAD(+), NADP(+) and their reduced forms in Paracoccus denitrificans. Sodium carbonate/hydrogencarbonate buffer (100 mM, pH 9.6) with the addition of beta-CD at a concentration of 10 mM was found to be an effective BGE for their separation within 20 min. Besides this, special attention was paid to the development of the procedure for the extraction of specific metabolites from the bacterium P. denitrificans. This procedure was not only optimised to achieve the highest metabolite yields but also to obtain a sample that was fully compatible with the online preconcetration strategy used. The developed methodology was finally applied in a study of the bacterium P. denitrificans at various stages of the active respiratory chain.
