In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM structures of Drosophila Dicer-1 that show how Dicer-1 and its partner Loqs-PB cooperate (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, and (4) following complete dicing and initial product release. Our reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer-1⋅Loqs-PB heterodimer. The Dicer-1 dsRBD and three Loqs-PB dsRBDs form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-miRNA cleavage shifts the dsRBDs and partially closes Dicer-1, which may promote product release. Our data suggest a model for how the Dicer-1⋅Loqs-PB complex affects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and product release.
- MeSH
- Drosophila genetika MeSH
- mikro RNA * genetika metabolismus MeSH
- proteiny Drosophily * genetika metabolismus MeSH
- proteiny vázající RNA metabolismus MeSH
- ribonukleasa III genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, N.I.H., Intramural MeSH
Pevná bismutová kapková elektroda (SBiDE – solid bismuth drop electrode) je nová pracovní elektroda, kterou od roku 2020 komerčně nabízí na českém trhu společnost Metrohm. Cílem této práce bylo ověřit použitelnost SBiDE při voltametrickém stanovení modelové organické látky reprezentující elektrochemicky redukovatelné biologicky aktivní sloučeniny – léčiva metronidazolu (antibiotika používaného k léčbě onemocnění způsobených gram-pozitivními i gram-negativními anaerobními bakteriemi). Podle dostupných informací se jedná o vůbec první publikovanou výzkumnou práci využívající SBiDE. Za optimálních podmínek (Brittonův-Robinsonův pufr o pH 12,0 byl použitý jako základní elektrolyt a povrch pracovní elektrody nebyl elektrochemicky regenerován) byla získána pomocí diferenční pulzní voltametrie (DPV) lineární kalibrační závislost metronidazolu v koncentračním rozmezí od 1 do 600 μmol l−1, přičemž dosažená mez detekce (LOD) byla 0,41 μmol l−1 a mez stanovitelnosti (LOQ) byla 1,4 μmol l−1. Cyklická voltametrie (CV) na SBiDE byla použita pro charakterizaci elektrodového děje při ireverzibilní katodické redukci metronidazolu. Nově vyvinutá DPV metoda byla rovněž úspěšně použita při stanovení metronidazolu v autentických vzorcích pitné vody (LOD = 1,8 μmol l−1 a LOQ = 5,8 μmol l−1) a v různých lékových formách (jako srovnávací analytická metoda byla použita UV-Vis spektrofotometrie).
Solid bismuth drop electrode (SBiDE) is a new working electrode commercially available on the Czech market since 2020 by the company Metrohm. The aim of this work was to verify the applicability of SBiDE for the voltammetric determination of a model organic substance representing electrochemically reducible biologically active compounds, namely, the drug metronidazole (an antibiotic used to treat diseases caused by both Gram-positive and Gram-negative anaerobic bacteria). To the best of our knowledge, this is the very first published research work using SBiDE. Under optimum conditions (Britton-Robinson buffer of pH 12.0 was used as the supporting electrolyte and the working electrode surface was not electrochemically regenerated), a linear calibration dependence of metronidazole was obtained using differential pulse voltammetry (DPV) in the concentration range from 1 to 600 μmol L–1, with the limits of detection (LOD) and quantification (LOQ) of 0.41 μmol L–1 and 1.4 μmol L–1, respectively. Cyclic voltammetry (CV) on SBiDE was used to characterize the electrode process of the irreversible reduction of metronidazole. The newly developed DPV method was also successfully applied for the determination of metronidazole in authentic drinking water samples (LOD = 1.8 μmol L−1 and LOQ = 5.8 μmol L−1) and in various dosage forms (UV-Vis spectrophotometry was used as a comparative analytical method).
Stagnant water bodies have generally received little attention regarding the presence of endocrine disruptive compounds, although they can integrate diverse pollutants from multiple different sources. Many compounds of anthropogenic as well as natural origin can contribute to the overall estrogenicity of surface waters and some of them can exhibit adverse effects on aquatic biota even in very low concentrations. This study focused on freshwater ponds and reservoirs affected by water blooms and determined the estrogenic activity of water by in vitro bioassay as well as concentrations of several important groups of estrogenic compounds (estrogenic hormones, alkylphenols, and phytoestrogens) by LC-MS/MS analyses. Estrogenic hormones were found at concentrations up to 7.1 ng.L-1, similarly to flavonoids, whose concentrations did not exceed 12.5 ng.L-1. Among alkylphenols, only bisphenol A and 4-tert-octylphenol were detected in levels reaching 100 ng.L-1 at maximum. Estrogenic activity of water samples varied from below the quantification limit to 1.95 ng.L-1. There does not seem to be any general causal link of the massive phytoplankton occurrence with the estrogenicity of water or concentration of phytoestrogens, since they showed no direct relationship with the phytoplankton abundance or composition across sites. The contribution of the analysed compounds to the estrogenic activity was calculated in three scenarios. In minimum scenario, just the compounds above quantification limit (LOQ) were taken into account and for most samples, only minor part (<6%) of the biological activity could be explained. In the mean and maximum scenarios, we included also compounds below LOQ into the calculations at the level of LOQ/2 and LOQ, respectively. In these cases, a considerable part of the estrogenic activity could be attributed to the possible presence of steroid estrogens below LOQ. However, for the samples with estrogenic activity greater than 1 ng.L-1, more than 50% of the estrogenic activity remained unexplained even in the maximum scenario. Probably other compounds or possible interactions between individual substances cause the estrogenic activity in these types of water bodies and in this case, the results of LC-MS/MS analyses cannot sufficiently predict the biological effects. A complex approach including bioassays is needed when assessing the estrogenicity of these types of surface waters.
- MeSH
- chemické látky znečišťující vodu analýza metabolismus MeSH
- chromatografie kapalinová MeSH
- endokrinní disruptory analýza metabolismus MeSH
- estrogeny analýza metabolismus MeSH
- fytoplankton chemie metabolismus MeSH
- sladká voda chemie MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ascorbic acid (AA) and dehydroascorbic acid (DHA) are small polar molecules difficult to be retained in conventional chromatographic RP systems. Hydrophilic interaction liquid chromatography (HILIC) using Obelisk R (100 x 3.2mm, 5 microm, Sielc) analytical column and isocratic elution by ammonium acetate buffer pH 4.2 was found to be successful at this task, while other tested HILIC columns--Obelisk N (100 x 3.2mm, 5 microm, Sielc) and Luna HILIC (100 x 3.0mm, 3 microm, Phenomenex) were unsuccessful for the purposes of analysis. Charged aerosol detection (CAD) has recently become a new alternative universal detection system in HPLC, and was extremely convenient for the simultaneous analysis of AA and DHA without the need of subtraction approach and oxidation/reduction step. CAD response was found linear in defined range in spite of the fact that CAD is designated as non-linear detection method. A simple and fast HILIC-CAD method was applied for the analysis of pharmaceutical preparations containing AA. Method validation was performed including parameters of precision, accuracy, linearity, limit of detection and limit of quantitation (LOQ). The method was fast, accurate and precise for both detectors with LOQ(AA) 5 microg/ml for UV detection and 10 microg/ml for CAD, respectively. DHA was detected only by CAD within tested concentration range with LOQ(DHA) 1 microg/ml.
Recreational use of the potent synthetic opioid 3,4- dichloro-N-(2-(dimethylamino)cyclohexyl)-N-methylbenzamide (U-47700) is rising, accompanied by increasingly frequent cases of serious intoxication. This article reports a case of near-fatal U-47700 intoxication. A man was found unconscious (with drug powder residues). After 40 h in hospital (including 12 h of supported ventilation), he recovered and was discharged. Liquid chromatography/high-resolution mass spectrometry (LC/HRMS) or gas chromatography/mass spectrometry (GC/MS) were used to detect and quantify substances in powders, serum and urine. Powders contained U-47700 and two synthetic cannabinoids. Serum and urine were positive for U-47700 (351.0 ng/mL), citalopram (
- MeSH
- benzamidy škodlivé účinky analýza MeSH
- benzodiazepiny analýza MeSH
- chromatografie kapalinová MeSH
- citalopram analýza MeSH
- dospělí MeSH
- hmotnostní spektrometrie MeSH
- lidé MeSH
- midazolam analýza MeSH
- odhalování abúzu drog MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
- předávkování léky * MeSH
- soudní toxikologie MeSH
- zakázané drogy škodlivé účinky analýza MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
- Geografické názvy
- Česká republika MeSH
The main goal of this work was determination of residues of the antibiotics ofloxacin (OFLO), norfloxacin (NOR), ciprofloxacin (CIPRO), and enrofloxacin (ENRO) in wastewater samples. The samples, after acidification to pH 4.5 and addition of EDTA, were extracted on an anion-exchange cartridge in tandem with an Oasis HLB cartridge. The LC-FD method, developed in previous studies, was based on application of a monolithic C(18) column. The limit of quantification (LOQ) of the method was 250 ng L(-1) for OFLO, 25 ng L(-1) for NOR and CIPRO, and 50 ng L(-1) for ENRO. Mean recovery ranged between 75 and 121% for OFLO, NOR, CIPRO, and ENRO. A total of 14 wastewater samples were analyzed; these were collected from four hospitals and from influent and effluent from a wastewater-treatment plant in Coimbra, Portugal, during spring and autumn. CIPRO was present in all the samples, NOR was detected second most often, followed by OFLO. ENRO was found at concentrations under the LOQ in five hospital samples, and the highest level was found in influent from the WWTP.
- MeSH
- antibakteriální látky analýza MeSH
- chemické látky znečišťující vodu analýza MeSH
- financování organizované MeSH
- fluorescence MeSH
- fluorochinolony analýza MeSH
- kalibrace MeSH
- nemocnice MeSH
- reprodukovatelnost výsledků MeSH
- roční období MeSH
- senzitivita a specificita MeSH
- vysokoúčinná kapalinová chromatografie metody přístrojové vybavení MeSH
- Geografické názvy
- Portugalsko MeSH
The use of contaminated raw materials can lead to the transfer of mycotoxins into the final product, including beer. This study describes the use of the commercially available immunoaffinity column 11+Myco MS-PREP® and UPLC-MS/MS for the determination of mycotoxins in pale lager-type beers brewed in Czech Republic and other European countries. The additional aim of the work was to develop, optimize and validate this analytical method. Validation parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ), precision and accuracy were tested. The calibration curves were linear with correlation coefficients (R2 > 0.99) for all mycotoxins under investigation. The LOD ranged from 0.1 to 50 ng/L and LOQ from 0.4 to 167 ng/L. Recoveries of the selected analytes ranged from 72.2 to 101.1%, and the relative standard deviation under conditions repeatability (RSDr) did not exceed 16.3% for any mycotoxin. The validated procedure was successfully applied for the analysis of mycotoxins in a total of 89 beers from the retail network. The results were also processed using advanced chemometric techniques and compared with similar published studies. The toxicological impact was taken into account.
Ochratoxin A (OTA) is nephrotoxic, hepatotoxic, immunotoxic, neurotoxic, reprotoxic, teratogenic, and carcinogenic (group 2B), being characterized by species and sex differences in sensitivity. Despite the fact that OTA is in some aspects a controversial topic, OTA is the most powerful renal carcinogen. The aim of this study was to make a small survey concerning OTA content in black tea, fruit tea, and ground roasted coffee, and to assess OTA transfer into beverages. OTA content was measured using a validated and accredited HPLC-FLD method with a limit of quantification (LOQ) of 0.35 ng/g. The OTA amount ranged from LOQ up to 250 ng/g in black tea and up to 104 ng/g in fruit tea. Black tea and fruit tea, naturally contaminated, were used to prepare tea infusions. The transfer from black tea to the infusion was 34.8% ± 1.3% and from fruit tea 4.1% ± 0.2%. Ground roasted coffee naturally contaminated at 0.92 ng/g was used to prepare seven kinds of coffee beverages. Depending on the type of process used, OTA transfer into coffee ranged from 22.3% to 66.1%. OTA intakes from fruit and black tea or coffee represent a non-negligible human source.
- MeSH
- čaj mikrobiologie MeSH
- káva mikrobiologie MeSH
- koncentrace vodíkových iontů MeSH
- kontaminace potravin analýza MeSH
- ochratoxiny analýza MeSH
- ovoce mikrobiologie MeSH
- potravinářská mikrobiologie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A simple, sensitive, specific, and cost-effective analytical methodology was developed for the analysis of human plasma samples spiked with imatinib by CZE with on-line UV detection in the context of Therapeutic Drug Monitoring. Several analytical conditions such as the ionic strength (I) and the pH of the BGE composed of citric acid and ε-amino caproic acid were studied in regards of the presence of sodium chloride (NaCl) in plasma samples (1% m/v). Computer simulations (Simul software) were used to confirm the experimental results and to understand imatinib electrophoretic behavior in the presence of NaCl. Furthermore, the advantages of adding ACN to the sample containing NaCl to combine efficient protein precipitation and on-line CZE stacking of imatinib were demonstrated. LOD and LOQ values of 48 and 191 ng/mL were obtained from plasma sample supernatant after protein precipitation with ACN, which is much lower than mean imatinib plasma level observed for patients treated by imatinib mesylate (about 1000 ng/mL). Good linearity was obtained in the concentration range 191-5000 ng/mL (R2 > 0.997). RSD of less than 1.68% and 2.60% (n = 6) for migration times and corrected peak areas, respectively, were observed at the LOQ.
- MeSH
- acetonitrily chemie MeSH
- chlorid sodný chemie MeSH
- elektroforéza kapilární metody MeSH
- imatinib mesylát krev MeSH
- lidé MeSH
- limita detekce MeSH
- lineární modely MeSH
- reprodukovatelnost výsledků MeSH
- software MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Twenty three strains of Penicillium expansum, as a predominant species, were isolated from 23 (92%) out of 25 grape samples of 17 different grape varieties. The results of the identification of P. expansum strains were confirmed by a PCR method. Most of the isolates of P. expansum (21/23, 91%), when tested for toxigenicity, were bi-toxigenic: they produced citrinin (CIT) and particularly high amounts of patulin (PAT). A validated UPLC-MS/MS method for the determination of PAT and CIT was applied. The limits of quantification (LOQ) for PAT and CIT in grape must and toxigenicity testing samples were 100 and 2 ng/g, respectively. The results of PAT and CIT quantification in 23 grape must samples demonstrated the occurrence of PAT in 10 (43%) grape must samples (mean: 171 ng/g; median: 50 ng/g; and range: 143-644 ng/g) and the occurrence of CIT in two (9%) grape must samples (mean: 1 ng/g; median: 1 ng/g; and range: 2.5-3.5 ng/g). This is the first report on the natural occurrence of CIT in grape must. A validated HPLC-UV-VIS method for the determination of PAT in wine samples was applied, and concentrations in all 23 wine samples were below the LOQ (<10 ng/g).
- MeSH
- citrinin analýza MeSH
- dietární expozice MeSH
- druhová specificita MeSH
- limita detekce MeSH
- patulin analýza MeSH
- Penicillium klasifikace izolace a purifikace MeSH
- polymerázová řetězová reakce MeSH
- reprodukovatelnost výsledků MeSH
- spektrofotometrie ultrafialová MeSH
- tandemová hmotnostní spektrometrie MeSH
- víno analýza MeSH
- Vitis chemie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
