Quantitative PCR (qPCR) is a widely used method for nucleic acid quantification of various pathogenic microorganisms. For absolute quantification of microbial load by qPCR, it is essential to create a calibration curve from accurately quantified quantification standards, from which the number of pathogens in a sample is derived. Spectrophotometric measurement of absorbance is a routine method for estimating nucleic acid concentration, however, it may be affected by presence of other potentially contaminating nucleic acids or proteins and salts. Therefore, absorbance measurement is not reliable for estimating the concentration of stock solutions of quantification standards, based on which they are subsequently diluted. In this study, we utilized digital PCR (dPCR) for absolute quantification of qPCR plasmid standards and thus detecting possible discrepancies in the determination of the plasmid DNA number of standards derived from UV spectrophotometry. The concept of dPCR utilization for quantification of standards was applied on 45 qPCR assays using droplet-based and chip-based dPCR platforms. Using dPCR, we found that spectrophotometry overestimated the concentrations of standard stock solutions in the majority of cases. Furthermore, batch-to-batch variation in standard quantity was revealed, as well as quantitative changes in standards over time. Finally, it was demonstrated that droplet-based dPCR is a suitable tool for achieving defined quantity of quantification plasmid standards and ensuring the quantity over time, which is crucial for acquiring homogenous, reproducible and comparable quantitative data by qPCR.

• Publikační typ
• časopisecké články MeSH

BACKGROUND: Blastocystis is a human gut symbiont of yet undefined clinical significance. In a set of faecal samples collected from asymptomatic children of six distant populations, we first assessed the community profiles of protist 18S rDNA and then characterized Blastocystis subtypes and tested Blastocystis association with the faecal bacteriome community. METHODS: Stool samples were collected from 244 children and young persons (mean age 11.3 years, interquartile range 8.1-13.7) of six countries (Azerbaijan 51 subjects, Czechia 52, Jordan 40, Nigeria 27, Sudan 59 and Tanzania 15). The subjects showed no symptoms of infection. Amplicon profiling of the 18S rDNA was used for verification that Blastocystis was the most frequent protist, whereas specific real-time PCR showed its prevalence and quantity, and massive parallel amplicon sequencing defined the Blastocystis subtypes. The relation between Blastocystis and the stool bacteriome community was characterized using 16S rDNA profiling. RESULTS: Blastocystis was detected by specific PCR in 36% (88/244) stool samples and was the most often observed faecal protist. Children from Czechia and Jordan had significantly lower prevalence than children from the remaining countries. The most frequent subtype was ST3 (49%, 40/81 sequenced samples), followed by ST1 (36%) and ST2 (25%). Co-infection with two different subtypes was noted in 12% samples. The faecal bacteriome had higher richness in Blastocystis-positive samples, and Blastocystis was associated with significantly different community composition regardless of the country (p < 0.001 in constrained redundancy analysis). Several taxa differed with Blastocystis positivity or quantity: two genera of Ruminococcaceae were more abundant, while Bifidobacterium, Veillonella, Lactobacillus and several other genera were undrerrepresented. CONCLUSIONS: Asymptomatic children frequently carry Blastocystis, and co-infection with multiple distinct subtypes is not exceptional. Prevalence and quantity of the organism clearly differ among populations. Blastocystis is linked to both faecal bacteriome diversity and its composition.

• MeSH
• asymptomatické infekce epidemiologie   MeSH
• Blastocystis klasifikace   genetika   izolace a purifikace   MeSH
• blastocystóza epidemiologie   parazitologie   MeSH
• dítě MeSH
• feces parazitologie   MeSH
• genetická variace MeSH
• lidé MeSH
• mladiství MeSH
• prevalence MeSH
• protozoální DNA genetika   MeSH
• ribozomální DNA genetika   MeSH
• střevní mikroflóra genetika   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Ázerbájdžán MeSH
• Československo MeSH
• Jordánsko MeSH
• Nigérie MeSH
• Súdán MeSH
• Tanzanie MeSH

A microtiter plate-based assay was developed for the automatic monitoring of degradation profile of the yellow-coloured nitrophenolic compounds. The method enables to reduce the intervals between measurements of substrate concentration to minutes and to overcome the problem of discontinuity of sampling typical for conventional methods. The concentrations of nitrophenolic compounds were calculated from the absorbance values determined automatically by BIOSCREEN C. Verification of the method was based on the comparison of results with the conventional HPLC method results. The values of the rate and saturation constants were comparable for both the microtiter plate-based assay and the conventional HPLC method. The automatic method described here seems to be efficient for the screening degradation studies, which requires the treatment of quantity of samples.

• MeSH
• Arthrobacter izolace a purifikace   metabolismus   růst a vývoj   MeSH
• bakteriologické techniky metody   přístrojové vybavení   MeSH
• biodegradace MeSH
• financování organizované MeSH
• katecholy metabolismus   MeSH
• kinetika MeSH
• kultivační média MeSH
• molekulární sekvence - údaje MeSH
• nitrofenoly metabolismus   MeSH
• půdní mikrobiologie MeSH
• Rhodococcus genetika   izolace a purifikace   klasifikace   metabolismus   MeSH
• sekvenční analýza DNA MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• hodnotící studie MeSH

The paper examines the interest of the commercial banks' stakeholders in Pillar 3 disclosures and their behaviour during the timing of serious market turbulence. The aim is to discover to which extent current banking regulation supports stakeholders' interest in the information required by regulators to be disclosed. The examined data consists of log files that were pre-processed using web mining techniques and from which were extracted frequent item sets by quarters and evaluated in terms of quantity. The authors have proposed a methodology to evaluate frequent item sets of web parts over a dedicated time. Based on the verification of applied methodology on two commercial banks, the results show that stakeholders' interest in disclosures is highest in the first quarter at each year and after turbulent times in 2009 their interests decreased. Moreover, the results suggest that stakeholders expressed higher interest than in regulatory required Pillar 3 information in the following group of information: Pillar3 related information, Annual reports, Information on Group. Following our results, the paper contributes to cover the gap in the research by analysing Pillar 3 disclosures and their compliance with regulatory requirements, which also increase the interest of the relevant stakeholders to conduce them as an effective market discipline tool.

• MeSH
• zveřejnění * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Metoda krevních skvrn (BPA) je validní forenzní metodou, spadající do metodik biologických stop, využívajících i trigonomické modely. Přes její historický vývoj byla vícekrát propracovaná až do současné formulace v celosvětově uznávané podobě standardního metodického listu. Jde o metodu, která umožňuje analyzovat dynamické a charakterové vlastnosti kapek krve po dopadu na pevnou plochu, kterou může být podlaha, stěny, strop, různé předměty, ale i šaty a eventuálně i tělo. Dle přítomností takovýchto krevních stop je jí možné využít v rekonstrukci trestních činů. Podmínkou jej jejich dobrá čitelnost. V naší práci jsme odzkoušeli validitu uvedené metody na experimentálním modelu s použitím střelné zbraně. Jsou srovnávané hodnoty výpočtu trajektorií délky dopadu, výšky výstřiku a vzdálenosti letu kapek krve ze vzdálenosti 1, 3, 5 a 10 metrů. Vycházelo se přitom ze dvou různých vstupních předpokladů. Prvním je známá hodnota vzdálenosti dopadu a uhel dopadu kapky krve a druhým známá hodnota výšky výstřiku a uhel dopadu kapky krve. Podle trigonomických vzorců byla pak vypočítaná buď vzdálenost dopadu vybrané kapky krve, nebo výška výstřiku vybrané kapky krve a bez možnosti ověření i vzdálenost trajektorie letu kapky krve. Výsledky dokumentují, že uvedená metoda je pro tyto potřeby jen orientační a vypočítané hodnoty se od reálních odchylují se zvětšující se vzdáleností výstřelu. Až na ojedinělé hodnoty jsou vypočítané výsledky vzdálenosti dopadu kapky krve pravidelně nižší než skutečné hodnoty a vypočítané hodnoty výšky výstřiku kapky krve pravidelně vyšší než skutečné hodnoty. Navzdory uvedeným nepřesnostem mají námi získané výsledky jistou výpovědní hodnotu. Jeví se užitečné využívat tuhle metodu při vyšetřování a ověřovaní průběhu jednotlivých činů v kriminalistické a forenzní praxi, jako doplňkový zdroj informací.

Bloodstain pattern analysis (BPA) is a valid forensic method which belongs to the category of biological methods using trigonomic models. Despite its development through the years, the method has been re-formulated a standard one and globally used, recognized in standard sheets. This method permits exact analysis of the dynamic and characteristic properties of bloodstains after impact on surfaces such as floors, walls, and ceilings, various exterior and interior items, and clothes. It is also possible to determine the characteristics of blood from the outer part of the body. According to the presence of blood and its quantity, it is also possible to use this method for verification of reconstruction of criminal acts, while being tested for its validity with primary conditions of preserved and readable traces of blood. Even though this method is not considered as the major one or the only one information obtained in this way can be used for judicial. In our research, we tested the validity of this method in an experimental model using firearms. We compared measurements of the lengths of trajectory of impact and the height of the blood sprayed upwards from a distance of 1, 3, 5 and 10 meters. The experiment was based on two main presumptions. The first was the knowledge of the value of the distance and the angle of impact of the bloodstain, the second, the ability of the blood to reach a certain height and the angle of its impact. In accordance with trigonometric formulas, both the impact of the selected distance of drops of blood, and the height of the selected bloodstain could be determined without any verification of the flight trajectory and the distance of bloodstains. The results indicate that the method for these requirements differs from the real values, while increasing the measurements with the indicated spot of the shot. Aside from the unique values which were calculated, other results of the impact of the distance of drops of bloodstain were considered of lower value, and the values concerning the height of the bloods stains after the shot higher than real values. In spite of the lack of total accuracy, we recommend using this method widely and more often for investigation and verification of individual acts in criminal and forensic practice.

• Klíčová slova
• balistika, trigonometrie, střelní zbraně, analýza krevních stop,
• MeSH
• biofyzikální jevy MeSH
• fyzikální jevy MeSH
• hydrodynamika MeSH
• krevní skvrny * MeSH
• lidé MeSH
• soudní balistika * statistika a číselné údaje   MeSH
• soudní lékařství MeSH
• střelné zbraně MeSH
• teoretické modely * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• hodnotící studie MeSH

ISO15189:2012 requires medical laboratories to document metrological traceability of their results. While the ISO17511:2003 standard on metrological traceability in laboratory medicine requires the use of the highest available level in the traceability chain, it recognizes that for many measurands there is no reference above the manufacturer's selected measurement procedure and the manufacturer's working calibrator. Some immunoassays, although they intend to measure the same quantity and may even refer to the same reference material, unfortunately produce different results because of differences in analytical selectivity as manufacturers select different epitopes and antibodies for the same analyte. In other cases, the cause is the use of reference materials, which are not commutable. The uncertainty associated with the result is another important aspect in metrological traceability implementation. As the measurement uncertainty on the clinical samples is influenced by the uncertainty of all steps higher in the traceability chain, laboratories should be provided with adequate and appropriate information on the uncertainty of the value assignment to the commercial calibrators that they use. Although the between-lot variation in value assignment will manifest itself as part of the long-term imprecision as estimated by the end-user, information on worst-case to be expected lot-lot variation has to be communicated to the end-user by the IVD provider. When laboratories use ancillary equipment that potentially could have a critical contribution to the reported results, such equipment needs verification of its proper calibration and criticality to the result uncertainty could be assessed by an approach based on risk analysis, which is a key element of ISO15189:2012 anyway. This paper discusses how the requirement for metrological traceability as stated in ISO15189 should be met by the medical laboratory and how this should be assessed by accreditation bodies.

• MeSH
• kalibrace MeSH
• konsensus * MeSH
• lidé MeSH
• nejistota MeSH
• referenční standardy MeSH
• řízení kvality MeSH
• technologie lékařská normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Nádory jsou geneticky a klinicky velmi různorodá onemocnění, a proto se v poslední době řada proteomických studií zabývá hledáním bio­markerů, které by usnadnily prognostiku, dia­gnostiku či léčbu onkologických onemocnění. Účinným nástrojem je hmotnostní spektrometrie umožňující identifikaci, kvantifikaci a charakterizaci bio­molekul v komplexních bio­logických vzorcích. Prvním krokem vedoucím k selekci bio­markerů je tzv. discovery proteomika, jejímž cílem je detailní analýza vzorků směřující ke zmapování a porovnání přítomných proteinů a výběru vhodných kandidátů na potenciální bio­markery. V dalším kroku proteomické analýzy probíhá verifikace vybraných bio­markerů tzv. cílenou (targeted) proteomikou, jejímž úkolem je ověření přítomnosti a kvantity daného proteinu v analyzovaných, přesně klinicky definovaných vzorcích. Předložený článek je zaměřen na popis různých typů metod vhodných pro kvantitativní analýzu proteinů využívající hmotnostní spektrometrií.

Cancers are genetically and clinically very heterogeneous diseases; therefore, various proteomic studies have been trying to find bio­markers which can facilitate prognosis, dia­gnosis or treatment of these oncological diseases. The mass spectrometry is an effective tool for identification, quantitation, and characterization of bio­molecules in the complex bio­logical samples. The first step suitable for selection of bio­markers called discovery proteomics provides a detailed analysis of the samples contributing to the identification of proteins, comparison of their presence in the samples, and selection of the convenient candidates for the prospective bio­markers. The next step of proteomics analysis is directed towards verification of chosen bio­markers with the approach called targeted proteomics. This technique evaluates presence and quantity of the proteins (bio­markers) in clinically precisely defined samples. This article focuses on the description of various approaches suitable for the quantitative analysis of the proteins connected with mass spectrometry. Key words: quantitative proteomics – mass spectrometry – protein – bio­marker – oncology – cancer This work was supported by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101) and by MH CZ – DRO (MMCI, 00209805). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers. Submitted: 20. 1. 2014 Accepted: 7. 4. 2014

• Klíčová slova
• kvantitativní proteomika, SILAC,
• MeSH
• hmotnostní spektrometrie * metody   MeSH
• izotopové značení MeSH
• lidé MeSH
• nádorové biomarkery * analýza   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorové buňky kultivované * MeSH
• nádorové proteiny * analýza   metabolismus   MeSH
• nádory diagnóza   MeSH
• proteomika MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

The annual effective doses of aircrew members often exceed the limit of 1 mSv for the public due to the increased level of cosmic radiation at the flight altitudes, and thus, it is recommended to monitor them. Aircrew dosimetry is usually performed using special computer programs mostly based on results of Monte Carlo simulations. Contemporary, detectors are used mostly for validation of these computer codes, verification of effective dose calculations and for research purposes. One of such detectors is active silicon semiconductor deposited energy spectrometer Liulin. Output quantities of measurement with the Liulin detector are the absorbed dose in silicon D and the ambient dose equivalent H*(10); to determine it, two calibrations are necessary. The purpose of this work was to develop a calibration methodology that can be used to convert signal from the detector to D independently on calibration performed at Heavy Ion Medical Accelerator facility in Chiba, Japan.

• MeSH
• absorpce radiace MeSH
• design vybavení MeSH
• kalibrace MeSH
• kosmické záření * MeSH
• křemík MeSH
• letadla přístrojové vybavení   MeSH
• lidé MeSH
• metoda Monte Carlo MeSH
• monitorování radiace přístrojové vybavení   MeSH
• nadmořská výška MeSH
• neutrony MeSH
• polovodiče MeSH
• pracovní expozice analýza   normy   MeSH
• radiační expozice MeSH
• radionuklidy MeSH
• sluneční aktivita MeSH
• těžké ionty MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• chemie MeSH
• heterotopická osifikace MeSH
• patologické procesy MeSH

• Konspekt
• Chemie. Mineralogické vědy
• NLK Obory
• chemie, klinická chemie
• anatomie
• NLK Publikační typ
• studie