In this paper, initial processing of biological material, cell separation algorithms and other procedures are discussed. For samples with initial infiltration of plasma cells > 5%, CD138 MicroBeads and Auto-Magnetic-Activated Cell Sorting program are used. Fluorescence-Activated Cell Sorting is used exclusively for cell populations with low-abundance; these samples are detected using fluorescently labeled antibodies only. Isolated plasma cells are further processed for molecular biological studies, for cytogenetics and protein analyses. Furthermore, this work examines the pitfalls of research related to multiple myeloma; some of them we have overcome, while the others are still problematic.

• MeSH
• banky biologického materiálu MeSH
• biomedicínský výzkum MeSH
• kostní dřeň patologie   MeSH
• lidé MeSH
• mnohočetný myelom diagnóza   MeSH
• odběr biologického vzorku MeSH
• plazmatické buňky klasifikace   imunologie   MeSH
• průtoková cytometrie metody   MeSH
• syndekan-1 analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• syndekan-1 MeSH

BACKGROUND: Acidic phytohormones are small molecules controlling many physiological functions in plants. A comprehensive picture of their profiles including the active forms, precursors and metabolites provides an important insight into ongoing physiological processes and is essential for many biological studies performed on plants. RESULTS: A high-throughput sample preparation method for liquid chromatography-tandem mass spectrometry determination of 25 acidic phytohormones classed as auxins, jasmonates, abscisates and salicylic acid was optimised. The method uses a small amount of plant tissue (less than 10 mg fresh weight) and acidic extraction in 1 mol/L formic acid in 10% aqueous methanol followed by miniaturised purification on reverse phase sorbent accommodated in pipette tips organised in a 3D printed 96-place interface, capable of processing 192 samples in one run. The method was evaluated in terms of process efficiency, recovery and matrix effects as well as establishing validation parameters such as accuracy and precision. The applicability of the method in relation to the amounts of sample collected from distantly related plant species was evaluated and the results for phytohormone profiles are discussed in the context of literature reports. CONCLUSION: The method developed enables high-throughput profiling of acidic phytohormones with minute amounts of plant material, and it is suitable for large scale interspecies studies.

• Klíčová slova
• 3D printing, Evolutionarily distant plant species, High-throughput, In-tip microSPE, Liquid chromatography, Mass spectrometry, Miniaturisation, Plant hormones,
• Publikační typ
• časopisecké články MeSH

Cytocentrifugation is a common technique for the capture of cells on microscopic slides. It usually requires a special cytocentrifuge or cytorotor and cassettes. In the study presented here, we tested the new concept of cytocentrifugation based on the threaded connection of the lid and the sample holder to ensure an adjustable flow of solutions through the filters and the collection of the filtered solutions in the reservoir during centrifugation. To test this concept, we developed a device for the preparation of cell samples on circular coverslips. The device was tested for the capture and sample processing of both eukaryotic and prokaryotic cells, cell nuclei, and mitochondria for microscopy analysis including image cytometry. Moreover, an efficient procedure was developed for capturing formaldehyde-fixed cells on non-treated coverslips without cell drying. The results showed that the tested arrangement enables the effective capture and processing of all of the tested samples and the developed device represents an inexpensive alternative to common cytocentrifuges, as only the paper filter is consumed during sample processing, and no special centrifuge, cytorotor, or cassette is necessary. As no additional system of solution removal is required during sample staining, the tested concept also facilitates the eventual automation of the staining procedure.

• Klíčová slova
• cytocentrifugation, microscopy, sample processing, staining,
• MeSH
• barvení a značení metody   MeSH
• centrifugace přístrojové vybavení   metody   MeSH
• cytologické techniky přístrojové vybavení   metody   MeSH
• lidé MeSH
• mikroskopie metody   MeSH
• odběr biologického vzorku metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The approaches to matrix effects determination and reduction in ultra-high performance supercritical fluid chromatography with mass spectrometry detection have been evaluated in this study using different sample preparation methods and investigation of different calibration models. Five sample preparation methods, including protein precipitation, liquid-liquid extraction, supported liquid extraction, and solid phase extraction based on both "bind and elute" and "interferent removal" modes, were optimized with an emphasis on the matrix effects and recovery of 8 forms of vitamin E, including α-, β-, γ-, and δ-tocopherols and tocotrienols, from plasma. The matrix effect evaluation included the use and comparison of external and internal calibration using three models, i.e., least square with no transformation and no weighting (1/x0), with 1/x2 weighting, and with logarithmic transformation. The calibration model with logarithmic transformation provided the lowest %-errors and the best fits. Moreover, the type of the calibration model significantly affected not only the fit of the data but also the matrix effects when evaluating them based on the comparison of calibration curve slopes. Indeed, based on the used calibration model, the matrix effects calculated from calibration slopes ranged from +92% to - 72% for α-tocopherol and from -77% to +19% in the case of δ-tocotrienol. Thus, it was crucial to calculate the matrix effect by Matuszewski's post-extraction approach at six concentration levels. Indeed, a strong concentration dependence was observed for all optimized sample preparation methods, even if the stable isotopically labelled internal standards (SIL-IS) were used for compensation. The significant differences between individual concentration levels and compounds were observed, even when the tested calibration range covered only one order of magnitude. In methods with wider calibration ranges, the inappropriate use of calibration slope comparison instead of the post-extraction addition approach could result in false negative results of matrix effects. In the selected example of vitamin E, solid-phase extraction was the least affected by matrix effects when used in interferent removal mode, but supported liquid extraction resulted in the highest recoveries. We showed that the calibration model, the use of a SIL-IS, and the analyte concentration level played a crucial role in the matrix effects. Moreover, the matrix effects can significantly differ for compounds with similar physicochemical properties and close retention times. Thus, in all bioanalytical applications, where different analytes are typically determined in one analytical run, it is necessary to carefully select the data processing in addition to the method for the sample preparation, SIL-IS, and chromatography.

• Klíčová slova
• Calibration curve, Electrospray ionization, Matrix effects, Post-extraction addition, Sample preparation, Single quadrupole mass spectrometry, Ultra-high performance supercritical fluid chromatography,
• MeSH
• extrakce na pevné fázi metody   MeSH
• hmotnostní spektrometrie * metody   MeSH
• kalibrace MeSH
• lidé MeSH
• superkritická fluidní chromatografie * metody   MeSH
• vitamin E * krev   analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• vitamin E * MeSH

An automated processing of convergent beam electron diffraction (CBED) patterns is presented. The proposed methods are used in an automated tool for estimating the thickness of transmission electron microscopy (TEM) samples by matching an experimental zone-axis CBED pattern with a series of patterns simulated for known thicknesses. The proposed tool detects CBED disks, localizes a pattern in detected disks and unifies the coordinate system of the experimental pattern with the simulated one. The experimental pattern is then compared disk-by-disk with a series of simulated patterns each corresponding to different known thicknesses. The thickness of the most similar simulated pattern is then taken as the thickness estimate. The tool was tested on [0 1 1] Si, [0 1 0] α-Ti and [0 1 1] α-Ti samples prepared using different techniques. Results of the presented approach were compared with thickness estimates based on analysis of CBED patterns in two beam conditions. The mean difference between these two methods was 4.1% for the FIB-prepared silicon samples, 5.2% for the electro-chemically polished titanium and 7.9% for Ar(+) ion-polished titanium. The proposed techniques can also be employed in other established CBED analyses. Apart from the thickness estimation, it can potentially be used to quantify lattice deformation, structure factors, symmetry, defects or extinction distance.

• Klíčová slova
• Automatization, CBED, Computer vision, TEM, Thickness estimation, Zone axis,
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Model samples of Campylobacter jejuni for polymerase chain reaction (PCR) were prepared by rapid and simple procedures consisting of centrifugation, proteinase K treatment, Chelex 100 treatment, and boiling lyses. A PCR based on specific amplification of the variable sequence of 16S rRNA gene was performed using Tth DNA polymerase and the PCR products were visualized by agarose gel electrophoresis. The assay allowed the detection of 10 CFU/mL C. jejuni in the physiological saline and 100 CFU/mL in the basic Park and Sanders broth.

• MeSH
• Campylobacter coli klasifikace   genetika   izolace a purifikace   MeSH
• Campylobacter jejuni klasifikace   genetika   izolace a purifikace   MeSH
• Campylobacter klasifikace   genetika   izolace a purifikace   MeSH
• chlorid sodný MeSH
• kultivační média MeSH
• lidé MeSH
• polymerázová řetězová reakce metody   MeSH
• potravinářská mikrobiologie * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorid sodný MeSH
• kultivační média MeSH

Cryo-electron microscopy has established as a mature structural biology technique to elucidate the three-dimensional structure of biological macromolecules. The Coulomb potential of the sample is imaged by an electron beam, and fast semi-conductor detectors produce movies of the sample under study. These movies have to be further processed by a whole pipeline of image-processing algorithms that produce the final structure of the macromolecule. In this chapter, we illustrate this whole processing pipeline putting in value the strength of "meta algorithms," which are the combination of several algorithms, each one with different mathematical rationale, in order to distinguish correctly from incorrectly estimated parameters. We show how this strategy leads to superior performance of the whole pipeline as well as more confident assessments about the reconstructed structures. The "meta algorithms" strategy is common to many fields and, in particular, it has provided excellent results in bioinformatics. We illustrate this combination using the workflow engine, Scipion.

• Klíčová slova
• Cryo-electron microscopy, Image processing, Scipion, Single particle,
• MeSH
• algoritmy * MeSH
• elektronová kryomikroskopie metody   MeSH
• makromolekulární látky ultrastruktura   MeSH
• molekulární biologie metody   MeSH
• počítačové zpracování obrazu metody   MeSH
• průběh práce MeSH
• výpočetní biologie MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• zobrazování trojrozměrné metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• makromolekulární látky MeSH

The growing technical standard of acquisition systems allows the acquisition of large records, often reaching gigabytes or more in size as is the case with whole-day electroencephalograph (EEG) recordings, for example. Although current 64-bit software for signal processing is able to process (e.g. filter, analyze, etc) such data, visual inspection and labeling will probably suffer from rather long latency during the rendering of large portions of recorded signals. For this reason, we have developed SignalPlant-a stand-alone application for signal inspection, labeling and processing. The main motivation was to supply investigators with a tool allowing fast and interactive work with large multichannel records produced by EEG, electrocardiograph and similar devices. The rendering latency was compared with EEGLAB and proves significantly faster when displaying an image from a large number of samples (e.g. 163-times faster for 75 × 10(6) samples). The presented SignalPlant software is available free and does not depend on any other computation software. Furthermore, it can be extended with plugins by third parties ensuring its adaptability to future research tasks and new data formats.

• MeSH
• časové faktory MeSH
• elektroencefalografie metody   MeSH
• elektrokardiografie metody   MeSH
• internet MeSH
• počítačové zpracování signálu * MeSH
• přístup k informacím MeSH
• software * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Spatial reference frames (RFs) play a key role in spatial cognition, especially in perception, spatial memory, and navigation. There are two main types of RFs: egocentric (self-centered) and allocentric (object-centered). Although many fMRI studies examined the neural correlates of egocentric and allocentric RFs, they could not sample the fast temporal dynamics of the underlying cognitive processes. Therefore, the interaction and timing between these two RFs remain unclear. Taking advantage of the high temporal resolution of intracranial EEG (iEEG), we aimed to determine the timing of egocentric and allocentric information processing and describe the brain areas involved. We recorded iEEG and analyzed broad gamma activity (50-150 Hz) in 37 epilepsy patients performing a spatial judgment task in a three-dimensional circular virtual arena. We found overlapping activation for egocentric and allocentric RFs in many brain regions, with several additional egocentric- and allocentric-selective areas. In contrast to the egocentric responses, the allocentric responses peaked later than the control ones in frontal regions with overlapping selectivity. Also, across several egocentric or allocentric selective areas, the egocentric selectivity appeared earlier than the allocentric one. We identified the maximum number of egocentric-selective channels in the medial occipito-temporal region and allocentric-selective channels around the intraparietal sulcus in the parietal cortex. Our findings favor the hypothesis that egocentric spatial coding is a more primary process, and allocentric representations may be derived from egocentric ones. They also broaden the dominant view of the dorsal and ventral streams supporting egocentric and allocentric space coding, respectively.

• Klíčová slova
• Allocentric, Egocentric, High-frequency gamma activity, Intracranial EEG, Reference frames, Spatial judgment,
• MeSH
• elektrokortikografie MeSH
• lidé MeSH
• magnetická rezonanční tomografie MeSH
• mínění fyziologie   MeSH
• prostorové vidění * MeSH
• vnímání prostoru * fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

For conducting questionnaire studies there exist generally recommended methods that were also respected during the project funded by the Grant Agency MoH CR - "Subjective approach of inhabitants of Ostrava to their health in association with their life-style, socio-economic status and education". The preparatory phase included the collection of literature and information on the investigated theme. The questionnaire had got five parts - A. General questions, B. Employment, C. Way of life, D. Health state and E. Personality. The validity of questionnaire was tested in the pre-research. In the main questionnaire study 3,000 questionnaires were sent. The total response rate was 21.1% (634 completed questionnaires). In the sample there were no differences in percentage rate by sex and age, but there are differences in the educational structure. After realizing the main questionnaire study, the repeatability study was carried out to find out the reliability of the answers. The response rate was 60.3% (181 questionnaires). The Kappa index and the total percentage of agreement were used for the evaluation of the repeatability study. The agreement was almost perfect and good in the total of 62.3 % of the questions. The quality of data was ensured by double data entry and by choosing the appropriate software. The selection of data for the complex analysis was based on the results of the repeatability study. On the basis of individual information from the questionnaire, new groups of individuals were generated. These groups of individuals were analysed further in relation to health and life-style by socio-economic factors in the models.

• MeSH
• automatizované zpracování dat MeSH
• lidé MeSH
• průzkumy a dotazníky * MeSH
• socioekonomické faktory * MeSH
• zdravotní stav * MeSH
• zdravotnické přehledy * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH