Optimal resolution
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Proteins, which, in their native conditions, sample a multitude of distinct conformational states characterized by high spatiotemporal heterogeneity, most often termed as intrinsically disordered proteins (IDPs), have become a target of broad interest over the past 15years. With the growing evidence of their important roles in fundamental cellular processes, there is an urgent need to characterize the conformational behavior of IDPs at the highest possible level. The unique feature of NMR spectroscopy in the context of IDPs is its ability to supply details of their structural and temporal alterations at atomic-level resolution. Here, we briefly review recently proposed NMR-based strategies to characterize transient states populated by IDPs and summarize the latest achievements and future prospects in methodological development. Because low chemical shift dispersion represents the major obstacle encountered when studying IDPs by nuclear magnetic resonance, particular attention is paid to techniques allowing one to approach the physical limits of attainable resolution.
Při získávání mikroskopických obrazů o velmi vysokém rozlišení metodou skladu výsledného obrazu z jednotlivých dílů jsme narazili na některé problémy. Mezi nimi byla nutnost refokusace hiezi jednotlivými dílky. S tím souvisely problémy se spojením obrazů, které si vzájemně zcela neodpovídaly a oblast spojení byla zřetelná. Byl vyvinut prograin překonávající některé problémy pri spojování obrazových dílků, který pracuje se všemi díly naráz a hledá optimální pořadí spojení dílů. Jednotlivé dílky spojuje strmým gradientem, který probíhá po náhodně generované křivce. Program dává dobré výsledky i pri spojení obrazů s pozadím či otvory ve snímané tkáni. Metoda postupného snímání a následné montáže obrazu byla využita i pro snímání sbírky kožnich lymfomů ve spolupráci s Institutem pro dermatologii Univerzitní nemocnice v Curychu. Takto vzniklá digitální sbírka je veřejně k dispozici jako kapitola šesté verze Hypertextového atlasu dermatopatologie na www.muni.cz/atlases.
Composing microscopic images of very high resolution from several paris posed some problems. One of them was the necessity to adjust the focusing level when moving fi'om one part to another. Re-focusing lead to problems with joining the image parts, which did not correspond exactly, and the area of image fusion was noticeable. A computer program was developed to overcome these problems. Our program worked with all the image parts together to find their optimal order for image fusion. Individual image parts were joined using a steep gradient running along a randomly generated curve. This method gave good results even in images with background or holes in the tissue. The method of composing large images from individual parts was used for digitizing the skin lymphoma collection of the Institute of Dermatology, University Hospital, Zurich. This collection of digital images is a part of the 6th version of Hypertext atlas of Dermatopathology at www.muni.cz/atlases.
Modern high resolution mass spectrometry offers unique identification capability in drug metabolism studies. In this work detailed imatinib metabolization in the plasma of patients with chronic myeloid leukemia is presented. The metabolites were separated by liquid chromatography on a C18 column with mass spectrometry detection via an Orbitrap Elite instrument (Thermo Scientific) based on exact mass measurement. A scan range of m/z 350-1200 resolution of 60,000 was applied (mass accuracy of 5ppm). The data were evaluated using the advanced software for mass spectrometry Mass Frontier and MetWorks. In all plasma samples, studied 90 metabolites in the concentration range of 0.0001-1μmol/L were identified by m/z values and confirmed by exact mass measurement of the MS(2) and MS(3) fragmentations. In order to achieve optimal clinical response and avoid toxicity, current therapeutic monitoring of parent drug is a useful tool for the individualization of treatment. Current high-resolution mass spectrometry possesses the potential to broaden this approach by monitoring number of potentially clinically relevant drug metabolites.
- MeSH
- chromatografie kapalinová metody MeSH
- chronická myeloidní leukemie krev farmakoterapie MeSH
- hmotnostní spektrometrie metody MeSH
- imatinib mesylát krev terapeutické užití MeSH
- lidé MeSH
- molekulová hmotnost MeSH
- protinádorové látky krev terapeutické užití MeSH
- software MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this short communication we report optimized procedures for the chiral separation of non-charged [6]helicene (1) and cationic derivative 1-butyl-3-(2-methyl[6]helicenyl)-imidazolium bromide (2) using high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) methods. The possibility of using capillary electrophoresis (CE) was also tested. The satisfactory results were obtained with SFC, where the highly selective resolution of four enantiopure 1 and 2 helicenes was achieved in a single run within 5min. The semi-preparative procedure for the isolation of P and M enantiomers of compound 2, including circular dichroism data, is reported here for the first time. The results could be used in further separations and analytical applications targeting carbohelicenes vs. positively charged helicene derivatives.
In this chapter, the basic principles and protocols of the electron microscopical detections of specific DNA and RNA sequences are described. We focused primarily on a comparison of various methods of electron microscopy in situ hybridization (EM-ISH) with respect to their sensitivity and the structural preservation of the sample with the aim of helping the readers select the appropriate hybridization protocol. As the post-embedding EM-ISH most frequently represents the optimal choice, the protocol for the post-embedding EM-ISH approach is described in detail. Concurrently, the alternative methods based on the enzymatic synthesis of the labeled nucleic acids chains that can be used for the detection of DNA or RNA molecules in situ are mentioned. In this respect, the technique enabling the enzymatic detection of the polyadenylated RNA sequences is described in detail.
May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FTNS) and Epstein (EPS) syndromes are rare autosomal dominant disorders with giant platelets and thrombocytopenia. Other manifestations of these disorders are combinations of the presence of granulocyte inclusions and deafness, cataracts and renal failure. Currently, MHA, SBS, FTNS and EPS are considered to be distinct clinical manifestation of a single illness caused by mutations of the MYH9 gene encoding the heavy chain of non-muscle myosin IIA (NMMHC-IIA). As the MYH9 gene has a high number of exons, it takes much time and material to use this method for the detection of MYH9 mutations. Recently, a new method has been introduced for scanning DNA mutations without the need for direct sequencing: high-resolution melting analysis (HRMA). Mutation detection with HRMA relies on the intercalation of the specific dye (LC Green plus) in double-strand DNA and fluorescence monitoring of PCR product melting profiles. In our study, we optimized the conditions and used HRMA for rapid screening of mutations in all MYH9 exons in seven affected individuals from four unrelated families with suspected MYH9 disorders. Samples identified by HRMA as positive for the mutation were analysed by direct sequencing. HRMA saved us over 85% of redundant sequencing.
- MeSH
- lidé MeSH
- missense mutace MeSH
- molekulární motory genetika chemie MeSH
- mutační analýza DNA metody MeSH
- těžké řetězce myosinu genetika chemie MeSH
- tranzitní teplota MeSH
- trombocytopenie genetika krev MeSH
- trombocyty patologie MeSH
- velikost buňky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
The objective of the presented study was to develop and optimize a simple, high-throughput method for the control of 32 mycotoxins (Fusarium and Alternaria toxins, aflatoxins, ergot alkaloids, ochratoxins, and sterigmatocystin) in beer. Due to the broad range of their physicochemical properties, the sample preparation step was simplified as much as possible to avoid analyte losses. The addition of acetonitrile to beer samples enabled precipitation of abundant matrix components. The clean-up efficiency was controlled by ambient mass spectrometry employing a direct analysis in real time (DART) ion source. For determination of analytes, ultra-high-performance liquid chromatography hyphenated with high-resolution mass spectrometry utilizing an orbitrap (U-HPLC-orbitrapMS) or time-of-flight (TOFMS) technology was used. Because of significantly better detection capabilities of the orbitrap technology, the U-HPLC-orbitrapMS method was chosen as a determinative step and fully validated. To compensate matrix effects, matrix-matched calibration was employed. The lowest calibration levels for most of the target mycotoxins ranged from 1 to 8 µg L(-1) beer and the recoveries of analytes were in range from 86 to 124%.
- MeSH
- acetonitrily chemie MeSH
- Fusarium MeSH
- hmotnostní spektrometrie metody MeSH
- kalibrace MeSH
- mykotoxiny analýza izolace a purifikace MeSH
- pivo analýza MeSH
- reprodukovatelnost výsledků MeSH
- rychlé screeningové testy MeSH
- senzitivita a specificita MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
PURPOSE OF THE STUDY Scapholunate dissociation is a clinically most frequently diagnosed form of carpal instability. The aim of this study was to compare high resolution MRI using a microscopic coil with direct MRI arthrography in patients with suspected scapholunate ligament lesions and compare the results with arthroscopy findings and Geissler's arthroscopy classification. MATERIAL AND METHODS A prospective study was carried out in 47 patients (average age, 30.7 years) with clinical symptoms of wrist instability from 2013 to 2014. The patients were examined with the MR device Philips Achieva 1.5T using a microscopic coil and subsequently by direct MR arthrography. The results of examination were evaluated independently by two groups of physicians using a modified arthroscopic classification. The results were verified arthroscopically. For evaluation, an adjusted Geissler's classification was used. The study was approved by the Multicentre Ethics Committee of the Faculty of Medicine in Brno and informed consent was obtained from each patient. RESULTS A total of The MRI examination was evaluated and included in the study in 44 patients (three were excluded for the presence of motion artefacts). Only 20 patients underwent arthroscopy. Examination with a microscopic coil correctly classified 14 of them; an accuracy of 70 % (95 % CI: 45.7 % - 88.1 %) and p = 0.021. Direct MR arthrography correctly classified 16 of 20 injured ligaments, i.e., an accuracy of 80 % (95 % CI: 56.3 % - 94.3 %) and p = 0.002. DISCUSSION Currently, the diagnosis of pathological changes in the wrist is made by routine MRI especially when there is the possibility of using sequences with high spatial resolution. Even though we achieved poorer results by native examination using these techniques, when they were compared with the results of direct MR arthrography, they were still better than those reported in the recent literature. CONCLUSION The optimal method for an examination algorithm of scapholunate ligament lesions is direct MR arthrography. In our study correct findings of direct MR arthrography using Geissler's classification were shown in 80 % of the patients. Key words: scapholunate ligament, scapholunate ligament lesion, direct MR arthrography, microscopic coil, Geissler's classification.
- MeSH
- artrografie metody MeSH
- artroskopie metody statistika a číselné údaje MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- magnetická rezonanční tomografie přístrojové vybavení MeSH
- poranění zápěstí diagnóza diagnostické zobrazování chirurgie MeSH
- prospektivní studie MeSH
- senzitivita a specificita MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
BACKGROUND: Axillary web syndrome (AWS) is one of the most prevalent and underrecognized disorders affecting breast cancer (BC) women. However, the optimal therapeutic strategy to manage AWS is far from being fully characterized. Therefore, this systematic review aims to provide a broad overview of the available rehabilitation treatments in this burdensome condition. METHODS: On 13 January 2022, PubMed, Scopus, Web of Science, Cochrane, and PEDro were systematically searched for clinical studies assessing rehabilitation interventions in post-surgical BC women with AWS. The outcomes analyzed were pain, AWS clinical resolution, upper limb function, and health-related quality of life (HR-QoL). RESULTS: The search identified 1115 records, of which 11 studies were included. A total of 174 patients were assessed (ages ranging from 37 and 66 years old). The interventions included manual lymphatic drainage, manual therapy, stretching, resistance training, mobilization techniques, and Kinesio tape. Positive improvements were reported in terms of pain relief (in 7 studies), AWS clinical resolution (in 9 studies), upper limb function (in 10 studies), and HR-QoL (in 2 studies). CONCLUSIONS: Our findings suggest that rehabilitation might be considered an effective therapeutic strategy in AWS patients. Further RCTs are needed to characterize the optimal rehabilitative interventions.
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
