The simulations of cells and microscope images thereof have been used to facilitate the development, selection, and validation of image analysis algorithms employed in cytometry as well as for modeling and understanding cell structure and dynamics beyond what is visible in the eyepiece. The simulation approaches vary from simple parametric models of specific cell components-especially shapes of cells and cell nuclei-to learning-based synthesis and multi-stage simulation models for complex scenes that simultaneously visualize multiple object types and incorporate various properties of the imaged objects and laws of image formation. This review covers advances in artificial digital cell generation at scales ranging from particles up to tissue synthesis and microscope image simulation methods, provides examples of the use of simulated images for various purposes ranging from subcellular object detection to cell tracking, and discusses how such simulators have been validated. Finally, the future possibilities and limitations of simulation-based validation are considered. © 2016 International Society for Advancement of Cytometry.

• MeSH
• algoritmy MeSH
• interpretace obrazu počítačem metody   MeSH
• lidé MeSH
• obrazová cytometrie metody   MeSH
• rozpoznávání automatizované metody   MeSH
• umělá inteligence MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

• MeSH
• deep learning * MeSH
• neuronové sítě MeSH
• počítačové zpracování obrazu * MeSH
• Publikační typ
• časopisecké články MeSH
• komentáře MeSH

PURPOSE: The purpose of this study was to investigate whether (44)Sc-labeled puromycin can be utilized for imaging of protein synthesis in vivo. METHODS: For micro-positron emission tomographic (μPET) studies, 20-25 MBq of [(44)Sc]-DOTA-puromycin was administered to tumor-bearing rats, and animals were scanned for 1 h dynamically. Results were further validated by dissecting organs and tissues of the animals after the measurement and in vitro blocking experiments using puromycin or cycloheximide to block protein synthesis. RESULTS: μPET images of tumor-bearing rats showed significant tumor uptake of [(44)Sc]-DOTA-puromycin and a clear-cut tumor visualization. In both blocking experiments, cellular uptake of [(44)Sc]-DOTA-puromycin ([(44)Sc]-DOTA-Pur) could be suppressed by blocking protein synthesis. CONCLUSIONS: We report for the first time successful μPET imaging with (44)Sc obtained from a (44)Ti/(44)Sc generator, as well as noninvasive μPET imaging of ribosomal activity, respectively protein synthesis, with a puromycin-based radiopharmaceutical and the direct correlation between cellular uptake of [(44)Sc]-DOTA-Pur and protein synthesis.

• MeSH
• heterocyklické sloučeniny monocyklické chemie   farmakokinetika   farmakologie   MeSH
• kinetika MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• molekulární zobrazování metody   MeSH
• nádorové buněčné linie MeSH
• pozitronová emisní tomografie MeSH
• proteiny analýza   chemie   metabolismus   MeSH
• proteosyntéza MeSH
• puromycin analogy a deriváty   farmakokinetika   farmakologie   MeSH
• skandium chemie   farmakokinetika   MeSH
• tkáňová distribuce MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• biologie buňky MeSH
• molekulární biologie MeSH
• Publikační typ
• monografie MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biologie
• cytologie, klinická cytologie

• MeSH
• biologie buňky MeSH
• molekulární biologie MeSH
• Publikační typ
• monografie MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biologie
• cytologie, klinická cytologie

The cell surface receptor claudin-4 (Cld-4) is upregulated in various tumours and represents an important emerging target for both diagnosis and treatment of solid tumours of epithelial origin. The C-terminal fragment of the Clostridium perfringens enterotoxin cCPE290-319 appears as a suitable ligand for targeting Cld-4. The synthesis of this 30mer peptide was attempted via several approaches, which has revealed sequential SPPS using three pseudoproline dipeptide building blocks to be the most efficient one. Labelling with fluorine-18 was achieved on solid phase using N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) and 4-[18F]fluorobenzoyl chloride as 18F-acylating agents, which was the most advantageous when [18F]SFB was reacted with the resin-bound 30mer containing an N-terminal 6-aminohexanoic spacer. Binding to Cld-4 was demonstrated via surface plasmon resonance using a protein construct containing both extracellular loops of Cld-4. In addition, cell binding experiments were performed for 18F-labelled cCPE290-319 with the Cld-4 expressing tumour cell lines HT-29 and A431 that were complemented by fluorescence microscopy studies using the corresponding fluorescein isothiocyanate-conjugated peptide. The 30mer peptide proved to be sufficiently stable in blood plasma. Studying the in vivo behaviour of 18F-labelled cCPE290-319 in healthy mice and rats by dynamic PET imaging and radiometabolite analyses has revealed that the peptide is subject to substantial liver uptake and rapid metabolic degradation in vivo, which limits its suitability as imaging probe for tumour-associated Cld-4.

• MeSH
• buňky HT-29 MeSH
• cílená molekulární terapie MeSH
• claudin-4 antagonisté a inhibitory   chemie   metabolismus   MeSH
• enterotoxiny chemická syntéza   chemie   farmakokinetika   farmakologie   MeSH
• izotopové značení MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• ligandy MeSH
• molekulární mimikry fyziologie   MeSH
• molekulární zobrazování MeSH
• myši nahé MeSH
• myši MeSH
• nádory farmakoterapie   MeSH
• potkani Wistar MeSH
• pozitronová emisní tomografie MeSH
• radioizotopy fluoru chemie   MeSH
• techniky syntézy na pevné fázi MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The approach for the detection of replicational activity in cells using 5-bromo-2'-deoxyuridine, a low concentration of hydrochloric acid and exonuclease III is presented in the study. The described method was optimised with the aim to provide a fast and robust tool for the detection of DNA synthesis with minimal impact on the cellular structures using image and flow cytometry. The approach is based on the introduction of breaks into the DNA by the low concentration of hydrochloric acid followed by the subsequent enzymatic extension of these breaks using exonuclease III. Our data showed that the method has only a minimal effect on the tested protein localisations and is applicable both for formaldehyde- and ethanol-fixed cells. The approach partially also preserves the fluorescence of the fluorescent proteins in the HeLa cells expressing Fluorescent Ubiquitin Cell Cycle Indicator. In the case of the short labelling pulses that disabled the use of 5-ethynyl-2'-deoxyuridine because of the low specific signal, the described method provided a bright signal enabling reliable recognition of replicating cells. The optimized protocol was also successfully tested for the detection of trifluridine, the nucleoside used as an antiviral drug and in combination with tipiracil also for the treatment of some types of cancer.

• MeSH
• bromodeoxyuridin metabolismus   MeSH
• buněčný cyklus * MeSH
• buňky A549 MeSH
• exodeoxyribonukleasy metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• HeLa buňky MeSH
• kyselina chlorovodíková farmakologie   MeSH
• lidé MeSH
• průtoková cytometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• buňky * MeSH
• molekulární biologie MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• molekulární biologie, molekulární medicína
• NLK Publikační typ
• učebnice vysokých škol

AIM: Puromycin has played an important role in our understanding of the eukaryotic ribosome and protein synthesis. It has been known for more than 40 years that this antibiotic is a universal protein synthesis inhibitor that acts as a structural analog of an aminoacyl-transfer RNA (aa-tRNA) in eukaryotic ribosomes. Due to the role of enzymes and their synthesis in situations of need (DNA damage, e.g., after chemo- or radiation therapy), determination of protein synthesis is important for control of antitumor therapy, to enhance long-term survival of tumor patients, and to minimize side-effects of therapy. Multiple attempts to reach this goal have been made through the last decades, mostly using radiolabeled amino acids, with limited or unsatisfactory success. The aim of this study is to estimate the possibility of determining protein synthesis ratios by using (68)Ga-DOTA-puromycin ((68)Ga-DOTA-Pur), [(3)H]tyrosine, and 2-fluoro-[(3)H]tyrosine and to estimate the possibility of different pathways due to the fluorination of tyrosine. METHODS: DOTA-puromycin was synthesized using a puromycin-tethered controlled-pore glass (CPG) support by the usual protocol for automated DNA and RNA synthesis following our design. (68)Ga was obtained from a (68)Ge/(68)Ga generator as described previously by Zhernosekov et al. (J Nucl Med 48:1741-1748, 2007). The purified eluate was used for labeling of DOTA-puromycin at 95°C for 20 min. [(3)H]Tyrosine and 2-fluoro-[(3)H]tyrosine of the highest purity available were purchased from Moravek (Bera, USA) or Amersham Biosciences (Hammersmith, UK). In vitro uptake and protein incorporation as well as in vitro inhibition experiments using cycloheximide to inhibit protein synthesis were carried out for all three substances in DU145 prostate carcinoma cells (ATCC, USA). (68)Ga-DOTA-Pur was additionally used for μPET imaging of Walker carcinomas and AT1 tumors in rats. Dynamic scans were performed for 45 min after IV application (tail vein) of 20-25 MBq (68)Ga-DOTA-Pur. RESULTS: No significant differences in the behavior of [(3)H]tyrosine and 2-fluoro-[(3)H]tyrosine were observed. Uptake of both tyrosine derivatives was decreased by inhibition of protein synthesis, but only to a level of 45-55% of initial uptake, indicating no direct link between tyrosine uptake and protein synthesis. In contrast, (68)Ga-DOTA-Pur uptake was directly linked to ribosomal activity and, therefore, to protein synthesis. (68)Ga-DOTA-Pur μPET imaging in rats revealed high tumor-to-background ratios and clearly defined regions of interest in the investigated tumors. SUMMARY: Whereas the metabolic pathway of (68)Ga-DOTA-Pur is directly connected with the process of protein synthesis and shows high tumor uptake during μPET imaging, neither [(3)H]tyrosine nor 2-fluoro-[(3)H]tyrosine can be considered useful for determination of protein synthesis.

• MeSH
• experimentální nádory metabolismus   MeSH
• heterocyklické sloučeniny monocyklické chemie   MeSH
• krysa rodu rattus MeSH
• pozitronová emisní tomografie MeSH
• proteosyntéza * účinky léků   MeSH
• puromycin diagnostické užití   MeSH
• radiofarmaka chemická syntéza   diagnostické užití   MeSH
• radioizotopy galia diagnostické užití   izolace a purifikace   MeSH
• tritium diagnostické užití   MeSH
• tyrosin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Nanomaterials are structures whose exceptionality is based on their large surface, which is closely connected with reactivity and modification possibilities. Due to these properties nanomaterials are used in textile industry (antibacterial textiles with silver nanoparticles), electronics (high-resolution imaging, logical circuits on the molecular level) and medicine. Medicine represents one of the most important fields of application of nanomaterials. They are investigated in connection with targeted therapy (infectious diseases, malignant diseases) or imaging (contrast agents). Nanomaterials including nanoparticles have a great application potential in the targeted transport of pharmaceuticals. However, there are some negative properties of nanoparticles, which must be carefully solved, as hydrophobic properties leading to instability in aqueous environment, and especially their possible toxicity. Data about toxicity of nanomaterials are still scarce. Due to this fact, in this work we focused on studying of the effect of magnetic nanoparticles (NPs) and modified magnetic nanoparticles (MNPs) on tobacco BY-2 plant cell suspension culture. We aimed at examining the effect of NPs and MNPs on growth, proteosynthesis - total protein content, thiols - reduced (GSH) and oxidized (GSSG) glutathione, phytochelatins PC2-5, glutathione S-transferase (GST) activity and antioxidant activity of BY-2 cells. Whereas the effect of NPs and MNPs on growth of cell suspension culture was only moderate, significant changes were detected in all other biochemical parameters. Significant changes in protein content, phytochelatins levels and GST activity were observed in BY-2 cells treated with MNPs nanoparticles treatment. Changes were also clearly evident in the case of application of NPs. Our results demonstrate the ability of MNPs to negatively affect metabolism and induce biosynthesis of protective compounds in a plant cell model represented by BY-2 cell suspension culture. The obtained results are discussed, especially in connection with already published data. Possible mechanisms of NPs' and MNPs' toxicity are introduced.

• MeSH
• antioxidancia metabolismus   MeSH
• buněčné kultury MeSH
• chromatografie MeSH
• fluorescenční mikroskopie MeSH
• fytochelatiny metabolismus   MeSH
• kovové nanočástice toxicita   MeSH
• proteosyntéza účinky léků   MeSH
• spektrofotometrie MeSH
• sulfhydrylové sloučeniny metabolismus   MeSH
• tabák účinky léků   růst a vývoj   metabolismus   MeSH
• viabilita buněk účinky léků   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• železité sloučeniny toxicita   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH