Generalized polarization
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Cell function is highly dependent on membrane structure, organization, and fluidity. Therefore, methods to probe the biophysical properties of biological membranes are required. Determination of generalized polarization (GP) values using Laurdan in fluorescence microscopy studies is one of the most widely-used methods to investigate changes in membrane fluidity in vitro and in vivo. In the last couple of decades, there has been a major increase in the number of studies using Laurdan GP, where several different methodological approaches are used. Such differences interfere with data interpretation inasmuch as it is difficult to validate if Laurdan GP variations actually reflect changes in membrane organization or arise from biased experimental approaches. To address this, we evaluated the influence of different methodological details of experimental data acquisition and analysis on Laurdan GP. Our results showed that absolute GP values are highly dependent on several of the parameters analyzed, showing that incorrect data can result from technical and methodological inconsistencies. Considering these differences, we further analyzed the impact of cell variability on GP determination, focusing on basic cell culture conditions, such as cell confluency, number of passages and media composition. Our results show that GP values can report alterations in the biophysical properties of cell membranes caused by cellular adaptation to the culture conditions. In summary, this study provides thorough analysis of the factors that can lead to Laurdan GP variability and suggests approaches to improve data quality, which would generate more precise interpretation and comparison within individual studies and among the literature on Laurdan GP.
Modern light microscopy imaging techniques have substantially advanced our knowledge about the ultrastructure of plant cells and their organelles. Laser-scanning microscopy and digital light microscopy imaging techniques, in general-in addition to their high sensitivity, fast data acquisition, and great versatility of 2D-4D image analyses-also opened the technical possibilities to combine microscopy imaging with spectroscopic measurements. In this review, we focus our attention on differential polarization (DP) imaging techniques and on their applications on plant cell walls and chloroplasts, and show how these techniques provided unique and quantitative information on the anisotropic molecular organization of plant cell constituents: (i) We briefly describe how laser-scanning microscopes (LSMs) and the enhanced-resolution Re-scan Confocal Microscope (RCM of Confocal.nl Ltd. Amsterdam, Netherlands) can be equipped with DP attachments-making them capable of measuring different polarization spectroscopy parameters, parallel with the 'conventional' intensity imaging. (ii) We show examples of different faces of the strong anisotropic molecular organization of chloroplast thylakoid membranes. (iii) We illustrate the use of DP imaging of cell walls from a variety of wood samples and demonstrate the use of quantitative analysis. (iv) Finally, we outline the perspectives of further technical developments of micro-spectropolarimetry imaging and its use in plant cell studies.
Collective rituals are biologically ancient and culturally pervasive, yet few studies have quantified their effects on participants. We assessed two plausible models from qualitative anthropology: ritual empathy predicts affective convergence among all ritual participants irrespective of ritual role; rite-of-passage predicts emotional differences, specifically that ritual initiates will express relatively negative valence when compared with non-initiates. To evaluate model predictions, images of participants in a Spanish fire-walking ritual were extracted from video footage and assessed by nine Spanish raters for arousal and valence. Consistent with rite-of-passage predictions, we found that arousal jointly increased for all participants but that valence differed by ritual role: fire-walkers exhibited increasingly positive arousal and increasingly negative valence when compared with passengers. This result offers the first quantified evidence for rite of passage dynamics within a highly arousing collective ritual. Methodologically, we show that surprisingly simple and non-invasive data structures (rated video images) may be combined with methods from evolutionary ecology (Bayesian Generalized Linear Mixed Effects models) to clarify poorly understood dimensions of the human condition.
- Publikační typ
- časopisecké články MeSH
Cationic lipids are used to deliver genetic material to living cells. Their proper biophysical characterization is needed in order to design and control this process. In the present work we characterize some properties of recently synthetized cationic lipophosphoramidates. The studied compounds share the same structure of their hydrophobic backbone, but differ in their hydrophilic cationic headgroup, which is formed by a trimethylammonium, a trimethylarsonium or a dicationic moiety. Dynamic light scattering and cryo-transmission electron microscopy proves that the studied lipophosphoramidates create stable unilamellar vesicles. Fluorescence of polarity probe, Laurdan, analyzed using time-dependent fluorescence shift method (TDFS) and generalized polarization (GP) gives important information about the phase, hydration and dynamics of the lipophosphoramidate bilayers. While all of the compounds produced lipid bilayers that were sufficiently fluid for their potential application in gene therapy, their polarity/hydration and mobility was lower than for the standard cationic lipid - DOTAP. Mixing cationic lipophosphoramidates with DOPC helps to reduce this difference. The structure of the cationic headgroup has an important and complex influence on bilayer hydration and mobility. Both TDFS and GP methods are suitable for the characterization of cationic amphiphiles and can be used for screening of the newly synthesized compounds.
- MeSH
- chorda dorsalis anatomie a histologie MeSH
- fylogeneze * MeSH
- histologické techniky MeSH
- meziobratlová ploténka anatomie a histologie MeSH
- meziobratlový kloub * anatomie a histologie MeSH
- mikrofotografie MeSH
- mikroskopie fázově kontrastní MeSH
- obratlovci * MeSH
- polarizační mikroskopie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
Východisko. Cílem šetření bylo získat údaje od etických komisí ustavených dle zákona č. 378/2007 Sb., o léčivech, týkající se jejich charakteristik, způsobu posuzování předkládané dokumentace, vydávání stanovisek a dalších aktivit souvisejících s jejich činností. Vyhodnocené údaje následně využít ke zmapování aktuálního stavu posuzování klinických hodnocení léčivých přípravků s důrazem na dodržování legislativy, a také k popisu rozdílů v činnosti jednotlivých etických komisí. Metody a výsledky. On-line dotazník, tvořený 36 otázkami, vyplnilo 66 etických komisí, což představuje 64 % ze 104 oslovených aktivních etických komisí. Všechny zúčastněné EK pokračují v činnosti i v roce 2011. Celkem 74 % sledovaných etických komisí vykonává činnost v souladu s legislativou. Etická doporučení vyplývající z mezinárodních úmluv a doporučení nad rámec české legislativy aplikuje 62 % komisí a čtvrtina etických komisí (26 %) porušuje některé ustanovení zákona. Závěr. Vzhledem k tomu, že jsme se mohly zaměřit pouze na formální stránku činnosti etických komisí, lze konstatovat, že jednotlivé etické komise se liší zejména v obecných charakteristikách, dále v podmínkách vytvořených k jejich činnosti a v některých procesních aspektech, což ale neznamená automaticky závažné porušení zákona a okamžitou potřebu nápravy. Zdá se ale, že ne všichni členové EK si dostatečně uvědomují také závažnost své činnosti v etickém nazírání na současný výzkum prováděný s lidskými subjekty.
Background. The goal of this work was to acquire data concerning characteristics, ways of assessing presented documentation, publishing attitudes and other related activities of ethics committees established according to the Act No. 378/2007 Coll., on Pharmaceuticals, and to use evaluated data subsequently for mapping actual state of assessing clinical trial projects with emphasis to observance of legislation, as well as description of differences between individual ethics committees. Methods and results. An on-line questionnaire, comprised of 36 questions, was filled in by 66 ethics committees. That is 64% out of 104 approached active ethics committees. All participating ethics committees continue working in 2011. 74% of the ethics committees work in accordance with the legislation. 62% ethics committees also apply ethical recommendations above the scope of law and only 26% ethics committees break some part of the legislation. Conclusions. Being able to focus only on a formal part of ethics committees’ activity, it is possible to say that individual ethics committees differ from each other particularly in the general characteristics, conditions made for their work and in some trial aspect, but it doesn’t automatically mean serious law breach and the immediate need of rectification. It seems that not all members of ethics committees realize adequately the seriousness of their actions from the ethical point of view at current research done with human subjects.
- Klíčová slova
- klinické hodnocení léčivých přípravků, správná klinická praxe,
- MeSH
- etické komise ekonomika normy zákonodárství a právo MeSH
- hodnocení léčiv MeSH
- interpretace statistických dat MeSH
- komise sledující výsledky klinických zkoušek normy zákonodárství a právo MeSH
- léčivé přípravky MeSH
- lidé MeSH
- průzkumy a dotazníky MeSH
- statistika jako téma MeSH
- Check Tag
- lidé MeSH
- Geografické názvy
- Česká republika MeSH
Chlorella and Stichococcus are morphologically simple airborne microalgae, omnipresent in terrestrial and aquatic habitats. The minute cell size and resistance against environmental stress facilitate their long-distance dispersal. However, the actual distribution of Chlorella- and Stichococcus-like species has so far been inferred only from ambiguous morphology-based evidence. Here we contribute a phylogenetic analysis of an expanded SSU and ITS2 rDNA sequence dataset representing Chlorella- and Stichococcus-like species from terrestrial habitats of polar, temperate and tropical regions. We aim to uncover biogeographical patterns at low taxonomic levels. We found that psychrotolerant strains of Chlorella and Stichococcus are closely related with strains originating from the temperate zone. Species closely related to Chlorella vulgaris and Muriella terrestris, and recovered from extreme terrestrial environments of polar regions and hot deserts, are particularly widespread. Stichococcus strains from the temperate zone, with their closest relatives in the tropics, differ from strains with the closest relatives being from the polar regions. Our data suggest that terrestrial Chlorella and Stichococcus might be capable of intercontinental dispersal; however, their actual distributions exhibit biogeographical patterns.
- MeSH
- biofilmy klasifikace MeSH
- Chlorella vulgaris klasifikace genetika růst a vývoj MeSH
- Chlorophyta klasifikace genetika růst a vývoj MeSH
- ekosystém MeSH
- fylogeneze MeSH
- fylogeografie MeSH
- mezerníky ribozomální DNA genetika MeSH
- studené klima * MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Antarktida MeSH
- Arktida MeSH
Molecular recognition mechanisms and kinetics of binding of ligands to buried active sites via access tunnels are not well understood. Fluorescence polarization enables rapid and non-destructive real-time quantification of the association between small fluorescent ligands and large biomolecules. In this study, we describe analysis of binding kinetics of fluorescent ligands resembling linear halogenated alkanes to haloalkane dehalogenases. Dehalogenases possess buried active sites connected to the surrounding solvent by access tunnels. Modification of these tunnels by mutagenesis has emerged as a novel strategy to tailor the enzyme properties. We demonstrate that the fluorescence polarization method can sense differences in binding kinetics originating from even single mutations introduced to the tunnels. The results show, strikingly, that the rate constant of the dehalogenase variants varied across seven orders of magnitude, and the type of ligand used strongly affected the binding kinetics of the enzyme. Furthermore, fluorescence polarization could be applied to cell-free extracts instead of purified proteins, extending the method's application to medium-throughput screening of enzyme variant libraries generated in directed evolution experiments. The method can also provide in-depth kinetic information about the rate-determining step in binding kinetics and reveals the bottlenecks of enzyme accessibility. Assuming availability of appropriate fluorescent ligand, the method could be applied for analysis of accessibility of tunnels and buried active sites of enzymes forming a covalent alkyl-enzyme intermediate during their catalytic cycle, such as α/β-hydrolases containing > 100 000 protein sequences based on the Pfam database.
- MeSH
- alkany chemie metabolismus MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- biokatalýza MeSH
- databáze proteinů MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- fluorescenční polarizace MeSH
- halogenované uhlovodíky chemie metabolismus MeSH
- hydrolasy chemie genetika metabolismus MeSH
- katalytická doména MeSH
- kinetika MeSH
- klonování DNA MeSH
- ligandy MeSH
- mutageneze cílená MeSH
- proteinové inženýrství * MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- rozpouštědla chemie MeSH
- rychlé screeningové testy MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
Fibrillar collagen in tendons and its natural development in rabbits are discussed in this paper. Achilles tendons from newborn (~7 days) to elderly (~38 months) rabbits were monitored in intact (n tendons=24) and microtome sectioned (n tendons=11) states with label-free second harmonic generation microscopy. After sectioning, the collagen fiber pattern was irregular for the younger animals and remained oriented parallel to the load axis of the tendon for the older animals. In contrast, the collagen fiber pattern in the intact samples followed the load axis for all the age groups. However, there was a significant difference in the tendon crimp pattern appearance between the age groups. The crimp amplitude (A) and wavelength (Λ) started at very low values (A=2.0±0.6 µm, Λ=19±4 µm) for the newborn animals. Both parameters increased for the sexually mature animals (>5 months old). When the animals were fully mature the amplitude decreased but the wavelength kept increasing. The results revealed that the microtome sectioning artifacts depend on the age of animals and that the collagen crimp pattern reflects the physical growth and development.
- MeSH
- Achillova šlacha cytologie růst a vývoj ultrastruktura MeSH
- biomechanika fyziologie MeSH
- extracelulární matrix fyziologie MeSH
- fibrilární kolageny metabolismus ultrastruktura MeSH
- konfokální mikroskopie MeSH
- králíci MeSH
- mikroskopie elektronová rastrovací MeSH
- pevnost v tahu fyziologie MeSH
- polarizační mikroskopie MeSH
- stárnutí fyziologie MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
