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The protein heterogeneity at the single-cell level has been recognized to be vital for an understanding of various life processes during animal development. In addition, the knowledge of accurate quantity of relevant proteins at cellular level is essential for appropriate interpretation of diagnostic and therapeutic results. Some low-copy-number proteins are known to play a crucial role during cell proliferation, differentiation, and also in apoptosis. The fate decision is often based on the concentration of these proteins in the individual cells. This is likely to apply also for caspases, cysteine proteases traditionally associated with cell death via apoptosis but recently being discovered also as important factors in cell proliferation and differentiation. The hypothesis was tested in bone-related cells, where modulation of fate from apoptosis to proliferation/differentiation and vice versa is particularly challenging, e.g., towards anti-osteoporotic treatments and anti-cancer strategies. An ultrasensitive and highly selective method based on bioluminescence photon counting was used to quantify activated caspase-3/7 in order to demonstrate protein-level heterogeneity in individual cells within one population and to associate quantitative measurements with different cell fates (proliferation, differentiation, apoptosis). The results indicate a gradual increase of caspase-3/7 activation from the proliferative status to differentiation (more than three times) and towards apoptosis (more than six times). The findings clearly support one of the putative key mechanisms of non-apoptotic functions of pro-apoptotic caspases based on fine-tuning of their activation levels.

Correct determination of the instantaneous level and changes of relevant proteins inside individual cells is essential for correct interpretation and understanding of physiological, diagnostic, and therapeutic events. Thus, single-cell analyses are important for quantification of natural cellular heterogeneity, which cannot be evaluated from averaged data of a cell population measurements. Here, we developed an original highly sensitive and selective instrumentation and methodology based on homogeneous single-step bioluminescence assay to quantify caspases and evaluate their heterogeneity in individual cells. Individual suspended cells are selected under microscope and reliably transferred into the 7 µL detection vials by a micromanipulator. The sensitivity of the method is given by implementation of photomultiplying tube with a cooled photocathode working in the photon counting mode. By optimization of our device and methodology, the limits of detection and quantitation were decreased down to 2.1 and 7.0 fg of recombinant caspase-3, respectively. These masses are lower than average amounts of caspase-3/7 in individual apoptotic and even non-apoptotic cells. As a proof of concept, the content of caspase-3/7 in single treated and untreated HeLa cells was determined to be 154 and 25 fg, respectively. Based on these results, we aim to use the technology for investigations of non-apoptotic functions of caspases.

We report the preparation and results of vibrational spectral analysis, which were obtained using both FT-IR and FT-Raman spectroscopy, for three 1-alkyl-1-methylpiperazine-1,4-diium salts (AMPSs), where alkyl is benzyl 4a, n-octadecyl 4b, and methyl 4c, respectively. These were prepared by multistep synthesis from piperazine. The acid base study of AMPSs was performed and corresponding acid base constants were obtained. Single crystals of AMPSs suitable for XRD-analysis were obtained and analyzed. The complete vibrational assignments of wavenumbers were made on the basis of a potential energy distribution. The HOMO and LUMO analysis was used to determine the charge transfer within the molecules. The calculated first hyperpolarizabilities of AMPSs 4a-4c are 48.34, 57.77 and 123.41 times that of urea. As can be seen from the MEP plots, the negative electrostatic potential regions are mainly localized over the chlorine and oxygen atoms for compounds 4a and 4b and chlorine and iodine atoms of compound 4c, and are possible sites for electrophilic attack. (C) 2015 Elsevier B.V. All rights reserved.

Background: Correct mastering of a basic Ollie jump is essential for development of other jumps in skateboarding. In scientific literature we can find a lack of scientifically proved knowledge that describes the difference in muscular activity on various levels of this jump performance. Objective: The aim of this study was to characterize muscular activity in the basic skateboard Ollie jump and to compare this activity with a more difficult modification of the switchstance Ollie jump (the same jump but changed position of limbs). Methods: Ten men experienced in skateboarding for several years, aged 20.0 ± 4.6 years participated in the study (height 1.79 ± 0.05 m, body mass 71.5 ± 4.1 kg). All subjects performed 3 measured Ollie jumps and after that 3 switchstance Ollie jumps. In case of the last-mentioned front and back lower limbs are switched. The observation of muscular activity was carried out by the Delsys Trigno electromyography system. The jump was divided (after video records) into four phases: preparatory, take-off, flight-up and landing. Mean amplitude of muscle activity was measured in following muscles: tibialis anterior, gastrocnemius medialis, rectus femoris, semitendinosus and gluteus medius. Comparison of muscle activity during Ollie and switchstance Ollie was performed by the Wilcoxon test in Statistica. Results: Significantly greater activity (p

We report the vibrational spectral analysis was carried out using FT-IR and FT-Raman spectroscopy for 1-(pyrid-4-yl)piperazine (PyPi). Single crystals of PyPi suitable for X-ray structural analysis were obtained. The acid-base properties are also reported. PyPi supported on a weak acid cation-exchanger in the single protonated form and this system can be used efficiently as the solid supported analogue of 4-N,N-dimethyl-aminopyridine. The complete vibrational assignments of wavenumbers were made on the basis of potential energy distribution. The HOMO and LUMO analysis is used to determine the charge transfer within the molecule and with the molecular electrostatic potential map was applied for the reactivity assessment of PyPi molecule toward proton, electrophiles and nucleopholes as well. The stability of the molecule arising from hyper-conjugative interaction and charge delocalization has been analyzed using NBO analysis. The calculated first hyperpolarizability of PyPi is 17.46 times that of urea. (C) 2013 Elsevier B.V. All rights reserved.

Introduction and aim: In the literature there is evidence that ballet dancers excessively load themedial edge of their foot during the gait in comparison with non-dancers [1]. It can be explained by the dancing positions (e.g. turnout) in which the dancers have a tendency to load the medial edge of foot with greater intensity [2]. The dancers transfer this stereotype into their everyday activities, such as gait. For a successful prevention of possible excessive foot inversion and eversion rehabilitation can be used. The aim of the present study is to determine the influence of rehabilitation intervention on the plantar pressure distribution in professional dancers. Materials andmethods: Seventeen professional ballet dancers (six men, 11 women; mean age: 25.2±5.2 years; mean height: 170.7±9.9 cm; mean weight: 60.0±12.3 kg) participated in the present study. Theirmean dancing experiencewas 16.1±4.8 years. The intensity of dancing training was 9h a day. Foot pressure data was collected using a 2m pressure plate (RSscan International, Olen, Belgium). Eight gait cycles were used for data analysis. Using the Footscan gait software (version 7.97) the foot was divided into ten parts. Dancerswere randomly divided into two groups – experimental (EG) (9) and control group (CG) (8). They underwent two measurements (before and after rehabilitation). The EG has been undergoing six weeks of rehabilitation, the CG has been without intervention. For rehabilitation we were using soft tissues treatment of foot and lower limb and methods based on the scientific principles of developmental kinesiology and the neurophysiological aspects of the maturing locomotor system. Results: The results showed significantly higher values (p