With the goal to investigate biological phenomena at a single-cell level, we designed, synthesized and tested a molecular probe based on Förster resonance energy transfer (FRET) between a highly luminescent quantum dot (QD) as a donor and a fluorophore or fluorescence quencher as an acceptor linked by a specific peptide. In principle, QD luminescence, effectively dissipated in the probe, is switched on after the cleavage of the peptide by a protease and the release of the quencher. We proposed a novel synthesis strategy of a probe. A two-step synthesis consists of: (i) Conjugation of CdTe QDs functionalized by -COOH groups of succinic acid on the nanoparticle surface with the designed specific peptide (GTADVEDTSC) using a ligand-exchange approach; (ii) A fast, high-yield reaction of amine-reactive succinimidyl group on the BHQ-2 quencher with N-terminal of the peptide. This way, any crosslinking between individual nanoparticles and any nonspecific conjugation bonds are excluded. The analysis of the product after the first step proved a high reaction yield and nearly no occurrence of unreacted QDs, a prerequisite of the specificity of our luminescent probe. Its parameters evaluated as Michaelis-Menten description of enzymatic kinetics are similar to products published by other groups. Our research is focused on the fluorescence microscopy analyses of biologically active molecules, such as proteolytic active caspases, playing important roles in cell signaling regulations in normal and diseased states. Consequently, they are attractive targets for clinical diagnosis and medical therapy. The ultimate goal of our work was to synthesize a new QD luminescent probe for a long-time quantitative monitoring of active caspase-3/7 distribution in apoptotic osteoblastic MC3T3-E1 cells treated with camptothecin. As a result of comparison, our synthetized luminescent probe provides longer imaging times of caspases than commercial products. The probe proved the stability of the luminescence signal inside cells for more than 14 days.
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.
- MeSH
- apoptóza * MeSH
- HeLa buňky MeSH
- kaspasa 3 MeSH
- kaspasy * MeSH
- lidé MeSH
- technologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Baletní tanečníci představují skupinu osob provádějících výkony na úrovni vrcholových sportovců, jejichž profesionální kariéra končí ve velmi nízkém věku. Tento fakt nás vedl k provedení výzkumu, který byl zaměřen na vliv dlouhodobého tanečního tréninku na každodenní stereotypy jedince. Dále nás zajímal efekt zvolené rehabilitační intervence, jako neodmyslitelné součásti prevence poranění a poúrazové péče. Výsledky naší práce potvrdily dopad každodenní tréninkové zátěže na zatížení nohy při provedení chůze u baletních tanečníků. Také prokázaly efekt terapie, který se projevil v optimalizaci zatížení na plosce nohy.
Ballet dancers represent a group of persons engaged in performances at the level of elite athletes whose professional career ends at a very young age. This fact led us to conduct the research, which was focused on the long-term influence of dance training on everyday stereotypes of individuals. Furthermore, we studied the effect of selected rehabilitation interventions, as a crucial part of preventing injuries and post-traumatic care. Our results confirmed the impact of daily training on the foot load during walking in ballet dancers. As well it demonstrated the effect of therapy, which resulted in the optimization of the foot load.
- Klíčová slova
- rehabilitační intervence, dynamická plantografie, Footscan,
- MeSH
- biomechanika MeSH
- chůze (způsob) * fyziologie MeSH
- dospělí MeSH
- lidé MeSH
- metatarzus fyziologie patofyziologie MeSH
- mladý dospělý MeSH
- noha (od hlezna dolů) * fyziologie patofyziologie MeSH
- pata fyziologie patofyziologie MeSH
- poranění nohy (od hlezna dolů) prevence a kontrola MeSH
- statistika jako téma MeSH
- studie případů a kontrol MeSH
- tanec * fyziologie MeSH
- techniky fyzikální terapie MeSH
- tělesná námaha MeSH
- zatížení muskuloskeletálního systému MeSH
- získané deformity nohy (od hlezna dolů) prevence a kontrola MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Ballet is an art that puts extreme demands on the dancer's musculoskeletal system and therefore significantly affects motor behavior of the dancers. The aim of our research was to compare plantar pressure distribution during stance phase of gait between a group of professional ballet dancers and non-dancers. Thirteen professional dancers (5 men, 8 women; mean age of 24.1 ± 3.8 years) and 13 nondancers (5 men, 8 women; mean age of 26.1 ± 5.3 years) participated in this study. Foot pressure analysis during gait was collected using a 2 m pressure plate. The participants were instructed to walk across the platform at a self-selected pace barefoot. Three gait cycles were necessary for the data analysis. The results revealed higher (p < 0.05) pressure peaks in medial edge of forefoot during gait for dancers in comparison with nondancers. Furthermore, differences in total foot loading and foot loading duration of rearfoot was higher (p < 0.05) in dancers as well. We can attribute these differences to long-term and intensive dancing exercises that can change the dancer's gait stereotype.
- MeSH
- chůze (způsob) fyziologie MeSH
- dospělí MeSH
- lidé MeSH
- mladý dospělý MeSH
- noha (od hlezna dolů) fyziologie MeSH
- tanec fyziologie MeSH
- tlak MeSH
- zatížení muskuloskeletálního systému MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
