The Superior Effect of Radiofrequency With Targeted Ultrasound for Facial Rejuvenation by Inducing Hyaluronic Acid Synthesis: A Pilot Preclinical Study.

Background: The level of dermal hyaluronic acid (HA) can be depleted by 75% at age 70. HA provides dermal hydration, volume, and thickness, making it a major component of the extracellular matrix. Restoration of dermal and epidermal HA can be achieved by combining radiofrequency (RF) energy and targeted ultrasound (TUS). The monopolar RF generates heat, with the TUS stimulating HA production. The heat induces a regenerative response in the skin, increasing the fibroblast activity and producing various extracellular matrix compounds, including HA. Objectives: To investigate the effect of the simultaneous application of RF + TUS or RF + US on the stimulation of HA production. Methods: Twelve animals underwent 4 treatments. Six were treated with transcutaneous RF + TUS and 6 with the combination RF + US. The opposite untreated side served as a control. Punch biopsies of the skin were taken at baseline, immediately posttreatment, 1 month, and 2 months posttreatment. The tissue was evaluated with real-time quantitative polymerase chain reaction (RT-qPCR), matrix-assisted laser desorption (MALDI) and time of flight (TOF), and confocal microscopy. Results: The RT-qPCR focused on assessing the production of has1 and has2, enzymes responsible for HA synthesis. RT-qPCR results of the RF + TUS group revealed a +98% and +45% increase in hyaluronic synthetase (HAS) 1 and HAS2 production after the treatments, respectively. The MALDI-TOF revealed a +224% increase in measured HA 2 months after the treatments. The changes were also visible in the confocal microscopy. The control group showed no significant (P > .05) results in either of the evaluation methods. Conclusions: Concurrent application of RF and TUS significantly enhances the natural regenerative processes in skin tissue.

Novel Förster Resonance Energy Transfer probe with quantum dot for a long-time imaging of active caspases inside individual cells.

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.

Miniaturized bioluminescence technology for single-cell quantification of caspase-3/7.

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.

A single-cell analytical approach to quantify activated caspase-3/7 during osteoblast proliferation, differentiation, and apoptosis.

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.

Standing balance of professional ballet dancers and non-dancers under different conditions.

Ballet training has been reported to positively influence balance ability. It is not entirely clear how improved balance ability manifests under standing conditions with different demands on postural control. The aim of the study was to compare balance of ballet dancers and non-dancers in a unipedal stance under different conditions. Twenty-five professional ballet dancers and twenty-five controls completed four unipedal standing balance tests: firm surface with eyes open and closed; foam mat surface with eyes open; and firm surface with eyes open immediately after performing ten 360° whole-body turns. The centre of pressure (COP) data were obtained with a force platform and the direction-specific standard deviations, velocities, and sample entropy of the COP displacement were computed. A three-way analysis of variance was used to compare groups, genders, and conditions. For standing immediately after performing ten turns, the postural sway parameters were significantly larger in the control group compared to the ballet dancers in both men and women. In this stance condition the values of postural sway and COP velocities in the control group were larger in the men compared to the women. For both genders in the control group all postural sway and COP velocity parameters were larger in standing with eyes closed and standing after performing 10 turns compared to standing with eyes open on both firm and foam surface. In the ballet dancers all COP velocity parameters were larger in standing with eyes closed compared to all other conditions. The results from the present study indicate that professional ballet dancers do not have a better general balance ability than untrained subjects.

Analysis of foot load during ballet dancers' gait.

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.

Acid-base properties, FT-IR, FT-Raman spectroscopy and computational study of 1-(pyrid-4-yl)piperazine.

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.