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Aim: mircoRNAs (miRNAs), small non-coding RNAs regulating gene expression, are stably secreted into the blood and circulating miRNAs (c-miRNAs) may play an important role in cell-cell communication. Furthermore, c-miRNAs might serve as novel biomarkers of the current vascular cell status. Here, we examined how the levels of three vascular c-miRNAs (c-miR-16, c-miR-21, c-miR-126) are acutely affected by different exercise intensities and volumes. Methods: 12 subjects performed 3 different endurance exercise protocols: 1. High-Volume Training (HVT; 130 min at 55% peak power output (PPO); 2. High-Intensity Training (HIT; 4 × 4 min at 95% PPO); 3. Sprint-Interval Training (SIT; 4 × 30 s all-out). c-miRNAs were quantified using quantitative real-time PCR with TaqMan probes at time points pre, 0', 30', 60', and 180' after each intervention. The expression of miR-126 and miR-21 was analyzed in vitro , in human coronary artery endothelial cells, human THP-1 monocytes, human platelets, human endothelial microparticles (EMPs) and human vascular smooth muscle cells (VSMCs). To investigate the transfer of miRNAs via EMPs, VSMCs were incubated with EMPs. Results: HVT and SIT revealed large increases on c-miR-21 [1.9-fold by HVT (cohen's d = 0.85); 1.5-fold by SIT (cohen's d = 0.85)] and c-miR-126 [2.2-fold by SIT (cohen's d = 1.06); 1.9-fold by HVT (cohen's d = 0.85)] post-exercise compared to pre-values, while HIT revealed only small to moderate changes on c-miRs-21 (cohen's d = -0.28) and c-miR-126 (cohen's d = 0.53). c-miR-16 was only slightly affected by SIT (1.4-fold; cohen's d = 0.57), HVT (1.3-fold; cohen's d = 0.61) or HIT (1.1-fold; cohen's d = 0.2). Further in vitro experiments revealed that miR-126 and miR-21 are mainly of endothelial origin. Importantly, under conditions of endothelial apoptosis, miR-126 and miR-21 are packed from endothelial cells into endothelial microparticles, which were shown to transfer miR-126 into target vascular smooth muscle cells. Conclusion: Taken together, we found that HVT and SIT are associated with the release of endothelial miRNAs into the circulation, which can function as intercellular communication devices regulating vascular biology.

Objective: To evaluate the results of total knee arthoplasty revisions performed in high complexity cases, with large bone defects or serious ligament deficiencies using a constrained implant hinge associated to a rotating tibial basis., Methods: We evaluated 11 patients in which we used the constrained implant hinge associated to rotating tibial basis, with minimum follow-up of two years. The indications for the procedure included instability, septic loosening, late postoperative infection without loosening and periprosthetic fracture. We evaluated the knee range of movement and functional outcomes by the Knee Society Score (KSS) e Knee Injury and Osteoarthritis Outcome Score (KOOS), besides the presence of complications., Results: All patients achieved 5(o) to 85(o) minimum range of motion at 1 year postoperatively and, in the present evaluation, KSS ranged from 67 to 95. Three patients had no complications until the last evaluation and two patients required implant revision., Conclusion: Despite the complications rate observed, the functional result were acceptable for most patients, and it proved being a viable alternative, especially for patients with low functional demand. Level of Evidence IV, Case Series.

The aroma compositions of oxidised sunflower oil, linseed oil and a blend thereof (85/15) were compared with frequently used indicators for primary and secondary lipid oxidation. Primary lipid oxidation was followed by the formation of conjugated dienes, secondary lipid oxidation by proponal and hexanal formation. Highest concentrations of conjugated dienes and propanal were measured in the linseed oil, followed by the blend and sunflower oil, respectively. Highest concentrations of hexanal were determined in the blend. At similar primary oxidation levels, volatile compounds of the oils were isolated in a model mouth system and subsequently analysed by gas chromatography/sniffing port analysis. Propanal, pentanal, 1-penten-3-one, hexanal, 1-pentanol, octanal, 1-octen-3-one, 1-octen-3-ol and (E,Z)-2,4-heptadienal possessed detectable odours. Comparing the three oils, most aroma compounds and greatest intensities were determined in the blend. Conjugated diene concentrations did not predict the formation of aroma compounds adequately. Although propanal and hexanal concentrations were reasonable indicators for aroma development in linseed and sunflower oil, respectively, neither of the indicators predicted aroma development in all three oils sufficiently.

The formation of odor active compounds resulting from initial lipid oxidation in sunflower oil-in-water emulsions was examined during storage at 60 degrees C. The emulsions differed in initial pH, that is, pH 3 and 6. The volatile compounds were isolated under mouth conditions and were analyzed by gas chromatography/sniffing port analysis. The lipid oxidation rate was followed by the formation of conjugated hydroperoxide dienes and headspace hexanal. The initial pH affected the lipid oxidation rate in the emulsions: the formation of conjugated diene hydroperoxides and the hexanal concentration in the static headspace were increased at pH 6. Pentanal, hexanal, 3-pentanol, and 1-octen-3-one showed odor activity in the emulsions after 6 days of storage, for both pH 3 and 6. Larger amounts of odor active compounds were released from the pH 6 emulsion with extended storage. It was shown that this increased release at pH 6 was not due to increased volatility because an increase in pH diminished the static headspace concentrations of added compounds in emulsions.

Semiempirical AM1 calculations have been carried out for beta-carotene and the three xanthophylls (zeaxanthin, canthaxanthin, and astaxanthin) containing oxygen functions (hydroxy/keto groups) found in the majority of natural pigment. The fully optimized geometries correspond well with the X-ray structures of beta-carotene and canthaxanthin and indicate that substitutions on the terminal rings have a minimal effect on the conformation of the chromophore. Twisting along the polyenic chain results from steric interaction involving methyl substituents, and a Ci point group can be proposed for the four investigated carotenoids. AM1 calculated excitation energies for the strongly allowed excited states can be compared to with the experimental absorption band in the visible region, considering solvent effect. Resonance Raman (RR) and Fourier transform (FT) Raman spectra of natural astaxanthin as well as astaxanthins specifically 13C labeled at the positions 12,12'; 13,13'; 14,14'; 15,15'; 15, and 20,20' were recorded. Furthermore the RR and FT Raman spectra of the asymmetric carotenoid 20-norastaxanthin are presented. The data reveal a substantial amount of information about the coupling between the different vibrations, and enabled an extensive experimental verification of the theoretical normal-coordinate analysis previously performed on polyenic molecules [J Raman Spectrosc 1983, 14, 310-321; Advances in Infrared and Raman Spectroscopy, Vol. 12, 1985, pp. 115-178; Spectrochim Acta 1996, 53, 381-392; Biochim Biophys Acta 1994, 1185, 188-196]. The results make up a very interesting dataset which allowed the interpretation and/or observation of several, hitherto never observed or not well understood, effects in the Raman spectra of the differently labeled astaxanthins.

Resonance Raman spectroscopy and quantum chemical calculations were used to investigate the molecular origin of the large redshift assumed by the electronic absorption spectrum of astaxanthin in alpha-crustacyanin, the major blue carotenoprotein from the carapace of the lobster, Homarus gammarus. Resonance Raman spectra of alpha-crustacyanin reconstituted with specifically 13C-labeled astaxanthins at the positions 15, 15,15', 14,14', 13,13', 12,12', or 20,20' were recorded. This approach enabled us to obtain information about the effect of the ligand-protein interactions on the geometry of the astaxanthin chromophore in the ground electronic state. The magnitude of the downshifts of the C==C stretching modes for each labeled compound indicate that the main perturbation on the central part of the polyene chain is not homogeneous. In addition, changes in the 1250-1400 cm(-1) spectral range indicate that the geometry of the astaxanthin polyene chain is moderately changed upon binding to the protein. Semiempirical quantum chemical modeling studies (Austin method 1) show that the geometry change cannot be solely responsible for the bathochromic shift from 480 to 632 nm of protein-bound astaxanthin. The calculations are consistent with a polarization mechanism that involves the protonation or another interaction with a positive ionic species of comparable magnitude with both ketofunctionalities of the astaxanthin-chromophore and support the changes observed in the resonance Raman and visible absorption spectra. The results are in good agreement with the conclusions that were drawn on the basis of a study of the charge densities in the chromophore in alpha-crustacyanin by solid-state NMR spectroscopy. From the results the dramatic bathochromic shift can be explained not only from a change in the ground electronic state conformation but also from an interaction in the excited electronic state that significantly decreases the energy of the pi-antibonding C==O orbitals and the HOMO-LUMO gap.

Selective isotope enrichment, 13C magic angle spinning (MAS) NMR, and semiempirical quantum chemical modeling, have been used to analyze ligand-protein interactions associated with the bathochromic shift of astaxanthin in alpha-crustacyanin, the blue carotenoprotein complex from the carapace of the lobster Homarus gammarus. Spectra of alpha-crustacyanin were obtained after reconstitution with astaxanthins labeled with 13C at positions 4,4', 12,12', 13,13', or 20,20'. The data reveal substantial downfield shifts of 4.9 and 7.0 ppm at positions 12 and 12' in the complex, respectively. In contrast, at the 13 and 13' positions, small upfield shifts of 1.9 ppm were observed upon binding to the protein. These data are in line with previously obtained results for positions 14,14' (3.9 and 6.8 ppm downfield) and 15,15' (0.6 ppm upfield) and confirm the unequal perturbation of both halves after binding of the chromophore. However, these results also show that the main perturbation is of symmetrical origin, since the chemical shift differences exhibit a similar pattern in both halves of the astaxanthin molecule. A small downfield shift of 2.4 ppm was detected for the 4 and 4' positions. Finally, the 20,20' methyl groups are shifted 0.4 ppm upfield by the protein. The full data set provides convincing evidence that charge polarization is of importance for the bathochromic shift. The NMR shifts are compared with calculated charge densities for astaxanthin subjected to variations in protonation states of the ring-functional groups, as models of ligand-protein interactions. Taking into account the color shift and other available optical data, the current model for the mechanisms of interaction with the protein was refined. The results point toward a mechanism in which the astaxanthin is charged and subject to strong electrostatic polarizations originating from both keto groups, most likely a double protonation.

MAS (magic angle spinning) 13C NMR has been used to study protein-chromophore interactions in alpha-crustacyanin, the blue astaxanthin-binding carotenoprotein of the lobster, Homarus gammarus, reconstituted with astaxanthins labelled with 13C at the 14,14' or 15,15' positions. Two signals are seen for alpha-crustacyanin containing [14,14'-13C2]astaxanthin, shifted 6.9 and 4.0 ppm downfield from the 134.1 ppm signal of uncomplexed astaxanthin in the solid state. With alpha-crustacyanin containing [15,15'-13C2]astaxanthin, one essentially unshifted broad signal is seen. Hence binding to the protein causes a decrease in electronic charge density, providing the first experimental evidence that a charge redistribution mechanism contributes to the bathochromic shift of the astaxanthin in alpha-crustacyanin, in agreement with inferences based on resonance Raman data [Salares, et al. (1979) Biochim. Biophys. Acta 576, 176-191]. The splitting of the 14 and 14' signals provides evidence for asymmetric binding of each astaxanthin molecule by the protein.