Determining collagen distribution in articular cartilage using contrast-enhanced micro-computed tomography.

OBJECTIVE: Collagen distribution within articular cartilage (AC) is typically evaluated from histological sections, e.g., using collagen staining and light microscopy (LM). Unfortunately, all techniques based on histological sections are time-consuming, destructive, and without extraordinary effort, limited to two dimensions. This study investigates whether phosphotungstic acid (PTA) and phosphomolybdic acid (PMA), two collagen-specific markers and X-ray absorbers, could (1) produce contrast for AC X-ray imaging or (2) be used to detect collagen distribution within AC. METHOD: We labeled equine AC samples with PTA or PMA and imaged them with micro-computed tomography (micro-CT) at pre-defined time points 0, 18, 36, 54, 72, 90, 180, 270 h during staining. The micro-CT image intensity was compared with collagen distributions obtained with a reference technique, i.e., Fourier-transform infrared imaging (FTIRI). The labeling time and contrast agent producing highest association (Pearson correlation, Bland-Altman analysis) between FTIRI collagen distribution and micro-CT -determined PTA distribution was selected for human AC. RESULTS: Both, PTA and PMA labeling permitted visualization of AC features using micro-CT in non-calcified cartilage. After labeling the samples for 36 h in PTA, the spatial distribution of X-ray attenuation correlated highly with the collagen distribution determined by FTIRI in both equine (mean ± S.D. of the Pearson correlation coefficients, r = 0.96 ± 0.03, n = 12) and human AC (r = 0.82 ± 0.15, n = 4). CONCLUSIONS: PTA-induced X-ray attenuation is a potential marker for non-destructive detection of AC collagen distributions in 3D. This approach opens new possibilities in development of non-destructive 3D histopathological techniques for characterization of OA.

Ionically self-assembled carboxymethyl cellulose/surfactant complexes for antistatic paper coatings.

We show that ionically self-assembled polyelectrolyte/surfactant complexes allow a facile route to tailor the electrical surface resistance of paper sheets for antistatic dissipative regime. We use anionic polyelectrolyte carboxymethyl cellulose (CMC) where cationic alkyltrimethylammonium chloride surfactants (C(n)TAC) with the alkyl chain lengths n=12, 14 or 16 methyl units are ionically complexed by precipitation from aqueous solutions. Such alkyl chains are sufficiently long to allow self-assembly in solid films after solvent evaporation. Short chain lengths, e.g., n=8, did not lead to precipitation. Small angle X-ray scattering indicates cylindrical self-assembly in bulk samples. Upon exposing bulk samples under humidity of 50% RH for 18 h, conductivity of ca. 10(-5) S/cm at room temperature is achieved based on AC-impedance analysis. Flexographic printing and spray coating were selected to conceptually test the feasibility as paper coatings and surface sheet resistances of ca. 10(9) Omega are reached. The results indicate that self-assembled polyelectrolyte/surfactant complexes can allow sufficient conductivity levels for antistatic paper coatings potentially due to protonic conductivity and suggest to develop processes and materials for realistic applications.

Influence of molecular weight on the phase behavior and structure formation of branched side-chain hairy-rod polyfluorene in bulk phase.

We report on an experimental study of the self-organization and phase behavior of hairy-rod pi -conjugated branched side-chain polyfluorene, poly[9,9-bis(2-ethylhexyl)-fluorene-2,7-diyl]-i.e., poly[2,7-(9,9-bis(2-ethylhexyl)fluorene] (PF2/6) -as a function of molecular weight (M(n)) . The results have been compared to those of phenomenological theory. Samples for which M(n) =3-147 kg/mol were used. First, the stiffness of PF2/6 , the assumption of the theory, has been probed by small-angle neutron scattering in solution. Thermogravimetry has been used to show that PF2/6 is thermally stable over the conditions studied. Second, the existence of nematic and hexagonal phases has been phenomenologically identified for lower and higher M(n) (LMW, M(n) < M(*)(n) and HMW, M(n) > M(*)(n) ) regimes, respectively, based on free-energy argument of nematic and hexagonal hairy rods and found to correspond to the experimental x-ray diffraction (XRD) results for PF2/6 . By using the lattice parameters of PF2/6 as an experimental input, the nematic-hexagonal transition has been predicted in the vicinity of glassification temperature (T(g)) of PF2/6 . Then, by taking the orientation parts of the free energies into account the nematic-hexagonal transition has been calculated as a function of temperature and M(n) and a phase diagram has been formed. Below T(g) of 80 degrees C only (frozen) nematic phase is observed for M(n)< M(*)(n) = 10(4) g/mol and crystalline hexagonal phase for M(n) > M(*)(n) . The nematic-hexagonal transition upon heating is observed for the HMW regime depending weakly on M(n) , being at 140-165 degrees C for M(n) > M(*)(n). Third, the phase behavior and structure formation as a function of M(n) have been probed using powder and fiber XRD and differential scanning calorimetry and reasonable semiquantitative agreement with theory has been found for M(n) >or=3 kg/mol. Fourth, structural characteristics are widely discussed. The nematic phase of LMW materials has been observed to be denser than high-temperature nematic phase of HMW compounds. The hexagonal phase has been found to be paracrystalline in the (ab0) plane but a genuine crystal meridionally. We also find that all these materials including the shortest 10-mer possess the formerly observed rigid five-helix hairy-rod molecular structure.

Characterization of apo and partially saturated states of calerythrin, an EF-hand protein from S. erythraea: a molten globule when deprived of Ca(2+).

Calerythrin, a four-EF-hand calcium-binding protein from Saccharopolyspora erythraea, exists in an equilibrium between ordered and less ordered states with slow exchange kinetics when deprived of Ca(2+) and at low temperatures, as observed by NMR. As the temperature is raised, signal dispersion in NMR spectra reduces, and intensity of near-UV CD bands decreases. Yet far-UV CD spectra indicate only a small decrease in the amount of secondary structure, and SAXS data show that no significant change occurs in the overall size and shape of the protein. Thus, at elevated temperatures, the equilibrium is shifted toward a state with characteristics of a molten globule. The fully structured state is reached by Ca(2+)-titration. Calcium first binds cooperatively to the C-terminal sites 3 and 4 and then to the N-terminal site 1, which is paired with an atypical, nonbinding site 2. EF-hand 2 still folds together with the C-terminal half of the protein, as deduced from the order of appearance of backbone amide cross peaks in the NMR spectra of partially Ca(2+)-saturated states.

Small-angle x-ray scattering studies of human breast tissue samples.

Small-angle x-ray scattering (SAXS) patterns are recorded from thin breast tissue samples containing healthy and cancerous regions. The SAXS patterns are compared with histo-pathological observations. The information available from SAXS is reviewed, and a model for scattering from collagen is presented. Scattering patterns of collagen at regions far from the tumours are essentially different from those at tumours. The axial period of collagen fibrils is 65.0 +/- 0.1 nm in healthy regions, and 0.3 nm larger in cancer-invaded regions. The average intensity of scattering from cancerous regions is an order of magnitude higher than the intensity from healthy regions. This is interpreted to arise from an increase of the specific surface area of the scatterers, which is due to a disruption of the molecular and supra-molecular structures in cancerous regions and invasion of new types of cells. The differences of the SAXS patterns are large and distinctive enough to suggest that these phenomena may be utilized in mammography.

X-ray scattering and electron cryomicroscopy study on the effect of carotenoid biosynthesis to the structure of Chlorobium tepidum chlorosomes.

Chlorosomes, the main antenna complexes of green photosynthetic bacteria, were isolated from null mutants of Chlorobium tepidum, each of which lacked one enzyme involved in the biosynthesis of carotenoids. The effects of the altered carotenoid composition on the structure of the chlorosomes were studied by means of x-ray scattering and electron cryomicroscopy. The chlorosomes from each mutant strain exhibited a lamellar arrangement of the bacteriochlorophyll c aggregates, which are the major constituents of the chlorosome interior. However, the carotenoid content and composition had a pronounced effect on chlorosome biogenesis and structure. The results indicate that carotenoids with a sufficiently long conjugated system are important for the biogenesis of the chlorosome baseplate. Defects in the baseplate structure affected the shape of the chlorosomes and were correlated with differences in the arrangement of lamellae and spacing between the lamellar planes of bacteriochlorophyll aggregates. In addition, comparisons among the various mutants enabled refinement of the assignments of the x-ray scattering peaks. While the main scattering peaks come from the lamellar structure of bacteriochlorophyll c aggregates, some minor peaks may originate from the paracrystalline arrangement of CsmA in the baseplate.

Structural variation of tracheids in Norway spruce (Picea abies [L.] Karst.).

The orientation of cellulose microfibrils in the cell wall and the shape and the dimensions of the cells of earlywood of four Norway spruce (Picea abies [L.] Karst.) stems grown in Finland were studied by X-ray diffraction and optical microscopy. The average microfibril angle (MFA) decreased and the diameter of the cell increased rapidly up to rings 5-10 from the pith and remained at the same level after that. The average MFA close to the pith was over 20 degrees and decreased to about 8 degrees after ring 10 from the pith. The average diameter of the cells was 35 microm in the outer rings. The shape of the cross section of the lumen changed from circular to rectangular from the pith to the bark. The tracheid length increased also as a function of the distance from the pith. The thickness of the cell wall varied between 2.8 and 3.5 microm. Automatic cell lumen and cell wall recognition procedures were developed for the analysis of the images of the cross sections of the cells.

One-dimensional optical reflectors based on self-organization of polymeric comb-shaped supramolecules.

We demonstrate that complexation of dodecylbenzenesulphonic acid, DBSA, to a diblock copolymer of polystyrene- block-poly(4-vinylpyridine), PS- block-P4VP, leads to polymeric supramolecules PS- block-P4VP(DBSA)y (y = 1.0, 1.5, and 2.0), which self-organize with a particularly large lamellar periodicity in excess of 1000 A. The structures consist of alternating PS and P4VP(DBSA)y layers, where the latter contains smaller internal structure, probably lamellar. The DBSA side chains are bonded to the pyridines by protonation and hydrogen bonding and they effectively plasticize the material. In this way relatively well-developed structures are obtained even without annealing or macroscopic alignment. Transmission and reflectance measurements show that a relatively narrow and incomplete bandgap exists for supramolecules of high molecular weight block copolymer at ca. 460 nm.

Lamellar organization of pigments in chlorosomes, the light harvesting complexes of green photosynthetic bacteria.

Chlorosomes of green photosynthetic bacteria constitute the most efficient light harvesting complexes found in nature. In addition, the chlorosome is the only known photosynthetic system where the majority of pigments (BChl) is not organized in pigment-protein complexes but instead is assembled into aggregates. Because of the unusual organization, the chlorosome structure has not been resolved and only models, in which BChl pigments were organized into large rods, were proposed on the basis of freeze-fracture electron microscopy and spectroscopic constraints. We have obtained the first high-resolution images of chlorosomes from the green sulfur bacterium Chlorobium tepidum by cryoelectron microscopy. Cryoelectron microscopy images revealed dense striations approximately 20 A apart. X-ray scattering from chlorosomes exhibited a feature with the same approximately 20 A spacing. No evidence for the rod models was obtained. The observed spacing and tilt-series cryoelectron microscopy projections are compatible with a lamellar model, in which BChl molecules aggregate into semicrystalline lateral arrays. The diffraction data further indicate that arrays are built from BChl dimers. The arrays form undulating lamellae, which, in turn, are held together by interdigitated esterifying alcohol tails, carotenoids, and lipids. The lamellar model is consistent with earlier spectroscopic data and provides insight into chlorosome self-assembly.

Switching supramolecular polymeric materials with multiple length scales

It was demonstrated that polymeric supramolecular nanostructures with several length scales allow straightforward tailoring of hierarchical order-disorder and order-order transitions and the concurrent switching of functional properties. Poly(4-vinyl pyridine) (P4VP) was stoichiometrically protonated with methane sulfonic acid (MSA) to form P4VP(MSA)1.0, which was then hydrogen-bonded to pentadecylphenol. Microphase separation, re-entrant closed-loop macrophase separation, and high-temperature macrophase separation were observed. When MSA and pentadecylphenol were complexed to the P4VP block of a microphase-separated diblock copolymer poly[styrene-block-(4-vinyl pyridine)], self-organized structures-in-structures were obtained whose hierarchical phase transitions can be controlled systematically. This microstructural control on two different length scales (in the present case, at 48 and 350 angstroms) was then used to introduce temperature-dependent transitions in electrical conductivity.

Binding of levosimendan, a calcium sensitizer, to cardiac troponin C.

Levosimendan is an inodilatory drug that mediates its cardiac effect by the calcium sensitization of contractile proteins. The target protein of levosimendan is cardiac troponin C (cTnC). In the current work, we have studied the interaction of levosimendan with Ca(2+)-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca(2+)-loaded regulatory domain of recombinant cTnC(C35S) was observed. The changes in the NMR spectra of the N-domain of full-length cTnC(C35S), due to the binding of levosimendan to the primary site, were indicative of a slow conformational exchange. In contrast, no binding of levosimendan to the regulatory domain of cTnC(A-Cys), where all the cysteine residues are mutated to serine, was detected. Moreover, it was shown that levosimendan was in fast exchange on the NMR time scale with a secondary binding site in the C-domain of both cTnC(C35S) and cTnC(A-Cys). The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca(2+)-saturated cTnC but show no domain-domain closure. The experiments were run in the absence of the reducing agent dithiothreitol and the preservative sodium azide (NaN(3)), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples.