Single Crystal FLIM Characterization of Clofazimine Loaded in Silica-Based Mesoporous Materials and Zeolites.

Clofazimine (CLZ) is an effective antibiotic used against a wide spectrum of Gram-positive bacteria and leprosy. One of its main drawbacks is its poor solubility in water. Silica based materials are used as drug delivery carriers that can increase the solubility of different hydrophobic drugs. Here, we studied how the properties of the silica framework of the mesoporous materials SBA-15, MCM-41, Al-MCM-41, and zeolites NaX, NaY, and HY affect the loading, stability, and distribution of encapsulated CLZ. Time-correlated single-photon counting (TCSPC) and fluorescence lifetime imaging microscopy (FLIM) experiments show the presence of neutral and protonated CLZ (1.3-3.8 ns) and weakly interacting aggregates (0.4-0.9 ns), along with H- and J-type aggregates (<0.1 ns). For the mesoporous and HY zeolite composites, the relative contribution to the overall emission spectra from H-type aggregates is low (<10%), while for the J-type aggregates it becomes higher (~30%). For NaX and NaY the former increased whereas the latter decreased. Although the CLZ@mesoporous composites show higher loading compared to the CLZ@zeolites ones, the behavior of CLZ is not uniform and its dynamics are more heterogeneous across different single mesoporous particles. These results may have implication in the design of silica-based drug carriers for better loading and release mechanisms of hydrophobic drugs.

Structure and biosynthesis of lecanopyrone, a naphtho[1,8-cd]pyran-3-one derivative from cultured lichen mycobionts of Lecanora leprosa.

From the cultures of spore-derived mycobionts of the lichen Lecanora leprosa a novel naphtho[1,8-cd]pyran-3-one derivative, lecanopyrone, was isolated. Its structure was determined by spectroscopic methods. The assembly pattern of acetate units in its biosynthesis was verified using sodium [1-13C]-acetate and sodium [1,2-13C2]-acetate.

Structure and short-time dynamics in suspensions of charged silica spheres in the entire fluid regime.

We present an experimental study of short-time diffusion properties in fluidlike suspensions of monodisperse charge-stabilized silica spheres suspended in dimethylformamide. The static structure factor S(q), the short-time diffusion function D(q), and the hydrodynamic function H(q) have been probed by combining x-ray photon correlation spectroscopy experiments with static small-angle x-ray scattering. Our experiments cover the full liquid-state part of the phase diagram, including de-ionized systems right at the liquid-solid phase boundary. We show that the dynamic data can be consistently described by the renormalized density fluctuation expansion theory of Beenakker and Mazur over a wide range of concentrations and ionic strengths. In accordance with this theory and Stokesian dynamics computer simulations, the measured short-time properties cross over monotonically, with increasing salt content, from the bounding values of salt-free suspensions to those of neutral hard spheres. Moreover, we discuss an upper bound for the hydrodynamic function peak height of fluid systems based on the Hansen-Verlet freezing criterion.

Synthesis, Raman, FT-IR, NMR spectroscopic data and antimicrobial activity of mixed aza-oxo-thia macrocyclic compounds.

Mixed aza-oxo-thia macrocyclic ligands 1,3,5,11,13,15-hexaaza-6,10,16,20-tetraoxo-8,18-dithia-2,3,4:12,13,14-dipyridine cyclocosane (L(1)); 1,3,5,12,14,16-hexeaza-6,11,17,22-tetraoxo-8,9,19,20-tetrathia-2,3,4:13,14,15-dipyridine cyclodocosane (L(2)); 1,3,5,13,15,17-hexaaza-6,12,18,24-tetraoxo-9,21-dithia-2,3,4:14,15,16-dipyridine cyclotetracosane (L(3)) and 1,3,5,14,16,18-hexaaza-6,13,19,26-tetraoxo-9,10,22,23-tetrathia-2,3,4:15,16,17-dipyridine cyclohexacosane (L(4)) were synthesised. The structural features of the ligands have been studied by elemental analyses, Raman, IR, (1)H and (13)C NMR spectroscopy. The antimicrobial activities of the ligands were evaluated using disk diffusion method in dimethyl sulfoxide (DMSO) as well as the minimal inhibitory concentration (MIC) dilution method, against nine bacteria. The obtained results from disk diffusion method were assessed in side-by-side comparison with those of penicillin G, ampicillin, cefotaxime, vancomycin, ofloxacin, and tetracycline well known antibacterial agents. The results from dilution procedure were compared with gentamycin as antibacterial and nystatin as antifungal. The antifungal activities are reported on five yeast cultures namely Candida albicans, Kluyveromyces fragilis, Rhodotorula rubra, Debaryomyces hansenii, and Hanseniaspora guilliermondii, and the results are referenced with nystatin, Ketoconazole, and clotrimazole, commercial antifungal agents. In most cases, the compounds show broad-spectrum (Gram(+) and Gram(-) bacteria) activities that were more active or equipotent to the antibiotic and antifungal agents in the comparison tests.

[EEG spectral coherence at patients submitted to tendon transfer surgery: study pre- and post-surgery]. / Coerência espectral do eletrencefalograma em pacientes submetidos a transposição tendinosa: estudo pré e pós-operatório.

Tendon transfer of the tibiliais posterior muscle is a surgical intervention widely employed in orthopedics for the correction of drop foot caused by leprosy. However, few models have proposed a thorough investigation of the brain plasticity phenomenon during tendon transfer. Thus, the present study aimed at analyzing EEG spectral coherence (SC) in patients submitted to tendon transfer of the tibiliais posterior muscle by Srinivasan's technique and quantitative EEG (EEGq). The sample consisted of four subjects with drop foot caused by leprosy. The SC parameter was evaluated in two experimental moments: pre and post-surgery. Results demonstrated a main moment effect for the C3-CZ electrode pair. Specifically, a significant increase in coherence values was observed. However, the ANOVA did not indicate a significant band/moment interaction. It can be assumed that coherence augmentation indicates that functional rehabilitation promoted by this specific surgery yields cortical alterations.

Coerência espectral do eletrencefalograma em pacientes submetidos a transposição tendinosa: estudo pré e pós-operatório / EEG spectral coherence at patients submitted to tendon transfer surgery: study pre and post surgery

A transferência tendinosa do músculo tibial posterior é intervenção cirúrgica bastante utilizada na ortopedia para correção do pé caído por seqüela de hanseníase. Poucos modelos propuseram investigações mais significativas sobre os fenômenos plásticos cerebrais nas transferências tendinosas. O presente estudo teve como objetivo analisar a coerência espectral (CE) na Eletroencefalografia (EEG) em pacientes submetidos a transferência do tendão do tibial posterior pela técnica de Srinivasan através da EEG quantitativa (EEGq). A amostra foi composta de quatro sujeitos com pé caído devido a seqüela de hanseníase. Os parâmetros de CE do EEG foram quantificados no momento pré e pós-operatório. Os resultados mostraram que houve efeito principal para o fator momento, significativo, no par de eletrodos C3-CZ, com aumento da coerência. Entretanto, os achados da ANOVA não revelaram interação significativa entre bandas versus momento. A recuperação funcional promovida por tal cirurgia parece gerar alterações corticais.

Reinvestigation of the method used to map the electronic structure of blue copper proteins by NMR relaxation.

A previous method for mapping the electron spin distribution in blue copper proteins by paramagnetic nuclear magnetic resonance (NMR) relaxation (Hansen DF, Led JJ, 2004, J Am Chem Soc 126:1247-1253) suggested that the blue copper site of plastocyanin from Anabaena variabilis (A.v.) is less covalent than those found for other plastocyanins by other experimental methods, such as X-ray absorption spectroscopy. Here, a detailed spectroscopic study revealed that the electronic structure of A.v. plastocyanin is similar to those of other plastocyanins. Therefore, the NMR approach was reinvestigated using a more accurate geometric structure as the basis for the mapping, in contrast to the previous approach, as well as a more complete spin distribution model including Gaussian-type natural atomic orbitals instead of Slater-type hydrogen-like atomic orbitals. The refinement results in a good agreement between the electron spin density derived from paramagnetic NMR and the electronic structure description obtained by the other experimental methods. The refined approach was evaluated against density functional theory (DFT) calculations on a model complex of the metal site of plastocyanin in the crystal phase. In general, the agreement between the experimental paramagnetic relaxation rates and the corresponding rates obtained by the DFT calculations is good. Small deviations are attributed to minor differences between the solution structure and the crystal structure outside the first coordination sphere. Overall, the refined approach provides a complementary experimental method for determining the electronic structure of paramagnetic metalloproteins, provided that an accurate geometric structure is available.

Exploratory multivariate spectroscopic study on human skin.

BACKGROUND/AIMS: Spectroscopy on human skin is a field that is being adopted increasingly because of its rapidity and high reproducibility. Infrared reflectance (IR), near-infrared reflectance (NIR), and fluorescence spectroscopy have previously been applied to human skin in vivo to compare healthy and sick skin, including skin cancer, atopy, and leprosy. Exploratory data analysis/chemometrics is a tool for evaluating multivariate data such as spectroscopic measurements. The objective of this study was to explore the spectral variance spanned by people with normal integument, and to demonstrate the advantages of multivariate analysis to skin research. METHODS: IR, NIR and fluorescence spectroscopy have been carried out in vivo on 216 volunteers' forearms before and after four tape strippings. The subjects were asked to fill in a questionnaire regarding factors suspected to influence the measurement results. Principal Component Analysis (PCA) was used to investigate whether the population can be divided into groups on the basis of their skin chemistry. Unless otherwise stated, the results are from the measurements prior to stripping. RESULTS: In contrast to IR and fluorescence spectra, NIR spectra proved able to detect gender differences. By use of PCA, classifications on male and female subjects were observed from the IR and NIR measurements, and as an indication from the fluorescence measurements. The NIR and fluorescence measurements varied between elderly and young subjects. The largest variance in the fluorescence landscapes was seen between pigmented and non-pigmented skin. No connection was found between the spectroscopic measurements and smoking or drinking habits. CONCLUSIONS: Future spectroscopic skin investigations should be balanced as regards to gender and age, as these can possibly affect the measurement results. Chemometrics proved to be superior to traditional attempts of interpreting the spectra.

Catalytic properties of lipoamide dehydrogenase from Mycobacterium smegmatis.

Lipoamide dehydrogenase from Mycobacterium smegmatis was purified to homogeneity over 60-fold. Of 20 amino acid residues identified at the amino terminus of the enzyme, 18 and 17 were identical to the sequences of Mycobacterium leprae and Pseudomonas fluorescens lipoamide dehydrogenases, respectively. The visible spectrum of the isolated enzyme was characteristic of a flavin in apolar environment. Reduction of the enzyme with dithionite results in the appearance of an absorbance shoulder at 530-550 nm, suggesting that reducing equivalents of the two-electron reduced enzyme reside predominantly on the redox-active disulfidedithiol. The kinetic mechanism of the forward (NAD+ reducing) and reverse (NADH oxidizing) reactions proved difficult to study due to severe substrate inhibition by NAD+ and NADH. The rate of lipoamide reduction was found to depend upon the NAD+/NADH ratio, with the reaction being activated at low ratios and inhibited at high ratios. The use of 3-acetylpyridine adenine dinucleotide allowed initial velocity kinetics to be performed and revealed that the kinetic mechanism is ping pong. In addition to catalyzing the reversible oxidation of dihydrolipoamide, the enzyme displayed high oxidase activity (30% of the lipoamide reduction rate), hydrogen and t-butyl peroxide reductase activity (10% of the lipoamide reduction rate), and both naphthoquinone and benzoquinone reduction (approximately 200% of the lipoamide reduction rate). The enzyme failed to catalyze the redox cycling of nitrocompounds, but could anaerobically reduce nitrofurazone. The lipoamide-reducing reaction was reversibly inactivated by sodium arsenite, but no decrease in diaphorase activity was observed under these conditions.

Kinetic studies on cytochrome c oxidase by combined epr and reflectance spectroscopy after rapid freezing.

1. Techniques and experiments are described concerned with the millisecond kinetics of EPT-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O2 or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy. 2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (less than 50 ms) approx. 0.5 electron equivalent per heme a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (less than 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a. 3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O2 in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O2 side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates. 4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms., whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO. 5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C.R., Hansen, R.E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477-2481). Both the low-spin (g=o; 2.2; 1.5) and slowly appearing high-spin (g=6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a3, when it is present in a state of interaction with EPR-undectectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.

Rapid identification of Candida albicans septicemia in man by gas-liquid chromatography.

Gas-liquid chromatography (GLC) was used to study normal serum and serum from patients with septicemia caused by a variety of bacteria and by Candida albicans. The gas chromatograms of seven sera from six patients with septicemia due to C. albicans were found to be significantly and reproducibly different from those of normal sera. Chromatograms of serum from 19 bacteremic patients were indistinguishable from normals. The major peaks present in chromatograms of normal sera were identified by GLC and mass spectroscopy as the methyl esters of palmitic, oleic, linoleic, and stearic acids. In addition to these peaks, serum from patients with candidemia contained abnormal peaks that were also present in cultures of C. albicans grown in normal serum and in washed C. albicans harvested from cultures in yeast nitrogen base broth. Chromatograms from 11 cases of mucosal candidates differed little from normal and were easily distinguished from those of fungemia patients. Chromatograms of serum from two of four patients with deep-invasive candidiasis were indistinguishable from those of fungemia and reverted to normal after infections were eradicated.