Preclinical Investigation of Methylene Blue-mediated Antimicrobial Photodynamic Therapy on Leishmania Parasites Using Real-Time Bioluminescence

Cutaneous leishmaniasis (CL) is a neglected disease that promotes destructive lesions. Difficulties in treatment are related to accessibility of drugs, resistance and toxicity. Antimicrobial photodynamic therapy (APDT) has been emerging as a promising treatment for CL. In this work, we evaluated methylene blue (MB)-mediated APDT (MB-APDT) on Leishmania amazonensis in vitro and in vivo by bioluminescence technique. In vitro, MB-APDT was performed using a red LED (λ = 660 ± 11 nm, 100 mW cm⁻²) and MB (100 µm ) at different light doses. In vivo, mice were infected and 4 weeks later, randomly divided into three groups: control, APDT 1 (single session) and APDT 2 (two sessions of MB-APDT). MB was used at 100 µm and energy dose was established at 150 J cm⁻². Parasite burden, lesion size and pain were evaluated weekly for 4 weeks. In vitro, lethal dose for 90% parasite inactivation was achieved at 48.8 J cm⁻². In vivo, although APDT 1 and APDT 2 groups have showed similar parasite burden after 4 weeks, two sessions were clinically better, especially considering the inflammatory process associated to CL. Our findings reinforce MB-APDT as a cost-effective treatment to combat CL.     

The evolutionary significance of the long variable arm in transfer RNA

The charging of amino acids by their cognate transfer RNA (tRNAs) is central to modern translation and links evolution of the genetic code with chronologies that describe the use of amino acids in an emergent protein world. Although it is commonly accepted that only few of the canonical amino acids were initially coded and charged by tRNA, the composition of this early group of amino acids has been controversial. To uncover evolutionary patterns embedded in the structure of these molecules, we conducted phylogenetic analyses of 42 structural characters scored from the cloverleaf secondary structures of 571 tRNAs from organisms belonging to the three domains of life, viruses, and bacteriophages. Results show that class II tRNA molecules containing a long variable arm, including tRNA^Sec, tRNA^Ser, tRNA^Tyr, and tRNA^Leu, were ancestral compared to those lacking this structural feature. This suggests that selenocysteine (Sec), serine (Ser), tyrosine (Tyr), and leucine (Leu) were among the first amino acids to be charged by their cognate tRNAs and that they may represent the first group of amino acids with functional specificities linked to modern biochemistry. Results also suggest that the stop codon UGA, which also codes for Sec, may be the oldest codon to have a modern functional role in the history of the genetic code. Finally, the charging of amino acids by cognate tRNAs appear to have occurred once the canonical cloverleaf structure was fully realized in evolution and before amino acid specificities and organismal domains of life diversified. © 2008 Wiley Periodicals, Inc. Complexity, 2009     

Phagocytosis of PLGA microparticles in rat peritoneal exudate cells: a time-dependent study.

With the purpose of enhancing the efficacy of microparticle-encapsulated therapeutic agents, in this study we evaluated the phagocytic ability of rat peritoneal exudate cells and the preferential location of poly(d,l-lactide-co-glycolic acid) (PLGA) microparticles inside these cells. The microparticles used were produced by a solvent evaporation method and were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Size distribution analysis using DLS and SEM showed that the particles were spherical, with diameters falling between 0.5 and 1.5 mum. Results from cell adhesion by SEM assay, indicated that the PLGA microparticles are not toxic to cells and do not cause any distinct damage to them as confirmed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Among the large variety of cell populations found in the peritoneal exudates (neutrophils, eosinophils, monocytes, and macrophages), TEM showed that only the latter phagocytosed PLGA microparticles, in a time-dependent manner. The results obtained indicate that the microparticles studied show merits as possible carriers of drugs for intracellular delivery.     

Fermi resonance effect and vibrational intensity in the weak charge-transfer complexes

Infrared and Raman spectra of naphthalene, tetrachlorophthalic anhydride, tetrabromophthalic anhydride and their respective weak charge-transfer complexes were recorded in solids. The spectral intensities of both donor and acceptor molecules were found to alter drastically upon complexation. The observed intensity changes might be attributed to the removal and formation of the Fermi resonance effect in the complexes.     

Kinetic Study of the Alkaline Hydrolysis of 1,n‐Bis(4‐cyanopyridinium)alkanes: Charge Density and New Conformational Effects on the Reactivity of 1,3‐Bis(4‐cyanopyridinium)propane

The alkaline hydrolysis reaction rates of 1,n‐bis(4‐cyanopyridinium)alkane derivatives C_nbis(CP)²⁺ with n = 3, 6, and 8 were studied and compared to the reaction rate of the N‐methyl‐4‐cyanopyridinium (MCP⁺). C₆bis(CP)²⁺ and C₈bis(CP)²⁺ obeyed the first‐order kinetic law. However for C₃bis(CP)²⁺ data fitted to a consecutive two‐step model reaction, the observed rate constants (k_obs) of C₈bis(CP)²⁺ and C₆bis(CP)²⁺ are approximately 50% and 100%, respectively, higher than those for MCP⁺, an effect mainly assigned to the higher charge density of these two derivatives. For C₃bis(CP)²⁺, the k_obs of the second (slow) step is almost twofold the value observed for C₆bis(CP)²⁺, whereas the first (fast) step is approximately six times higher. As for MCP, the hydrolysis of C_nbis(CP)²⁺ generates pyridone (P^o) and carbamidopyridinium (A⁺) units. For C₃bis(CP)²⁺, however at pHs above 11.5, one additional product is formed. From the existence of the new product and the kinetic evidence, a “sandwiched‐type” complex with the OH^? inserted between the rings is proposed. This structural effect in the C₃bis(CP)²⁺ due to the conformational effect justifies the (i) two kinetic steps, (ii) high rate constants, (iii) high P^o/A⁺ ratios, (iv) observed temperature and salt effects, and (v) the formation of the new product.     

Interaction of small amounts of bovine serum albumin with phospholipid monolayers investigated by surface pressure and atomic force microscopy

The influence of small amounts of bovine serum albumin (BSA) (nM concentration) on the lateral organization of phospholipid monolayers at the air-water interface and transferred onto solid substrates as one-layer Langmuir-Blodgett (LB) films was investigated. The kinetics of adsorption of BSA onto the phospholipid monolayers was monitored with surface pressure isotherms in a Langmuir trough, for the zwitterionic dipalmitoylphosphatidyl ethanolamine (N,N-dimethyl-PE) and the anionic dimyristoylphosphatidic acid (DMPA). A monolayer of N,N-dimethyl-PE or DMPA incorporating BSA was transferred onto a solid substrate using the Langmuir-Blodgett technique. Atomic force microscopy (AFM) images of one-layer LB films displayed protein-phospholipid domains, whose morphology was characterized using dynamic scaling theories to calculate roughness exponents. For DMPA-BSA films the surface is characteristic of self-affine fractals, which may be described with the Kardar-Parisi-Zhang (KPZ) equation. On the other hand, for N,N-dimethyl-PE-BSA films, the results indicate a relatively flat surface within the globule. The height profile and the number and size of globules varied with the type of phospholipid. The overall results, from kinetics of adsorption on Langmuir monolayers and surface morphology in LB films, could be interpreted in terms of the higher affinity of BSA to the anionic DMPA than to the zwitterionic N,N-dimethyl-PE. Furthermore, the effects from such small amounts of BSA in the monolayer point to a cooperative response of DMPA and N,N-dimethyl-PE monolayers to the protein.     

An analytical application of the electrocatalysis of the iodate reduction at tungsten oxide films

Films of non-stoichiometric tungsten oxides have been deposited onto glassy carbon surfaces by electrodeposition from acidic W(VI) solutions and the chemical stability of these oxides was investigated by using the electrochemical quartz crystal microbalance. At these modified surfaces, rotating disc electrode voltammetric experiments indicated that iodate is electrocatalytically reduced in a mass-transport controlled process. The influence of the film thickness on the response to iodate was investigated and the results indicated a linear relationship between catalytic current and film thickness for relatively thin oxide layers. The modified electrode was employed successfully as an amperometric sensor for iodate in a flow injection apparatus. The linear response of the developed method is extended from 5 μmol L⁻¹ to 5 mmol L⁻¹ iodate with a limit of detection (signal-to-noise = 3) of 1.2 μmol L⁻¹. The repeatability of the method for 41 injections of a 1 mmol L⁻¹ iodate solution was 0.8% and the throughput was determined as 123 h⁻¹. Interference from other oxidant anions such as nitrate and nitrite was not noticeable, whereas bromate and chlorate interfere at slight levels. The method was used in the determination of the iodate content in table salt samples.     

Phenol removal from aqueous solution by adsorption and ion exchange mechanisms onto polymeric resins

The removal of phenol from aqueous solution was evaluated by using a nonfunctionalized hyper-cross-linked polymer Macronet MN200 and two ion exchange resins, Dowex XZ (strong anion exchange resin) and AuRIX 100 (weak anion exchange). Equilibrium experimental data were fitted to the Langmuir and Freundlich isotherms at different pHs. The Langmuir model describes successfully the phenol removal onto the three resins. The extent of the phenol adsorption was affected by the pH of the solution; thus, the nonfunctionalized resin reported the maximum loading adsorption under acidic conditions, where the molecular phenol form predominates. In contrast both ion exchange resins reported the maximum removal under alkaline conditions where the phenolate may be removed by a combined effect of both adsorption and ion exchange mechanisms. A theoretical model proposed in the literature was used to fit the experimental data and a double contribution was observed from the parameters obtained by the model. Kinetic experiments under different initial phenol concentrations and under the best pH conditions observed in the equilibrium experiments were performed. Two different models were used to define the controlling mechanism of the overall adsorption process: the homogeneous particle diffusion model and the shell progressive model fit the kinetic experimental data and determined the resin phase mechanism as the rate-limiting diffusion for the phenol removal. Resins charged after the kinetic experiments were further eluted by different methods. Desorption of nonfunctionalized resin was achieved by using the solution (50% v/v) of methanol/water with a recovery close to 90%. In the case of the ion exchange resins the desorption process was performed at different pHs and considering the effect of the competitive ion Cl⁻. The desorption processes were controlled by the ion exchange mechanism for Dowex XZ and AuRIX 100 resins; thus, no significant effect for the addition of Cl⁻ under acidic conditions was observed, while under alkaline conditions the total recovery increased, specially for Dowex XZ resin.     

Phase transitions in Langmuir monolayer of long chain attached fluorescein studied by second harmonic generation technique

Optical second harmonic generation (SHG) measurements coupled with Π-A isotherms have been shown to be helpful, for the following and comprehension of orientational orders and phase transitions in Langmuir monolayers. Using the SHG-(Π-A) measurements, monolayers of 5-hexadecanoylaminofluorescein on the water surface were examined by monolayer compression. The phase transitions were noticeably revealed. Dependence of the square root of the intensities polarizations quotient in the molecules surface density, allowed establishing tilting orientation alignment phases. In addition, change in the monolayer symmetry C_∞v→C_2v as it goes through the LE–LC phase transition, was clearly recognized. It was concluded that a possible change in β is taking place due to aggregate formation.     

The intramandibular gland of leaf-cutting ants (Atta sexdens rubropilosa Forel 1908)

The eusociality developed in Hymenoptera and Isoptera is driven by an efficient interaction between exocrine glands and jointed appendages, both in close interaction with the environment. In this context, the mandible of ants plays an important role, since, in addition to being the main jointed appendage, it possess glandular functions. As an example we might name the two glands associated with the mandible: the mandibular and the intramandibular glands. The intramandibular gland is found inside the mandible and consists of a hypertrophied secretory epithelium and secretory cells in the mandible's lumen. The secretion of the secretory epithelium is liberated through intracuticular ducts that open at the base of hairs at the mandible's surface. The secretion of the intramandibular gland (epithelium and secretory cells) reacted positively to tests for the detection of polysaccharides and proteins, thus suggesting that it consists of glycoproteins. The ultrastructure of the secretory epithelium presents variations related to the developmental stage of the individual, showing a large number of ribosomes and microvilli close to the cuticle in young individuals, while in the older specimens it was possible to note the formation of an intracellular reservoir. These variations of secretory epithelium, as also the interaction between the cellular groups inside the mandible, are important information about this gland in leaf-cutting ants.