André Amaral

Dose-dependency of Low-energy HeNe Laser Effect in Regeneration of Skeletal Muscle in Mice

Abstract. We evaluated the effect on mice skeletal muscle regeneration of different doses (2.6, 8.4, and 25 J/cm2) of HeNe laser (λ 632.8 nm; power, 2.6 mW; spot size, 0.007 cm2) applied directly to intact skin of injured muscle. Muscle injury was induced in both right and left Tibialis anterior (TA) muscles by ACL myotoxin (5 mg/kg). Right TA muscles were irradiated daily for 5 days while contralateral muscles received a sham treatment. Only the 2.6 J/cm2 dose resulted in changes such as increased mitochondrial density and muscle fibre in the TA muscles as compared to sham groups (3280±704 µm2 versus 2110±657 µm2, p=0.02). We concluded that the HeNe effect on mouse muscle regeneration is dose-specific: only 2.6 J/cm2 increased muscle fibre area and mitochondrial density.

Systemic skeletal muscle necrosis induced by crotoxin

Systemic skeletal muscle necrosis induced by crotoxin, the major component of the venom of Crotalus durissus terrificus, was investigated. Mice received an intramuscular injection of crotoxin (0.35mg/kg body weight) into the right tibialis anterior (TA) muscles, which were evaluated 3h, 24h and 3 days later. Control mice were injected with saline. Right and left TAs, gastrocnemius, soleus and right masseter and longissimus dorsi were removed and frozen. Histological sections were stained with Toluidine Blue or incubated for acidic phosphatase reaction. Three and 24h after the injection, signals of muscle fiber injury were found: (a) in the injected TA muscles; (b) in both right and contralateral soleus and red gastrocnemius; and (c) in the masseter muscles. Contralateral TA, longissimus dorsi and white gastrocnemius muscles were not injured. In conclusion, crotoxin induced a systemic and selective muscle injury in muscles or muscle regions composed by oxidative muscle fibers.

Chemical, spectroscopic characterization, and in vitro antibacterial studies of a new gold(I) complex with N-acetyl-L-cysteine

A new gold(I) complex with N-acetyl-L-cysteine was synthesized and characterized by chemical and spectroscopic techniques. The elemental and thermal analyses of the solid compound fit to the composition AuC5H8NO3S · 0.75H2O. Solid-state 13C-nuclear magnetic resonance (SSNMR) and infrared (IR) analyses indicate the coordination of the ligand to Au(I) through sulfur. The insolubility of the complex in both polar and non-polar solvents supports a polymeric structure. The antibacterial activity of the complex was evaluated by antibiogram assays using the disc diffusion method. The compound showed effective antibacterial activity against Staphylococcus aureus (Gram positive) and Escherichia coli (Gram negative) bacterial cells.

Expression of an active recombinant lysine 49 phospholipase A2 myotoxin as a fusion protein in bacteria

ACL myotoxin (ACLMT) is a K49 phospholipase A(2)-like protein isolated from the venom of the snake Agkistrodon contortrix laticinctus (broad-banded copperhead) that induces necrosis of skeletal muscle. We have previously cloned and sequenced the cDNA coding for ACLMT from a venom gland cDNA library. In order to perform structure and function studies, we have developed an expression system for production of ACLMT as a fusion protein with maltose binding protein (MBP) from the periplasm of bacteria, using the pMAL-p2 expression vector. The cDNA coding for the mature toxin without the signal peptide was amplified by PCR and subcloned into the pMAL-p2 vector. The new plasmid (pMAL-MT) was used to transform BL21(DE3) E. coli cells. Culture of transformed cells induced with IPTG led to the expression of a 60 kDa fusion protein which strongly reacts with anti-native ACLMT antibodies. The fusion protein was purified from the bacterial periplasm by affinity chromatography in an amylose column and by gel filtration. The purified fusion protein (MBP-rACLMT) was able to induce necrosis of skeletal muscle of mice very similar to that caused by the native myotoxin.

Characterization of bilayer bacterial cellulose membranes with different fiber densities: a promising system for controlled release of the antibiotic ceftriaxone

This work describes the synthesis of bilayer bacterial cellulose membranes (BCs) produced by Gluconacetobacter hansenii ATCC 23769 in culture media with different carbon sources (sugarcane molasses, syrup and fructose) as well as their retention capacity and sustained release of the antibacterial agent ceftriaxone. Scanning electronic microscopy analysis showed that BCs produced in all culture media exhibit a double layer and three-dimensional fiber network obtained in only one step. Elemental and thermogravimetric analyses, Fourier transform infrared spectroscopy and X-ray diffraction show that the BC membranes are composed of pure cellulose. In particular, the BC produced in sugarcane molasses medium presented a three-dimensional network structure of the bilayer with high-density fiber entangling, which was responsible for the largest holding capacity and sustained release of the antibiotic ceftriaxone in relation to Staphylococcus aureus bacterial strains. This behavior shows the potential of applying such BC membranes in wound dressings as a sustained support to release different antibiotics to treat skin infections.

Transparent nanostructured cellulose acetate films based on the self assembly of PEO-b-PPO-b-PEO block copolymer.

In this study fabrication and characterization of transparent nanostructured composite films based on cellulose triacetate (CTA) and poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (EPE) triblock copolymer were presented. The effect of the addition of EPE triblock copolymer on the thermal stability, morphology, and mechanical properties of cellulose triacetate films was investigated. The triblock EPE was chosen since PEO blocks interact favorably with CTA, whereas, PPO blocks remain immiscible which provokes a microphase separation. This allows to obtain EPE/CTA composite films with ordered microphase-separated structures where PPO spherical microdomains are well-dispersed in PEO/CTA matrix by simple solvent-evaporation process. During this process, PEO block chains selectively interact with CTA by strong interpolymer hydrogen-bonding while PPO block microseparated. The addition even 40wt% of EPE leads to nanostructured EPE/CTA composite. The cytotoxicity assay of CTA and EPE/CTA composite films confirm non-toxic character of designed transparent nanostructured composites based on sustainable matrices.

Fabrication of Biocompatible, Functional, and Transparent Hybrid Films Based on Silk Fibroin and Epoxy Silane for Biophotonics

In this work we explored the fabrication of flexible and transparent hybrids of silk fibroin (SF) and epoxy-modified siloxane for photonic applications. It is well-known that regenerated SF solutions can form free-standing films with high transparency. Although SF has a restricted number of chemically reactive side groups, the main issues of as-cast pristine SF films regard the high solubility into aqueous media, brittleness, and low thermal stability. The design of SF films with enhanced functionality but high transparency triggers new opportunities on a broader range of applications in biophotonics. Here we present a simple, functional, yet remarkably versatile hybrid material derived from silica sol-gel process based on SF protein and (3-glycidyloxypropyl)trimethoxysilane (GPTMS), an organically modified silicon-alkoxide owning a reactive terminal epoxy group. Specifically, we investigated the effect of the addition of GPTMS into SF solutions on the processability, morphology, crystallinity, and mechanical and optical properties of the resulting hybrid films. Highly transparent (ca. 90%) and flexible free-standing hybrid films were achieved. Cell viability assays revealed that the hybrid films are noncytotoxic to rat osteoblast cells even at high GPTMS content (up to 70 wt %). The hybrid films showed enhanced thermal stability and were rich in organic (epoxy) and inorganic (silanol) functional groups according to the content of GPTMS. We also evaluated the successful preparation of high-quality optical red emissive SF hybrid films by loading YVO4:Eu3+ nanoparticles at low concentration (<5 wt %). A meaningful description of the hybrid film structure is reported from the combination of scanning electron and atomic force microscopies, vibrational spectroscopy, solid-state NMR, and X-ray diffraction analyses.