Carbon nanoparticles for gene transfection in eukaryotic cell lines.

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.

The effects of low laser irradiation on angiogenesis in injured rat tibiae.

The influence of He-Ne laser radiation on the formation of new blood vessels in the bone marrow compartment of a regenerating area of the mid-cortical diaphysis of the tibiae of young adult rats was studied. A small hole was surgically made with a dentistry burr in the tibia and the injured area received a daily laser therapy over 7 or 14 days transcutaneously starting 24 h from surgery. Incident energy density dosages of 31.5 and 94.5 Jcm(-2) were applied during the period of the tibia wound healing investigated. Light microscopic examination of histological sections of the injured area and quantification of the newly-formed blood vessels were undertaken. Low-level energy treatment accelerated the deposition of bone matrix and histological characteristics compatible with an active recovery of the injured tissue. He-Ne laser therapy significantly increased the number of blood vessels after 7 days irradiation at an energy density of 94.5 Jcm(-2), but significantly decreased the number of vessels in the 14-day irradiated tibiae, independent of the dosage. These effects were attributed to laser treatment, since no significant increase in blood vessel number was detected between 8 and 15 non-irradiated control tibiae. Molecular mechanisms involved in low-level laser therapy of angiogenesis in post-traumatic bone regeneration needs further investigation.

Low-power laser irradiation improves histomorphometrical parameters and bone matrix organization during tibia wound healing in rats.

The influence of daily energy doses of 0.03, 0.3 and 0.9 J of He-Ne laser irradiation on the repair of surgically produced tibia damage was investigated in Wistar rats. Laser treatment was initiated 24 h after the trauma and continued daily for 7 or 14 days in two groups of nine rats (n=3 per laser dose and period). Two control groups (n=9 each) with injured tibiae were used. The course of healing was monitored using morphometrical analysis of the trabecular area. The organization of collagen fibers in the bone matrix and the histology of the tissue were evaluated using Picrosirius-polarization method and Masson's trichrome. After 7 days, there was a significant increase in the area of neoformed trabeculae in tibiae irradiated with 0.3 and 0.9 J compared to the controls. At a daily dose of 0.9 J (15 min of irradiation per day) the 7-day group showed a significant increase in trabecular bone growth compared to the 14-day group. However, the laser irradiation at the daily dose of 0.3 J produced no significant decrease in the trabecular area of the 14-day group compared to the 7-day group, but there was significant increase in the trabecular area of the 15-day controls compared to the 8-day controls. Irradiation increased the number of hypertrophic osteoclasts compared to non-irradiated injured tibiae (controls) on days 8 and 15. The Picrosirius-polarization method revealed bands of parallel collagen fibers (parallel-fibered bone) at the repair site of 14-day-irradiated tibiae, regardless of the dose. This organization improved when compared to 7-day-irradiated tibiae and control tibiae. These results show that low-level laser therapy stimulated the growth of the trabecular area and the concomitant invasion of osteoclasts during the first week, and hastened the organization of matrix collagen (parallel alignment of the fibers) in a second phase not seen in control, non-irradiated tibiae at the same period. The active osteoclasts that invaded the regenerating site were probably responsible for the decrease in trabecular area by the fourteenth day of irradiation.

Shadow-angle method for anisotropic and weakly absorbing films.

A method for determining the optical properties of a film on an isotropic substrate is proposed. The method is based on the existence of two specific incidence angles in the angular interference pattern of the p-polarized light where oscillations of the reflection coefficient cease. The first of these angles, theta(B1), is the well-known Abelès angle, i.e., the ambient-film Brewster angle, and the second angle theta(B2) is the film-substrate Brewster angle. In the conventional planar geometry and in a vacuum ambient there is a rigorous constraint epsilon(1) + epsilon > epsilon(1)epsilon on the film and the substrate dielectric permittivities epsilon(1) and epsilon, respectively, for the existence of the second angle theta(B2.) The limitation may be removed in an experiment by use of a cylindrical lens as an ambient with epsilon(0) > 1, so that both angles become observable. This, contrary to general belief, allows one to adopt the conventional Abelès method not only for films with epsilon(1) close to the substrate's value epsilon but also for any value of epsilon(1). The method, when applied to a wedge-shaped film or to any film of unknown variable thickness, permits one to determine (i) the refractive index of a film on an unknown substrate, (ii) the vertical and the horizontal optical anisotropies of a film on an isotropic substrate, (iii) the weak absorption of a moderately thick film on a transparent or an absorbing isotropic substrate.

Anisotropic protective coating for brewster angle windows.

We propose what we believe is a new type of dielectric anisotropic coating of arbitrary thickness that can protect Brewster angle windows without degrading their optical quality. Such a coating may be fabricated as a multilayer two-component structure. The parameters of the structure, i.e., the dielectric permittivities of the components and their concentrations, are calculated. For ZnSe windows two examples of anisotropic coatings are presented. The optical quality of the multilayer films does not depend on their precise thickness, which makes them less sensitive to surface damage.

Observation of geometric structure of collagen molecules by atomic force microscopy.

Atomic force microscopy was used to study the geometric structure of collagen fibrils and molecules of rat calcanean tendon tissues. The authors found that the diameter of the fibrils ranged from 124 to 170 nm, and their geometric form suggested a helical winding with spectral period from 59.4 to 61.7 nm, close to the band dimensions reported by electron microscopy. At high magnification, the surface of these bands revealed images that probably correspond to the almost crystalline array of collagen molecules, with the triple helix structure almost visible. The typical helix width is 1.43 nm, with main periods of 1.15 and 8.03 nm, very close to the dimensions reported by X-ray diffraction.

Three polarization reflectometry methods for determination of optical anisotropy.

Three novel methods for the determination of optical anisotropy are proposed and tested. The first, the special points method, may be applied to any uniaxially anisotropic medium and is based on the measurement of s- and p-polarized light reflectances under near-normal or grazing angles (or both) and of the Brewster angle. The second method is based on the use of the Azzam universal relationship between the Fresnel s- and p-reflection coefficients. For a flat surface and an isotropic medium, the Azzam combination of coefficients becomes zero and thus is independent of the incidence angle, whereas for a uniaxial or biaxial anisotropic sample it acquires a certain angular dependence, which may be used to determine the anisotropy of the sample. Finally, for those cases in which the anisotropy of the material of a film deposited on an isotropic substrate is itself of interest, a third method, the interference method, is suggested. This technique makes use of the different dependences of s- and p-polarized beam optical path-length changes on the variation of the angle of incidence.

Observation of baker's yeast strains used in biotransformation by atomic force microscopy.

Different strains of baker's yeast (Saccharomyces cerevisiae) were imaged with an atomic force microscope (AFM). The images of uncoated and nonfixed samples were reproducible with high-constrast and nanometer-resolution. Molecules from the polysaccharide surface of the cell wall were pictured and the distance of atoms was measured. The preparation of samples was easy, suggesting that AFM is a useful tool in this type of analyses.