Enhanced visualization of histological samples with an adjustable RGB contrast system with application for tissue used in photodynamic therapy

The analysis of histological sections has long been a valuable tool in the pathological studies. The interpretation of tissue conditions, however, relies directly on visual evaluation of tissue slides, which may be difficult to interpret because of poor contrast or poor color differentiation. The Chromatic Contrast Visualization System (CCV) combines an optical microscope with electronically controlled light-emitting diodes (LEDs) in order to generate adjustable intensities of RGB channels for sample illumination. While most image enhancement techniques rely on software post-processing of an image acquired under standard illumination conditions, CCV produces real-time variations in the color composition of the light source itself. The possibility of covering the entire RGB chromatic range, combined with the optical properties of the different tissues, allows for a substantial enhancement in image details. Traditional image acquisition methods do not exploit these visual enhancements which results in poorer visual distinction among tissue structures. Photodynamic therapy (PDT) procedures are of increasing interest in the treatment of several forms of cancer. This study uses histological slides of rat liver samples that were induced to necrosis after being exposed to PDT. Results show that visualization of tissue structures could be improved by changing colors and intensities of the microscope light source. PDT-necrosed tissue samples are better differentiated when illuminated with different color wavelengths, leading to an improved differentiation of cells in the necrosis area. Due to the potential benefits it can bring to interpretation and diagnosis, further research in this field could make CCV an attractive technique for medical applications.

Cellular and tissue effects induced by photogemA (R) and red LED in photodynamic therapy

In order to consider the photodynamic therapy (PDT) as a clinical treatment for candidosis, it is necessary to know its cytotoxic effect on normal cells and tissues. Therefore, this study evaluated the toxicity of PDT with PhotogemA (R) associated with red light-emitting diode (LED) on L929 and MDPC-23 cell cultures and healthy rat palatal mucosa. In the in vitro experiment, the cells (30000 cells/cm(2)) were seeded in 24-well plates for 48 h, incubated with PhotogemA (R) (50, 100, or 150 mg/l) and either irradiated or not with a red LED source (630 +/- 3 nm; 75 or 100 J/cm(2); 22 mW/cm(2)). Cell metabolism was evaluated by the MTT assay (ANOVA and Dunnet`s post hoc tests; p < 0.05) and cell morphology was examined by scanning electron microscopy. In the in vivo evaluation, PhotogemA (R) (500 mg/l) was applied to the palatal mucosa of Wistar rats during 30 min and exposed to red LED (630 nm) during 20 min (306 J/cm(2)). The palatal mucosa was photographed for macroscopic analysis at 0, 1, 3, and 7 days posttreatment and subjected to histological analysis after sacrifice of the rats. For both cell lines, there was a statistically significant decrease of the mitochondrial activity (90-97%) for all PhotogemA (R) concentrations associated with red LED regardless of the energy density. However, in the in vivo evaluation, the PDT-treated groups presented intact mucosa with normal characteristics both macroscopically and histologically. From these results, it may be concluded that the association of PhotogemA (R) and red LED caused severe toxic effects on normal cell cultures, characterized by the reduction of mitochondrial activity and morphological alterations, but did not cause damage to the rat palatal mucosa in vivo.

Electrospinning of Hyperbranched Poly-L-Lysine/Polyaniline Nanofibers for Application in Cardiac Tissue Engineering

Electrospun polyaniline nanofibers are one of the most promising materials for cardiac tissue engineering due to their tunable electroactive properties. Moreover, the biocompatibility of polyaniline nanofibes can be improved by grafting of adhesive peptides during the synthesis. In this paper, we describe the biocompatible properties and cardiomyocytes proliferation on polyaniline electrospun nanofibers modified by hyperbranched poly-L-lysine dendrimers (HPLys). The microstructure characterization of the HPLys/polyaniline nanofibers was carried out by scanning electron microscopy (SEM). It was observed that the application of electrical current stimulates the differentiation of cardiac cells cultured on the nanofiber scaffolds. Both electroactivity and biocompatibility of the HPLys based nanofibers suggest the use this material for culture of cardiac cells and opens the possibility of using this material as a biocompatible electroactive 3-D matrix in cardiac tissue engineering.

Early Changes in Gene Expression Induced by Tobacco Smoke: Evidence for the Importance of Estrogen within Lung Tissue

Lung cancer is the leading cause of cancer deaths in the United States, surpassing breast cancer as the primary cause of cancer-related mortality in women. The goal of the present study was to identify early molecular changes in the lung induced by exposure to tobacco smoke and thus identify potential targets for chemoprevention. Female A/J mice were exposed to either tobacco smoke or HEPA-filtered air via a whole-body exposure chamber (6 h/d, 5 d/wk for 3, 8, and 20 weeks). Gene expression profiles of lung tissue from control and smoke-exposed animals were established using a 15K cDNA microarray. Cytochrome P450 1b1, a phase I enzyme involved in both the metabolism of xenobiotics and the 4-hydroxylation of 17 beta-estradiol (E(2)), was modulated to the greatest extent following smoke exposure. A panel of 10 genes were found to be differentially expressed in control and smoke-exposed lung tissues at 3, 8, and 20 weeks (P < 0.001). The interaction network of these differentially expressed genes revealed new pathways modulated by short-term smoke exposure, including estrogen metabolism. In addition, E(2) was detected within murine lung tissue by gas chromatography-coupled mass spectrometry and immunohistochemistry. Identification of the early molecular events that contribute to lung tumor formation is anticipated to lead to the development of promising targeted chemopreventive therapies. In conclusion, the presence of E2 within lung tissue when combined with the modulation of cytochrome P450 1b1 and other estrogen metabolism genes by tobacco smoke provides novel insight into a possible role for estrogens in lung cancer. Cancer Prev Res; 3(6); 707-17. (C) 2010 AACR.

Identification of genes associated with local aggressiveness and metastatic behavior in soft tissue tumors

Soft tissue tumors represent a group of neoplasia with different histologic and biological presentations varying from benign, locally confined to very aggressive and metastatic tumors. The molecular mechanisms responsible for such differences are still unknown. The understanding of these molecular alterations mechanism will be critical to discriminate patients who need systemic treatment from those that can be treated only locally and could also guide the development of new drugs` against this tumors. Using 102 tumor samples representing a large spectrum of these tumors, we performed expression profiling and defined differentially expression genes that are likely to be involved in tumors that are locally aggressive and in tumors with metastatic potential. We described a set of 12 genes (SNRPD3, MEGF9, SPTAN-1, AFAP1L2, ENDOD1, SERPIN5, ZWINTAS, TOP2A, UBE2C, ABCF1, MCM2, and ARL6IP5) showing opposite expression when these two conditions were compared. These genes are mainly related to cell-cell and cell-extracellular matrix interactions and cell proliferation and might represent helpful tools for a more precise classification and diagnosis as well as potential drug targets.

Tissue-, substrate-, and site-specific characteristics of mitochondrial reactive oxygen species generation

Reactive oxygen species are a by-product of mitochondrial oxidative phosphorylation, derived from a small quantity of superoxide radicals generated during electron transport. We conducted a comprehensive and quantitative study of oxygen consumption, inner membrane potentials, and H(2)O(2) release in mitochondria isolated from rat brain, heart, kidney, liver, and skeletal muscle, using various respiratory substrates (alpha-ketoglutarate, glutamate, succinate, glycerol phosphate, and palmitoyl carnitine). The locations and properties of reactive oxygen species formation were determined using oxidative phosphorylation and the respiratory chain modulators oligomycin, rotenone, myxothiazol, and antimycin A and the Uncoupler CCCP. We found that in mitochondria isolated from most tissues incubated under physiologically relevant conditions, reactive oxygen release accounts for 0.1-0.2% of O(2) consumed. Our findings support an important participation of flavoenzymes and complex III and a substantial role for reverse electron transport to complex I as reactive oxygen species sources. Our results also indicate that succinate is an important substrate for isolated mitochondrial reactive oxygen production in brain, heart, kidney, and skeletal muscle, whereas fatty acids generate significant quantities of oxidants in kidney and liver. Finally, we found that increasing respiratory rates is an effective way to prevent mitochondrial oxidant release under many, but not all, conditions. Altogether, our data uncover and quantify many tissue-, substrate-, and site-specific characteristics of mitochondrial ROS release. (C) 2009 Elsevier Inc. All rights reserved.

Purification and partial characterization of an aminopeptidase from the midgut tissue of Dysdercus peruvianus

The surface of midgut cells in Hemiptera is ensheathed by a lipoprotein membrane (the perimicrovillar membrane), which delimits a closed compartment with the microvillar membrane, the so-called perimicrovillar space. In Dysdercus peruvianus midgut perimicrovillar space a soluble aminopeptidase maybe involved in the digestion of oligopeptides and proteins ingested in the diet. This D. peruvianus aminopeptidase was purified to homogeneity by ion-exchange chromatography on an Econo-Q column, hydrophobic interaction chromatography on phenyl-agarose column and preparative polyacrylamide gel electrophoresis. The results suggested that there is a single molecular species of aminopeptidase in D. peruvianus midgut. Molecular mass values for the aminopeptidase were estimated to be 106 kDa (gel filtration) and 55 kDa (SDS-PAGE), suggesting that the enzyme occurs as a dimer under native conditions. Kinetic data showed that D. peruvianus aminopeptidase hydrolyzes the synthetic substrates LpNA, RpNA, A beta NA and AsnMCA (K(m)s 0.65, 0.14, 0.68 and 0.74 mM, respectively). The aminopeptidase activity upon LpNA was inhibited by EDTA and 1,10-phenanthroline, indicating the importance of metal ions in enzyme catalysis. One partial sequence of BLAST-identified aminopeptidase was found by random sequencing of the D. peruvianus midgut cDNA library. Semi-quantitative RT-PCR analysis showed that the aminopeptidase genes were expressed throughout the midgut epithelium, in the epithelia of V1, V2 and V3. Malphigian tubules and fat body, but it was not expressed in the salivary glands. These results are important in furthering our understanding of the digestive process in this pest species. (c) 2010 Elsevier Inc. All rights reserved.

1,4-Dioxane enhances properties and biocompatibility of polyanionic collagen for tissue engineering applications

Polyanionic collagen obtained from bovine pericardial tissue submitted to alkaline hydrolysis is an acellular matrix with strong potential in tissue engineering. However, increasing the carboxyl content reduces fibril formation and thermal stability compared to the native tissues. In the present work, we propose a chemical protocol based on the association of alkaline hydrolysis with 1,4-dioxane treatment to either attenuate or revert the drastic structural modifications promoted by alkaline treatments. For the characterization of the polyanionic membranes treated with 1,4-dioxane, we found that (1) scanning electron microscopy (SEM) shows a stronger reorientation and aggregation of collagen microfibrils; (2) histological evaluation reveals recovering of the alignment of collagen fibers and reassociation with elastic fibers; (3) differential scanning calorimetry (DSC) shows an increase in thermal stability; and (4) in biocompatibility assays there is a normal attachment, morphology and proliferation associated with high survival of the mouse fibroblast cell line NIH3T3 in reconstituted membranes, which behave as native membranes. Our conclusions reinforce the ability of 1,4-dioxane to enhance the properties of negatively charged polyanionic collagen associated with its potential use as biomaterials for grafting, cationic drug- or cell-delivery systems and for the coating of cardiovascular devices.

Ixolaris, a tissue factor inhibitor, blocks primary tumor growth and angiogenesis in a glioblastoma model

Background: The expression levels of the clotting initiator protein Tissue Factor (TF) correlate with vessel density and the histological malignancy grade of glioma patients. Increased procoagulant tonus in high grade tumors (glioblastomas) also indicates a potential role for TF in progression of this disease, and suggests that anticoagulants could be used as adjuvants for its treatment. Objectives: We hypothesized that blocking of TF activity with the tick anticoagulant Ixolaris might interfere with glioblastoma progression. Methods and results: TF was identified in U87-MG cells by flow-cytometric and functional assays (extrinsic tenase). In addition, flow-cytometric analysis demonstrated the exposure of phosphatidylserine in the surface of U87-MG cells, which supported the assembly of intrinsic tenase (FIXa/FVIIIa/FX) and prothrombinase (FVa/FXa/prothrombin) complexes, accounting for the production of FXa and thrombin, respectively. Ixolaris effectively blocked the in vitro TF-dependent procoagulant activity of the U87-MG human glioblastoma cell line and attenuated multimolecular coagulation complexes assembly. Notably, Ixolaris inhibited the in vivo tumorigenic potential of U87-MG cells in nude mice, without observable bleeding. This inhibitory effect of Ixolaris on tumor growth was associated with downregulation of VEGF and reduced tumor vascularization. Conclusion: Our results suggest that Ixolaris might be a promising agent for anti-tumor therapy in humans.

Determination of cadmium and lead in cetacean Dolphinidae tissue from the coast of Bahia state in Brazil by GFAAS

In the present study, cadmium and lead in the muscle, lung, liver and kidney of dolphins (Sotalia guianensis and Stenella clymene) of the Bahia coast in the northwest of Brazil were determined by graphite furnace atomic absorption spectrometry. Samples were digested using a diluted oxidant mixture (HNO(3) + H(2)O(2)) with a microwave heating program performed in five steps. The optimized temperatures and chemical modifier for the pyrolysis and atomization were 700 degrees C, 1400 degrees C and Pd plus Mg for Cd, and 900 degrees C, 1800 degrees C and NH(4)H(2)PO(4) for Pb, respectively. Characteristic masses and limits of detections (n = 20, 3 sigma) for Cd and Pb were 1.6 and 9.0 pg and 0.82 ng g(-1) and 0.50 ng g(-1), respectively. Repeatability ranged from 0.87 to 8.22% for Cd and 4.31 to 8.09% for Pb. The found concentrations presented no statistical differences at the 95% confidence level when compared with the ICP OES methods. Addition and recovery tests were also performed and the results ranged between 87 and 112% for both elements. Samples of cetacean Dolphinidae (S. guianensis and S. clymene) were analyzed, and the higher concentrations ranged from 0.09 to 46.2 mu g g(-1) for Cd and 0.04 to 0.47 mu g g(-1) for Pb in liver, and from 0.133 to 277 mu g g(-1) for Cd in the kidney. (C) 2010 Elsevier By. All rights reserved.

Conserved tissue expression signatures of intronic noncoding RNAs transcribed from human and mouse loci

It has been postulated that noncoding RNAs (ncRNAs) are involved in the posttranscriptional control of gene expression, and may have contributed to the emergence of the complex attributes observed in mammalians. We show here that the complement of ncRNAs expressed from intronic regions of the human and mouse genomes comprises at least 78,147 and 39,660 transcriptional units, respectively. To identify conserved intronic sequences expressed in both humans and mice, we used custom-designed human cDNA microarrays to separately interrogate RNA from mouse and human liver, kidney, and prostate tissues. An overlapping tissue expression signature was detected for both species, comprising 198 transcripts; among these, 22 RNAs map to intronic regions with evidence of evolutionary conservation in humans and mice. Transcription of selected human-mouse intronic ncRNAs was confirmed using strand-specific RT-PCR. Altogether, these results support an evolutionarily conserved role of intronic ncRNAs in human and mouse, which are likely to be involved in the fine tuning of gene expression regulation in different mammalian tissues. (C) 2008 Elsevier Inc. All rights reserved.