Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets

Background: Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of alpha v beta 3-integrin and low levels of RHOC. Methods: Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified. Results: We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library. Conclusion: This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

Differential expression of 12 histone deacetylase (HDAC) genes in astrocytomas and normal brain tissue: class II and IV are hypoexpressed in glioblastomas

Background: Glioblastoma is the most lethal primary malignant brain tumor. Although considerable progress has been made in the treatment of this aggressive tumor, the clinical outcome for patients remains poor. Histone deacetylases (HDACs) are recognized as promising targets for cancer treatment. In the past several years, HDAC inhibitors (HDACis) have been used as radiosensitizers in glioblastoma treatment. However, no study has demonstrated the status of global HDAC expression in gliomas and its possible correlation to the use of HDACis. The purpose of this study was to evaluate and compare mRNA and protein levels of class I, II and IV of HDACs in low grade and high grade astrocytomas and normal brain tissue and to correlate the findings with the malignancy in astrocytomas. Methods: Forty-three microdissected patient tumor samples were evaluated. The histopathologic diagnoses were 20 low-grade gliomas (13 grade I and 7 grade II) and 23 high-grade gliomas (5 grade III and 18 glioblastomas). Eleven normal cerebral tissue samples were also analyzed (54 total samples analyzed). mRNA expression of class I, II, and IV HDACs was studied by quantitative real-time polymerase chain reaction and normalized to the housekeeping gene beta-glucuronidase. Protein levels were evaluated by western blotting. Results: We found that mRNA levels of class II and IV HDACs were downregulated in glioblastomas compared to low-grade astrocytomas and normal brain tissue (7 in 8 genes, p < 0.05). The protein levels of class II HDAC9 were also lower in high-grade astrocytomas than in low-grade astrocytomas and normal brain tissue. Additionally, we found that histone H3 (but not histone H4) was more acetylated in glioblastomas than normal brain tissue. Conclusion: Our study establishes a negative correlation between HDAC gene expression and the glioma grade suggesting that class II and IV HDACs might play an important role in glioma malignancy. Evaluation of histone acetylation levels showed that histone H3 is more acetylated in glioblastomas than normal brain tissue confirming the downregulation of HDAC mRNA in glioblastomas.

Novel Primate-Specific Genes, RMEL 1, 2 and 3, with Highly Restricted Expression in Melanoma, Assessed by New Data Mining Tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma.

Glut1 and Glut3 as Potential Prognostic Markers for Oral Squamous Cell Carcinoma

We associated clinical-pathological features of 142 OSCC with the expression pattern of GLUT1 and GLUT3 in order to estimate their prognostic value. Methods: Clinical-pathological features and overall survival data of 142 patients with Oral Squamous Cell Carcinoma (OSCC) were retrospectively reviewed from A. C. Camargo hospital records. A tissue microarray (TMA) was built for the immunohistochemical (IHC) analysis of GLUT 1 and GLUT 3. IHC results were evaluated according to the staining pattern and number of positive cells. Results: GLUT 1 was over expressed in 50.3% of OSSC cases showing membrane staining pattern. However, nuclear expression was observed in 49.7% of the analyzed cases. GLUT 3 over expression was detected in 21.1% of OSCC cases. The pattern of GLUT 1 expression showed significant association with alcohol consumption (p = 0.004). Positive cell membrane GLUT 3 protein expression was associated with advanced clinic-staging of tumours (p = 0.005) as well as with vascular embolization (p = 0.005). Positive expression of GLUT 3 was associated with unfavorable free-disease survival (p = 0.021). Conclusion: GLUT1 and GLUT3 protein expression evaluated by immunohistochemistry are, significantly, indicators of poor prognosis outcome in oral squamous cell carcinoma, probably due to the enhanced glycolytic metabolism of more aggressive neoplastic cells.

Epigenetic Silencing of CRABP2 and MX1 in Head and Neck Tumors

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease affecting the epithelium of the oral cavity, pharynx and larynx. Conditions of most patients are diagnosed at late stages of the disease, and no sensitive and specific predictors of aggressive behavior have been identified yet. Therefore, early detection and prognostic biomarkers are highly desirable for a more rational management of the disease. Hypermethylation of CpG islands is one of the most important epigenetic mechanisms that leads to gene silencing in tumors and has been extensively used for the identification of biomarkers. In this study, we combined rapid subtractive hybridization and microarray analysis in a hierarchical manner to select genes that are putatively reactivated by the demethylating agent 5-aza-2'-deoxycytidine (5Aza-dC) in HNSCC cell lines (FaDu, UM-SCC-14A, UM-SCC-17A, UM-SCC-38A). This combined analysis identified 78 genes, 35 of which were reactivated in at least 2 cell lines and harbored a CpG island at their 5' region. Reactivation of 3 of these 35 genes (CRABP2, MX1, and SLC15A3) was confirmed by quantitative real-time polymerase chain reaction (PCR; fold change, >= 3). Bisulfite sequencing of their CpG islands revealed that they are indeed differentially methylated in the HNSCC cell lines. Using methylation-specific PCR, we detected a higher frequency of CRABP2 (58.1% for region 1) and MX1 (46.3%) hypermethylation in primary HNSCC when compared with lymphocytes from healthy individuals. Finally, absence of the CRABP2 protein was associated with decreased disease-free survival rates, supporting a potential use of CRABP2 expression as a prognostic biomarker for HNSCC patients.

Nuclear morphometry in cytopathology: a prognostic indicator for canine cutaneous mast cell tumors

Twenty-nine canine cutaneous mast cell tumors (MCTs) were morphometrically analyzed with regard to mean nuclear area (MNA) using cytopathology smears. The results showed a correlation between MNA and survival. When graded into 2 morphometrically different groups, there were statistically significant differences among high- and low-grade MCTs, regarding both Romanowsky-type stain and hematoxylin and eosin. Cytomorphometry could also separate histologic grade II tumors with better prognosis from the more aggressive MCTs. The results indicated that nuclear morphometry on cytopathology preparations can predict the biological behavior of cutaneous MCTs in dogs in an independent manner, yielding a rapid and reproducible diagnosis, which renders the method useful for veterinary oncology.

Deep weathering and landscape evolution in a tropical plateau

The thick weathering profiles of humid tropical areas are an important, yet generally neglected, source of information on landscape evolution. Six complete profiles of the weathering mantle were sampled by drilling on the three stepped levels of the Campos do Jordao Plateau, on the NW flank of the Continental Rift of Southeastern Brazil. Mineralogical and micromorphological analyses of drill core samples, complemented by geochemical interpretations and by previous data on the upper saprolite, indicate continuity of a general lateritic trend during the entire process of mantle formation. Lateritization phases of different intensity were defined and considered to reflect adjustment to changes in environmental conditions created by the gradual uplift of the plateau to its present position. Older and more superficial materials related to intense lateritic weathering are characterized by allitization with direct formation of gibbsite from silicates, probably related to tropical climates existing immediately before the formation of the continental rift, during the Paleogene, and also before any significant increase in altitude. Monosialitization phase with general kaolinitization and restricted indirect formation of gibbsite from silicates could be associated to less aggressive climates that followed the Neogene (Miocene?) accentuation of uplift rates along the continental rift. The changes produced by uplift in the tropical climate eventually favored the development of a podzolization trend in soils above 1800m. (C) 2011 Elsevier BM. All rights reserved.

Prevalence, magnitude, and methods of rapid weight loss among judo competitors

ARTIOLI, G. G., B. GUALANO, E. FRANCHINI, F. B. SCAGLIUSI, M. TAKESIAN, M. FUCHS, and A. H. LANCHA. Prevalence, Magnitude, and Methods of Rapid Weight Loss among Judo Competitors. Med. Sci. Sports Exerc., Vol. 42, No. 3, pp. 436-442, 2010. Purpose: To identify the prevalence, magnitude, and methods of rapid weight loss among judo competitors. Methods: Athletes (607 males and 215 females; age = 19.3 +/- 5.3 yr, weight = 70 +/- 7.5 kg, height = 170.6 +/- 9.8 cm) completed a previously validated questionnaire developed to evaluate rapid weight loss in judo athletes, which provides a score. The higher the score obtained, the more aggressive the weight loss behaviors. Data were analyzed using descriptive statistics and frequency analyses. Mean scores obtained in the questionnaire were used to compare specific groups of athletes using, when appropriate, Mann-Whitney U-test or general linear model one-way ANOVA followed by Tamhane post hoc test. Results: Eighty-six percent of athletes reported that have already lost weight to compete. When heavyweights are excluded, this percentage rises to 89%. Most athletes reported reductions of up to 5% of body weight (mean +/- SD: 2.5 +/- 2.3%). The most weight ever lost was 2%-5%, whereas a great part of athletes reported reductions of 5%-10% (mean +/- SD: 6 +/- 4%). The number of reductions underwent in a season was 3 +/- 5. The reductions usually occurred within 7 +/- 7 d. Athletes began cutting weight at 12.6 +/- 6.1 yr. No significant differences were found in the score obtained by male versus female athletes as well as by athletes from different weight classes. Elite athletes scored significantly higher in the questionnaire than nonelite. Athletes who began cutting weight earlier also scored higher than those who began later. Conclusions: Rapid weight loss is highly prevalent in judo competitors. The level of aggressiveness in weight management behaviors seems to not be influenced by the gender or by the weight class, but it seems to be influenced by competitive level and by the age at which athletes began cutting weight.

Bidimensional codes recorded on an oxide glass surface using a continuous wave CO(2) laser

Surface heat treatment in glasses and ceramics, using CO(2) lasers, has attracted the attention of several researchers around the world due to its impact in technological applications, such as lab-on-a-chip devices, diffraction gratings and microlenses. Microlens fabrication on a glass surface has been studied mainly due to its importance in optical devices (fiber coupling, CCD signal enhancement, etc). The goal of this work is to present a systematic study of the conditions for microlens fabrications, along with the viability of using microlens arrays, recorded on the glass surface, as bidimensional codes for product identification. This would allow the production of codes without any residues (like the fine powder generated by laser ablation) and resistance to an aggressive environment, such as sterilization processes. The microlens arrays were fabricated using a continuous wave CO(2) laser, focused on the surface of flat commercial soda-lime silicate glass substrates. The fabrication conditions were studied based on laser power, heating time and microlens profiles. A He-Ne laser was used as a light source in a qualitative experiment to test the viability of using the microlenses as bidimensional codes.

FOREIGN INVESTMENT AND THE BRAZILIAN REAL ESTATE MARKET

This article discusses the main aspects of the Brazilian real estate market in order to illustrate if it would be attractive for a typical American real estate investor to buy office-building portfolios in Brazil. The article emphasizes: [i] - the regulatory frontiers, comparing investment securitization, using a typical American REIT structure, with the Brazilian solution, using the Fundo de Investimento Imobiliario - FII; [ii] - the investment quality attributes in the Brazilian market, using an office building prototype, and [iii] - the comparison of [risk vs. yield] generated by an investment in the Brazilian market, using a FII, benchmarked against an existing REIT (OFFICE SUB-SECTOR) in the USA market. We conclude that investing dollars exchanged for Reais [the Brazilian currency] in a FII with a triple A office-building portfolio in the Sao Paulo marketplace will yield an annual income and a premium return above an American REIT investment. The highly aggressive scenario, along with the strong persistent exchange rate detachment to the IGP-M variations, plus instabilities affecting the generation of income, and even if we adopt a 300-point margin for the Brazil-Risk level, demonstrates that an investment opportunity in the Brazilian market, in the segment we have analyzed, outperforms an equivalent investment in the American market.

The electrochemical behaviour of Ti-13Nb-13Zr alloy in various solutions

The electrochemical behaviour of a near-beta Ti-13Nb-13Zr alloy for the application as implants was investigated in various solutions. The electrolytes used were 0.9 wt% NaCl solution, Hanks` solution and a culture medium known as minimum essential medium (MEM) composed of salts, vitamins and amino acids, all at 37 degrees C. The electrochemical behaviour was investigated by the following electrochemical techniques: open circuit potential measurements as a function of time, electrochemical impedance spectroscopy (EIS) and determination of polarisation curves. The obtained results showed that the Ti alloy was passive in all electrolytes. The EIS results were analysed using an equivalent electrical circuit representing a duplex structure oxide layer, composed of an inner barrier layer, mainly responsible for the alloy corrosion resistance, and an outer and porous layer that has been associated to osteointegration ability. The properties of both layers were dependent on the electrolyte used. The results suggested that the thickest porous layer is formed in the MEM solution whereas the impedance of the barrier layer formed in this solution was the lowest among the electrolytes used. The polarisation curves showed a current increase at potentials around 1300 mV versus saturated calomel electrode (SCE), and this increase was also dependent on the electrolyte used. The highest increase in current density was also associated to the MEM solution suggesting that this is the most aggressive electrolyte to the Ti alloy among the three tested solutions.

Secondary metabolites produced by Propionicimonas sp (ENT-18) induce histological abnormalities in the sclerotia of Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a highly aggressive pathogen that causes great economic losses, especially in temperate climates. Several biological control agents are available, but actinobacteria have seldom been used to control this fungus. Our objective was to evaluate the efficiency and ultrastructural effects of the secondary metabolites produced by the ant-associated actinobacterium Propionicimonas sp. ENT-18 in controlling the sclerotia of S. sclerotiorum. We demonstrated total inhibition of sclerotia treated with 62.5 mu g/10 mu l of an ethyl acetate extract of compounds produced by ENT-18, and calculated an LC(50) of 1.69 mu g/sclerotia. Histological and ultrastructural analysis indicated that the cells of the treated sclerotia were severely damaged, suggesting direct action of the biomolecule(s) produced by the actinobacterium ENT-18 on the cell structure of the medullae and rind cell wall. This is the first report demonstrating a novel property of Propionicimonas sp.-antifungal activity against S. sclerotiorum.

Effective Tityus serrulatus anti-venom produced using the Ts1 component

Scorpion stings are a public health problem in Brazil, with most incidents involving the species Tityus serrulatus. Some T serrulatus toxins may act as immunogens for the production of a specific anti-venom, but many of the component toxins remain poorly characterized. Here, we describe the immunological characteristics of the toxin Ts1 (also known as TsVII and Ts-gamma) and evaluate production of neutralizing antibodies against the crude venom of T serrulatus. Recombinant Ts1 with one copy (Ts1((1))) or two copies in tandem (Ts1((2))) was expressed in BL21 (DE3) cells. Rabbits and mice were immunized with the recombinant proteins (inclusion bodies) and then tested for production of neutralizing antibodies. Neutralization assays showed that anti-Ts1((1)) and anti-Ts1((2)) protected animals challenged with T serrulatus crude venom and native Ts1 Thus, Ts1 could be used in a mixed ""cocktail"" of immunogens for T serrulatus anti-venom production. (C) 2008 Elsevier Ltd. All rights reserved.

Nestmate recognition in a stingless bee: does the similarity of chemical cues determine guard acceptance?

The ability to discriminate nestmates from non-nestmates is critical to the maintenance of the integrity of social insect colonies. Guard workers compare the chemical cues of an incoming individual with their internal template to determine whether the entrant belongs to their colony. In contrast to honeybees, Apis mellifera, stingless bees have singly mated queens and, therefore, are expected to have a higher chemical homogeneity in their colonies. We tested whether aggressive behaviour of Frieseomelitta varia guards towards nestmate and non-nestmate foragers reflects chemical similarities and dissimilarities, respectively, of cuticular hydrocarbon profiles. We also introduced individuals of Lestrimelitta limao, an obligatory robber species, to test the ability of guards to react effectively to intruders from other taxa. We verified that foraging nestmates were almost invariably accepted, while heterospecific and conspecific non-nestmates were rejected at relatively high rates. However, non-nestmate individuals with higher chemical profile similarity were likely to be accepted by guards. We conclude that guards compare the chemical cuticular blend of incoming individuals and make acceptance decisions according to the similarity of the compounds between the colonies. (c) 2007 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Trypanosoma cruzi: Effects of social stress in Calomys callosus a natural reservoir of infection

Social environment can represent a major source of stress affecting cortisol and/or corticosterone levels, thereby altering the immune response. We have investigated the effects of social isolation on the development of Trypanosoma cruzi infection in female Calomys callosus, a natural reservoir of this protozoan parasite. Animals were divided in groups of five animals each. The animals of one group were kept together in a single cage. In a second group, four females were kept together in a cage with one male. In the final group, five individuals were kept isolated in private cages. The isolated animals showed body weight reduction, decreased numbers of peritoneal macrophages, lower global leucocytes counts, smaller lytic antibody percentage and a significantly higher level of blood parasites compared to the other animals. Their behavior was also altered. They were more aggressive than grouped females, or females exposed to the presence of a male. These results suggest that isolation creates a distinct social behavior in which immunity is impaired and pathogenesis is enhanced. (C) 2008 Elsevier Inc. All rights reserved.