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.

ADAM23 Negatively Modulates alpha(v)beta(3) Integrin Activation during Metastasis

The ADAM23 gene is frequently silenced in different types of tumors, and, in breast tumors, silencing is correlated with tumor progression, suggesting that it might be associated with the acquisition of a metastatic phenotype. ADAM23 exerts its function mainly through the disintegrin domain, because its metalloprotease domain is inactive. Analysis of ADAM23 binding to integrins has revealed a specific interaction with alpha(v)beta(3) integrin mediated by the disintegrin domain. Altered expression of alpha(v)beta(3) integrin has been observed in different types of tumors, and expression of this integrin in the activated form has been shown to promote metastasis formation. Here, we investigated the possibility that interaction between ADAM23 and alpha(v)beta(3) integrin might negatively modulate alpha(v)beta(3) activation during metastatic progression. ADAM23 expression was knocked down using short hairpin RNA in the MDA-MB-435 cell line, which has been extensively used as a model for alpha(v)beta(3) integrin activation. Ablation of ADAM23 enhanced alpha(v)beta(3) integrin activation by at least 2- to 4-fold and ADAM23 knockdown cells showed enhanced migration and adhesion to classic alpha(v)beta(3) integrin ligands. Ablation of ADAM23 expression also enhanced pulmonary tumor cell arrest in immunodeficient mice. To complement our findings with clinical evidence, we showed that silencing of ADAM23 gene by DNA promoter hypermethylation in a collection of 94 primary breast tumors was significantly associated with lower distant metastases-free and disease-specific survivals and was an independent prognostic factor for poor disease outcome. Our results strongly support a functional role of ADAM23 during metastatic progression by negatively modulating alpha(v)beta(3) integrin activation. [Cancer Res 2009;69(13):5546-52]

Integration pattern of HIV-1 based lentiviral vector carrying recombinant coagulation factor VIII in Sk-Hep and 293T cells

293T and Sk-Hep-1 cells were transduced with a replication-defective self-inactivating HIV-1 derived vector carrying FVIII cDNA. The genomic DNA was sequenced to reveal LTR/human genome junctions and integration sites. One hundred and thirty-two sequences matched human sequences, with an identity of at least 98%. The integration sites in 293T-FVIIIDB and in Sk-Hep-FVIIIDB cells were preferentially located in gene regions. The integrations in both cell lines were distant from the CpG islands and from the transcription start sites. A comparison between the two cell lines showed that the lentiviral-transduced DNA had the same preferred regions in the two different cell lines.

Genomic islands in the pathogenic filamentous fungus Aspergillus fumigatus

We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility ( het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer ( HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated ""gene dumps'' and, perhaps, simultaneously, as "" gene factories''.

CpG-Induced Th1-Type Response in the Downmodulation of Early Development of Allergy and Inhibition of B7 Expression on T Cells of Newborn Mice

Several differences have been described between neonatal and adult immune responses. The predisposition in early life to Th2-type response or tolerance makes it a susceptible period for infections and allergic sensitization. The aim of this work was to evaluate the effects of CpG-containing oligodeoxynucleotides on neonatal and adult immunization with ovalbumin and Blomia tropicalis extract and compare the CpG effects on B and T cells of neonatal and adult mice. Mice that received CpG showed reduced immunoglobulin E (IgE) antibody production in both neonatal and adult periods, in parallel to increased IgG2a antibody levels. We observed that spleen cells of mice that received CpG in early life produced increased amounts of interferon-gamma upon anti-CD3 stimulation. Negative regulation of IgE response was more pronounced in adult than neonate mice; further, CpG decreased anaphylactic antiovalbumin IgG1 only in adults. Also, an upregulation of toll-like receptor 9 expression was detected in adult B cells, but not in neonatal, upon CpG stimuli. Neonatal B cells showed enhanced interleukin (IL)-10 expression and decreased IL-6 levels than adult B cells in response to CpG. When we analyzed in vitro activation of CD4+ T cells, an increased expression of B7 molecules on T cells in neonates was suppressed by CpG. Altogether, we verified qualitative and quantitative evidences regarding CpG effect on neonatal and adult allergens immunizations, which points to the importance of understanding neonatal immune system to establish immunomodulatory strategies for prevention of allergic diseases.

Mycobacterium tuberculosis Culture Filtrate Proteins plus CpG Oligodeoxynucleotides Confer Protection to Mycobacterium bovis BCG-Primed Mice by Inhibiting Interleukin-4 Secretion

Culture filtrate proteins (CFP) are potential targets for tuberculosis vaccine development. We previously showed that despite the high level of gamma interferon (IFN-gamma) production elicited by homologous immunization with CFP plus CpG oligodeoxynucleotides (CFP/CpG), we did not observe protection when these mice were challenged with Mycobacterium tuberculosis. In order to use the IFN-gamma-inducing ability of CFP antigens, in this study we evaluated a prime-boost heterologous immunization based on CFP/CpG to boost Mycobacterium bovis BCG vaccination in order to find an immunization schedule that could induce protection. Heterologous BCG-CFP/CpG immunization provided significant protection against experimental tuberculosis, and this protection was sustained during the late phase of infection and was even better than that conferred by a single BCG immunization. The protection was associated with high levels of antigen-specific IFN-gamma and interleukin-17 (IL-17) and low IL-4 production. The deleterious role of IL-4 was confirmed when IL-4 knockout mice vaccinated with CFP/CpG showed consistent protection similar to that elicited by BCG-CFP/CpG heterologous immunization. These findings show that a single dose of CFP/CpG can represent a new strategy to boost the protection conferred by BCG vaccination. Moreover, different immunological parameters, such as IFN-gamma and IL-17 and tightly regulated IL-4 secretion, seem to contribute to the efficacy of this tuberculosis vaccine.

CpG-ODN combined with Neospora caninum lysate, but not with excreted-secreted antigen, enhances protection against infection in mice

CpG oligodeoxynucleotides (ODN) have shown to be potent immunoadjuvants for several pathogens, but there is limited information concerning their use in immunization protocols against neosporosis. This study aimed to evaluate the potential of CpG-ODN combined with Neosporar lysate antigen (NLA) or excreted-secreted antigen (NcESA) to induce protective immune response against Neospora caninum infection in mice. C57BL/6 mice were vaccinated subcutaneously three times at 2-week intervals with NLA, NLA+CpG, NcESA, NcESA+CpG, CpG (adjuvant control) or PBS (infection control). Serological assays showed an increased specific IgG2a response in animals immunized with either antigen plus adjuvant and elevated levels of the IgG1 isotype in those vaccinated with antigens alone. Splenocyte proliferative responses upon antigen stimulation were higher in groups immunized with NLA OF NcESA combined with CpG, showing increased IL-12 levels. Also, mice vaccinated with NcESA or NcESA+CpG demonstrated higher IFN-gamma levels and IFN-gamma/IL-10 ratio. After lethal challenge, mice immunized with NLA+CpG or NLA had lower Morbidity score and body weight changes in comparison to other groups, and animals did not succumb during acute infection. In contrast, NcESA+CpG or NcESA groups exhibited the highest morbidity scores, body weight impairment and mortality rates, associated with greatest brain parasite burden and inflammation. In conclusion, CpG-ODN was able to induce a Th1-type humoral immune response with predominant IgG2a levels for either NLA or NcESA, but resulting in an effective Th1-driven cellular immune response and total Protection only when combined with NLA. Vaccination with NcESA alone or combined with CpG resulted in a strong cellular immune response associated with high levels of IFN-gamma and inflammation, rendering mice more susceptible to parasite challenge. (C) 2009 Elsevier Ltd. All rights reserved.

E-cadherin abnormalities resulting from CPG methylation promoter in metastatic and nonmetastatic oral cancer

Background. This study aims to compare the alterations in the methylation profiles of E-cadherin in oral cancer, especially in tumors with lowest metatastic potential. Methods. Nine oral verrucous carcinomas (VCs), 20 oral well-differentiated squamous cell carcinomas without lymph node involvement (SCC-pNO), and 17 with lymph node involvement (SCC-pN+) were analyzed using methylation-specific polymerase chain reaction and immunohistochemical expression of E-cadherin gene. Results. The immunohistochemical expression of E-cadherin in VC was significantly higher (p = .016) when compared with SCC-pNO and SCC-pN+ groups. The E-cadherin gene methylation was not correlated with its abnormal immunohistochemical expression in VC and SCC-pNO. All tumors of the SCC-pN+ group with unmethylated E-cadherin gene showed significant loss of E-cadherin immunoexpression (p = .044). Conclusions. The E-cadherin gene methylation presence in tumors with lowest invasive and metastatic potential, such as VC, suggests the early involvement of this epigenetic event in the multistep progression of the oral carcinogenesis. (c) 2007 Wiley Periodicals, Inc.

Palisada flagellifera (Ceramiales, Rhodophyta) from the Canary Islands, Spain: a new record for the eastern Atlantic Ocean based on morphological and molecular evidence

Palisada flagellifera (Ceramiales, Rhodophyta) is recorded for the first time in the eastern Atlantic Ocean off Tenerife, La Gomera, La Palma and Fuerteventura, Canary Islands, Spain. The specimens were collected in 2006-2009 growing from the lower intertidal to subtidal zones to 2 m depth at sites exposed to wave action. The species possesses a palisade-like arrangement of cortical cells in cross section, lacks secondary pit connections between them, and has tetrasporangia produced by three fertile pericentral cells (the third and the fourth additional and the second that becomes fertile), and a right-angled arrangement of tetrasporangia. Gametangia were not observed. The phylogenetic relationships were inferred by analyses of the chloroplast-encoded rbcL gene sequences from 46 taxa. The Canarian and Brazilian P. flagellifera specimens formed a highly supported clade with a low level of genetic variation in the rbcL sequences (0.02-0.04%), confirming that they are the same taxonomic entity. This study expands the geographical distribution of P. flagellifera to the eastern Atlantic Ocean.

Laterally transferred genomic islands in Xanthomonadales related to pathogenicity and primary metabolism

Lateral gene transfer (LGT) is considered as one of the drivers in bacterial genome evolution, usually associated with increased fitness and/or changes in behavior, especially if one considers pathogenic vs. non-pathogenic bacterial groups. The genomes of two phytopathogens, Xanthomonas campestris pv. campestris and Xanthomonas axonopodis pv. citri, were previously inspected for genome islands originating from LGT events, and, in this work, potentially early and late LGT events were identified according to their altered nucleotide composition. The biological role of the islands was also assessed, and pathogenicity, virulence and secondary metabolism pathways were functions highly represented, especially in islands that were found to be recently transferred. However, old islands are composed of a high proportion of genes related to cell primary metabolic functions. These old islands, normally undetected by traditional atypical composition analysis, but confirmed as product of LGT by atypical phylogenetic reconstruction, reveal the role of LGT events by replacing core metabolic genes normally inherited by vertical processes.

Growth of EuTe islands on SnTe by molecular beam epitaxy

Semiconductor magnetic quantum dots are very promising structures, with novel properties that find multiple applications in spintronic devices. EuTe is a wide gap semiconductor with NaCl structure, and strong magnetic moments S=7/2 at the half filled 4f(7) electronic levels. On the other hand, SnTe is a narrow gap semiconductor with the same crystal structure and 4% lattice mismatch with EuTe. In this work, we investigate the molecular beam epitaxial growth of EuTe on SnTe after the critical thickness for island formation is surpassed, as a previous step to the growth of organized magnetic quantum dots. The topology and strain state of EuTe islands were studied as a function of growth temperature and EuTe nominal layer thickness. Reflection high energy electron diffraction (RHEED) was used in-situ to monitor surface morphology and strain state. RHEED results were complemented and enriched with atomic force microscopy and grazing incidence X-ray diffraction measurements made at the XRD2 beamline of the Brazilian Synchrotron. EuTe islands of increasing height and diameter are obtained when the EuTe nominal thickness increases, with higher aspect ratio for the islands grown at lower temperatures. As the islands grow, a relaxation toward the EuTe bulk lattice parameter was observed. The relaxation process was partially reverted by the growth of the SnTe cap layer, vital to protect the EuTe islands from oxidation. A simple model is outlined to describe the distortions caused by the EuTe islands on the SnTe buffer and cap layers. The SnTe cap layers formed interesting plateau structures with easily controlled wall height, that could find applications as a template for future nanostructures growth. (C) 2010 Elsevier B.V. All rights reserved.

Velocity-dependent KAM islands

We revisit the non-dissipative time-dependent annular billiard and we consider the chaotic dynamics in two planes of conjugate variables in order to describe the behavior of the growth, or saturation, of the mean velocity of an ensemble of particles. We observed that the changes in the 4-d phase space occur without changing any parameter. They occur depending on where the initial conditions start. The emerging KAM islands interfere in the behavior of the particle dynamics especially in the Fermi acceleration mechanism. We show that Fermi acceleration can be suppressed, without dissipation, even considering the non-dissipative energy context. (C) 2011 Published by Elsevier Ltd.

TEM, XRD and AFM study of poly(o-ethoxyaniline) films: new evidence for the formation of conducting islands

The existence of conducting islands in polyaniline films has long been proposed in the literature, which would be consistent with conducting mechanisms based on hopping. Obtaining direct evidence of conducting islands, however, is not straightforward. In this paper, conducting islands were visualized in poly(o-ethoxyaniline) (POEA) films prepared at low pH, using Transmission Electron Microscopy (TEM) and atomic force spectroscopy (AFS). The size of the islands varied between 67 and 470 angstrom for a pH=3.0, with a larger average being obtained with AFS, probably due to the finite size effect of the atomic force microscopy tip. In AFS, the conducting islands were denoted by regions with repulsive forces due to the double-layer forces. On the basis of X-ray diffraction (XRD) patterns for POEA in the powder form, we infer that the conducting islands are crystalline, and therefore a POEA film is believed to consist of conducting islands dispersed in an insulating, amorphous matrix. From conductivity measurements we inferred the charge transport to be governed by a typical quasi-one dimensional variable range hopping (VRH) mechanism.

Sr and Nd isotope composition of the metamorphic, sedimentary and ultramafic xenoliths of Lanzarote (Canary Islands): Implications for magma sources

The lavas produced by the Timanfaya eruption of 1730-1736 (Lanzarote, Canary Islands) contain a great many sedimentary and metamorphic (metasedimentary), and mafic and ultramafic plutonic xenoliths. Among the metamorphosed carbonate rocks (calc-silicate rocks [CSRs]) are monomineral rocks with forsterite or wollastonite, as well as rocks containing olivine +/- orthopyroxene +/- clinopyroxene +/- plagioclase: their mineralogical compositions are identical to those of the mafic (gabbros) and ultramafic (dunite, wherlite and lherzolite) xenoliths. The (87)Sr/(16)Sr (around 0.703) and (143)Nd/(144)Nd (around 0.512) isotope ratios of the ultramafic and metasedimentary xenoliths are similar, while the (147)Sm/(144)Nd ratios show crustal values (0.13-0.16) in the ultramafic xenoliths and mantle values (0.18-0.25) in some CSRs. The apparent isotopic anomaly of the metamorphic xenoliths can be explained in terms of the heat source (basaltic intrusion) inducing strong isotopic exchange ((87)Sr/(86)Sr and (143)Nd/(144)Nd) between metasedimentary and basaltic rocks. Petrofabric analysis also showed a possible relationship between the ultramafic and metamorphic xenoliths. (C) 2009 Elsevier B.V. All rights reserved.