Serological investigation of protozoan pathogens (Trypanosoma cruzi, Toxoplasma gondii and Neospora caninum) in opossums from southern Brazil.

South American opossums of the order Didelphimorphia are considered sentinels for zoonotic infections and environmental diseases, such as for Chagas disease caused by Trypanosoma cruzi. Nevertheless, there is a paucity of data regarding protozoan diseases such Toxoplasma gondii and Neospora caninum in Neotropical marsupials; despite these pathogens have been considered threats to some marsupial species. The aim of this study was to determine whether Didelphis albiventris and Philander frenatus opossums from southern Brazil had been previously exposed to T. cruzi, T. gondii or N. caninum. Opossum samples were obtained by live-trapping of free-ranging animals and collection at wildlife rehabilitation centers in Rio Grande do Sul (RS) state, Brazil. The detection of anti-T. cruzi and anti-T. gondii antibodies was performed by indirect hemagglutination and anti-N. caninum antibodies by competitive enzyme-linked immunosorbent assay. In total, samples were collected from 76 marsupials from nine municipalities in RS state, including 69 D. albiventris (white-eared opossum), and seven were P. frenatus (southern four-eyed opossum). For T. cruzi, 11% were seropositive, and for T. gondii 26% were seropositive. None of the marsupials sampled here were seropositive for N. caninum. Risk factor analysis showed that free-living animals were about five-fold more likely to be infected by T. gondii than were rescued animals. Our study showed the exposure of Neotropical marsupials (D. albiventris and P. frenatus) to protozoan pathogens T. cruzi and T. gondii, while no evidence of N. caninum exposure was found. The set of results presented here may have an effect on ecology and conservation of the studied species and may also indicate possible sentinels of these pathogen circulation.

Novel Gyrovirus genomes recovered from free-living pigeons in Southern Brazil.

Wild birds carry a number of infectious agents, some of which may have pathogenic potential for the host and others species, including humans. Domestic pigeons (Columba livia) are important targets of study since these increasingly cohabit urban spaces, being possible spillover sources of pathogens to humans. In the present study, two genomes (PiGyV_Tq/RS/Br and PiGyV_RG/RS/Br), representative of Gyrovirus genus, family Anelloviridae, were detected in sera of free-living pigeons collected in Southern Brazil. The genomes exhibit less than 50% identity to previously described members of Gyrovirus genus, suggesting that they constitute a new viral species circulating in pigeons, to which the name "pigeon gyrovirus (PiGyV)" is proposed. The current study characterizes these two PiGyV genomes which, to date, are the first gyrovirus species identified in domestic pigeons.

Correction to: Columbid circoviruses detected in free ranging pigeons from Southern Brazil: insights on PiCV evolution.

Unfortunately, the word "evolution" was found missing in title of the original article which is corrected here by this erratum. The original article has been corrected.

Columbid circoviruses detected in free ranging pigeons from Southern Brazil: insights on PiCV evolution.

Pigeon circovirus (PiCV) is taxonomically classified as a member of the Circovirus genus, family Circoviridae. The virus contains a single stranded DNA genome of approximately 2 kb, with minor length variations among different isolates. The occurrence of PiCV infections in pigeons (Columba livia) has been documented worldwide over the past 20 years; however, in Brazil there were still no reports on PiCV detection. This study identifies seven PiCV genomes recovered from domestic pigeons of South Brazil through high-throughput sequencing and shows a high frequency of PiCV infection, through quantitative real-time PCR. Phylogenetic classification was performed by maximum likelihood analysis of the full genomes, ORF V1 (Rep) and ORF C1 (Cap). The results show that either full genome or Cap based analysis allowed PiCV classification into five major clades (groups A to E), where Brazilian sequences were classified as A, C or D. Recombination analyses were carried out with Simplot and RDP4 and the results show that both Rep and Cap ORFs contain several recombination hotspots, pointing to an important role for such events in PiCV evolution.

Beclin 1 and autophagy are required for the tumorigenicity of breast cancer stem-like/progenitor cells.

Malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew; these cells are known as cancer stem-like cells (CSCs) or tumor-initiating cells. Primitive mammary CSCs/progenitor cells can be propagated in culture as floating spherical colonies termed 'mammospheres'. We show here that the expression of the autophagy protein Beclin 1 is higher in mammospheres established from human breast cancers or breast cancer cell lines (MCF-7 and BT474) than in the parental adherent cells. As a result, autophagic flux is more robust in mammospheres. We observed that basal and starvation-induced autophagy flux is also higher in aldehyde dehydrogenase 1-positive (ALDH1(+)) population derived from mammospheres than in the bulk population. Beclin 1 is critical for CSC maintenance and tumor development in nude mice, whereas its expression limits the development of tumors not enriched with breast CSCs/progenitor cells. We found that decreased survival in autophagy-deficient cells (MCF-7 Atg7 knockdown cells) during detachment does not contribute to an ultimate deficiency in mammosphere formation. This study demonstrates that a prosurvival autophagic pathway is critical for CSC maintenance, and that Beclin 1 plays a dual role in tumor development.

Assessment of transformed properties in vitro and of tumorigenicity in vivo in primary keratinocytes cultured for epidermal sheet transplantation.

Epidermal keratinocytes are used as a cell source for autologous and allogenic cell transplant therapy for skin burns. The question addressed here is to determine whether the culture process may induce cellular, molecular, or genetic alterations that might increase the risk of cellular transformation. Keratinocytes from four different human donors were investigated for molecular and cellular parameters indicative of transformation status, including (i) karyotype, (ii) telomere length, (iii) proliferation rate, (iv) epithelial-mesenchymal transition, (v) anchorage-independent growth potential, and (vi) tumorigenicity in nude mice. Results show that, despite increased cell survival in one keratinocyte strain, none of the cultures displayed characteristics of cell transformations, implying that the culture protocol does not generate artefacts leading to the selection of transformed cells. We conclude that the current protocol does not result in an increased risk of tumorigenicity of transplanted cells.

Evaluation of tumorigenic risk of tissue-engineered oral mucosal epithelial cells by using combinational examinations.

Recently, oral mucosal epithelial cells were proposed as a cell source of the autologous cell transplant therapy for corneal trauma or disease. The question addressed is to know if the biological conditions of grafting could induce certain cellular, molecular, and genetic alterations that might increase the risk of mutations and possibly of cellular transformation. Recent progress in cancer research enables us to depict the generation mechanisms and basic characteristics of human cancer cells from molecular, cytological, and biological aspects. The aim of this study is to evaluate the risk of tumorigenicity of the oral mucosal epithelial culture process in order to mitigate that risk, if any, before clinical application. Oral mucosal epithelial cells from three different human donors were investigated by combinational examinations to detect possible tumorigenic transformation. We investigated (i) clonogenic and karyology types, (ii) the validation of proliferation rate, (iii) the epithelial-mesenchymal transition, (iv) anchorage-independent growth potential, and (v) tumorigenicity on nude mice. Results show that the culture process used in this study presents no risk of tumorigenicity.

TWISTing an embryonic transcription factor into an oncoprotein.

Over the past decade, the reactivation of TWIST embryonic transcription factors has been described as a frequent event and a marker of poor prognosis in an impressive array of human cancers. Growing evidence now supports the premise that these cancers hijack TWIST's embryonic functions, granting oncogenic and metastatic properties. In this review, we report on the history and recent breakthroughs in understanding TWIST protein functions and the emerging role of the associated epithelial-mesenchymal transition (EMT) in tumorigenesis. We then broaden the discussion to address the general contribution of reactivating embryonic programs in cancerogenesis.

[Role of the epithelial-mesenchymal transition during tumor progression]. / Rôle de la transition épithéliomésenchymateuse dans la progression tumorale.

The epithelial-mesenchymal transition (EMT) is a morphogenetic program that converts epithelial into mesenchymal cells during the embryonic development. This mechanism is frequently reactivated during tumor progression and provides cells with motility and invasive capabilities favoring the metastatic dissemination from epithelial tumors. Various EMT-inducing transcription factors, such as the TWIST proteins, were also shown to inhibit oncogene-induced fail-safe programs (senescence and apoptosis), thereby promoting the progression from benign to malignant stages. Altogether, these observations suggest that EMT could play an important role in favoring both tumor development and metastatic dissemination.

A new set of monoclonal antibodies directed to proline-rich and central regions of p53.

The p53 protein can adopt several conformations in cells--"latent," "active," or mutant--depending on cellular stress or mutations of the TP53 gene. Today, only a few antibodies discriminating these conformations are available. We produced three new anti-p53 monoclonal antibodies (MAbs) directed against epitopes of human p53. The H53C1 MAb recognizes an epitope located at the N-terminal part of the central region of p53 and can discriminate mutant from wild-type conformation. The H53C2 and H53C3 MAbs are against different epitopes within the proline-rich region of p53. Moreover, the H53C2 epitope is located in the second negative regulatory domain of p53 between residues 80 and 93. These MAbs can be used as new tools to study and modulate the cellular functions of p53.

Identification of four families of yCCR4- and Mg2+-dependent endonuclease-related proteins in higher eukaryotes, and characterization of orthologs of yCCR4 with a conserved leucine-rich repeat essential for hCAF1/hPOP2 binding.

BACKGROUND: The yeast yCCR4 factor belongs to the CCR4-NOT transcriptional regulatory complex, in which it interacts, through its leucine-rich repeat (LRR) motif with yPOP2. Recently, yCCR4 was shown to be a component of the major cytoplasmic mRNA deadenylase complex, and to contain a fold related to the Mg2+-dependent endonuclease core. RESULTS: Here, we report the identification of nineteen yCCR4-related proteins in eukaryotes (including yeast, plants and animals), which all contain the yCCR4 endonuclease-like fold, with highly conserved CCR4-specific residues. Phylogenetic and genomic analyses show that they form four distinct families, one of which contains the yCCR4 orthologs. The orthologs in animals possess a leucine-rich repeat domain. We show, using two-hybrid and far-Western assays, that the human member binds to the human yPOP2 homologs, i.e. hCAF1 and hPOP2, in a LRR-dependent manner. CONCLUSIONS: We have identified the mammalian orthologs of yCCR4 and have shown that the human member binds to the human yPOP2 homologs, thus strongly suggesting conservation of the CCR4-NOT complex from yeast to human. All members of the four identified yCCR4-related protein families show stricking conservation of the endonuclease-like catalytic motifs of the yCCR4 C-terminal domain and therefore constitute a new family of potential deadenylases in mammals.

Relationships of the antiproliferative proteins BTG1 and BTG2 with CAF1, the human homolog of a component of the yeast CCR4 transcriptional complex: involvement in estrogen receptor alpha signaling pathway.

We have reported previously the physical interaction of B-cell translocation gene proteins (BTG)1 and BTG2 with the mouse protein CAF1 (CCR4-associated factor 1) and suggested that these proteins may participate, through their association with CAF1, in transcription regulation. Here we describe the in vitro and in vivo association of these proteins with hPOP2, the human paralog of hCAF1. The physical and functional relationships between the BTG proteins and their partners hCAF1 and hPOP2 were investigated to find out how these interactions affect cellular processes, and in particular transcription regulation. We defined their interaction regions and examined their expression in various human tissues. We also show functional data indicating their involvement in estrogen receptor alpha (ERalpha)-mediated transcription regulation. We found that BTG1 and BTG2, probably through their interaction with CAF1 via a CCR4-like complex, can play both positive or negative roles in regulating the ERalpha function. In addition, our results indicate that two LXXLL motifs, referred to as nuclear receptor boxes, present in both BTG1 and BTG2, are involved in the regulation of ERalpha-mediated activation.

P53 but not p16INK4a induces growth arrest in retinoblastoma-deficient hepatocellular carcinoma cells.

BACKGROUND/AIM: Both p16INK4a and p53 proteins are negative regulators of the cell cycle. In human hepatocellular carcinomas (HCC), the loss of function of p53, retinoblastoma (pRb) and pl6INK4a genes by different mechanisms has been largely documented, but their hepatocellular effects are poorly known. We compared the growth-inhibitory effects of p16INK4a and p53 proteins in Hep3B cell line-derived clones. METHODS: Cells were transfected with inducible p16INK4a and p53 expression vectors, and stable clones were analyzed for transgene expression by Western blotting and immunoperoxidase staining. Effects on cell growth were analyzed by in vitro growth assay, thymidine incorporation and flow cytometry. Biochemical effects of p53 were tested by Northern blotting of p21Cip1 transcripts and by Western blotting of p21Cip1, mdm-2, bax, cyclin-dependent kinase 2 and cyclin E proteins. The pRb protein was studied by Western blotting and immunoprecipitation assays. RESULTS: The induction of p16INK4a protein expression did not affect in vitro growth of cells. In contrast, p53 protein in its wild-type conformation provoked a growth arrest accompanied by transactivation of p21Cip1 gene and accumulation of p21Cip1, bax and mdm-2 proteins. p53-induced growth arrest was due to a cell cycle arrest at the G1/S transition, probably mediated by p21Cip1 protein, which inhibits cyclin-dependent kinase 2/cyclin E complexes. CONCLUSIONS: The lack of detectable pRb protein and resistance of cells to p16TNK4a strongly suggest that p53 is able to arrest the growth of HCC cells by a mechanism independent of "p53-retinoblastoma pathway". These findings are applicable to HCC with abberrations of both p53 and pRb genes, and may not represent the universal effects of p53 in hepatic cells.

BTG gene expression in the p53-dependent and -independent cellular response to DNA damage.

Exposure of mammalian cells to genotoxic agents evokes a complex cellular response. An ordered series of molecular events is necessary to sense DNA damage, transduce the signal, and ultimately delay the cell cycle or trigger apoptosis. Recently, we have shown that BTG2/TIS21 gene expression was induced in response to DNA damage through a p53-dependent pathway. This gene belongs to a newly identified family of structurally related genes whose other known human members are BTG1, BTG3, and Tob. To define the respective involvement of these four related genes in the cellular response to DNA damage, we studied their expression in human cell lines after a variety of genotoxic treatments. Our results demonstrated that were BTG1, BTG2/TIS21, and Tob genes the DNA damage--inducible genes. However, BTG2/TIS21 appeared to be the only p53-transcriptional target gene. We speculate that BTG proteins may play a coordinate role in a general transduction pathway that is induced in response to DNA damage. It has been previously described that recombinant BTG1 and BTG2/TIS21 can physically interact with PRMT1, an arginine methyl transferase, suggesting that BTG1 and BTG2/TIS21 induction may lead to posttranslational modifications of cellular proteins. In support of this hypothesis, we showed that the endogenous induction of BTG1 and BTG2 after genotoxic treatment was correlated with a modulation of protein methylation.

The leukemia-associated protein Btg1 and the p53-regulated protein Btg2 interact with the homeoprotein Hoxb9 and enhance its transcriptional activation.

BTG1 and BTG2 belong to a family of functionally related genes involved in the control of the cell cycle. As part of an ongoing attempt to understand their biological functions, we used a yeast two-hybrid screening to look for possible functional partners of Btg1 and Btg2. Here we report the physical and functional association between these proteins and the homeodomain protein Hoxb9. We further show that Btg1 and Btg2 enhance Hoxb9-mediated transcription in transfected cells, and we report the formation of a Hoxb9.Btg2 complex on a Hoxb9-responsive target, and the fact that this interaction facilitates the binding of Hoxb9 to DNA. The transcriptional activity of the Hoxb9.Btg complex is essentially dependent on the activation domain of Hoxb9, located in the N-terminal portion of the protein. Our data indicate that Btg1 and Btg2 act as transcriptional cofactors of the Hoxb9 protein, and suggest that this interaction may mediate their antiproliferative function.

Hepatocarcinoma-specific mutant p53-249ser induces mitotic activity but has no effect on transforming growth factor beta 1-mediated apoptosis.

Mutations affecting the p53 gene abrogate its tumor suppressor activity. It is, however, unclear whether such mutations can generate mutant p53 proteins with an intrinsic transforming ability. More importantly, the mechanism(s) by which they exert such activity is unknown. We report here that p53-deficient hepatoma cells (Hep3B) transfected with mutant p53-249ser (codon 249 Arg-->Ser) acquire a new phenotype with an increased in vitro survival and mitotic activity. However, such a phenotypic change is not sufficient to cause a major shift in the poor tumorigenic potential of these cells. This is apparently due to transforming growth factor beta 1-mediated apoptotic death of Hep3B cells which is not affected by the expression of p53-249ser.