Hypermethylation-Mediated Silencing of CIDEA, MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies.

Gene expression is controlled by epigenetic deregulation, a hallmark of cancer. The DNA methylome of canine diffuse large B-cell lymphoma (cDLBCL), the most frequent malignancy of B-lymphocytes in dog, has recently been investigated, suggesting that aberrant hypermethylation of CpG loci is associated with gene silencing. Here, we used a multi-omics approach (DNA methylome, transcriptome and copy number variations) combined with functional in vitro assays, to identify putative tumour suppressor genes subjected to DNA methylation in cDLBCL. Using four cDLBCL primary cell cultures and CLBL-1 cells, we found that CiDEA, MAL and PCDH17, which were significantly suppressed in DLBCL samples, were hypermethylated and also responsive (at the DNA, mRNA and protein level) to pharmacological unmasking with hypomethylating drugs and histone deacetylase inhibitors. The regulatory mechanism underneath the methylation-dependent inhibition of those target genes expression was then investigated through luciferase and in vitro methylation assays. In the most responsive CpG-rich regions, an in silico analysis allowed the prediction of putative transcription factor binding sites influenced by DNA methylation. Interestingly, regulatory elements for AP2, MZF1, NF-kB, PAX5 and SP1 were commonly identified in all three genes. This study provides a foundation for characterisation and experimental validation of novel epigenetically-dysregulated pathways in cDLBCL.

Development and Validation of the Acute PNeumonia Early Assessment Score for Safely Discharging Low-Risk SARS-CoV-2-Infected Patients from the Emergency Department.

A continuous demand for assistance and an overcrowded emergency department (ED) require early and safe discharge of low-risk severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients. We developed (n = 128) and validated (n = 330) the acute PNeumonia early assessment (aPNea) score in a tertiary hospital and preliminarily tested the score on an external secondary hospital (n = 97). The score's performance was compared to that of the National Early Warning Score 2 (NEWS2). The composite outcome of either death or oral intubation within 30 days from admission occurred in 101 and 28 patients in the two hospitals, respectively. The area under the receiver operating characteristic (AUROC) curve of the aPNea model was 0.86 (95% confidence interval (CI), 0.78-0.93) and 0.79 (95% CI, 0.73-0.89) for the development and validation cohorts, respectively. The aPNea score discriminated low-risk patients better than NEWS2 at a 10% outcome probability, corresponding to five cut-off points and one cut-off point, respectively. aPNea's cut-off reduced the number of unnecessary hospitalizations without missing outcomes by 27% (95% CI, 9-41) in the validation cohort. NEWS2 was not significant. In the external cohort, aPNea's cut-off had 93% sensitivity (95% CI, 83-102) and a 94% negative predictive value (95% CI, 87-102). In conclusion, the aPNea score appears to be appropriate for discharging low-risk SARS-CoV-2-infected patients from the ED.

G-Quadruplex Modulation of SP1 Functional Binding Sites at the KIT Proximal Promoter.

The regulation of conformational arrangements of gene promoters is a physiological mechanism that has been associated with the fine control of gene expression. Indeed, it can drive the time and the location for the selective recruitment of proteins of the transcriptional machinery. Here, we address this issue at the KIT proximal promoter where three G-quadruplex forming sites are present (kit1, kit2 and kit*). On this model, we focused on the interplay between G-quadruplex (G4) formation and SP1 recruitment. By site directed mutagenesis, we prepared a library of plasmids containing mutated sequences of the WT KIT promoter that systematically exploited different G4 formation attitudes and SP1 binding properties. Our transfection data showed that the three different G4 sites of the KIT promoter impact on SP1 binding and protein expression at different levels. Notably, kit2 and kit* structural features represent an on-off system for KIT expression through the recruitment of transcription factors. The use of two G4 binders further helps to address kit2-kit* as a reliable target for pharmacological intervention.

DNA elements for constitutive androstane receptor- and pregnane X receptor-mediated regulation of bovine CYP3A28 gene.

The regulation of cytochrome P450 3A (CYP3A) enzymes is established in humans, but molecular mechanisms of its basal and xenobiotic-mediated regulation in cattle are still unknown. Here, ~10 kbp of the bovine CYP3A28 gene promoter were cloned and sequenced, and putative transcription factor binding sites were predicted. The CYP3A28 proximal promoter (PP; -284/+71 bp) contained DNA elements conserved among species. Co-transfection of bovine nuclear receptors (NRs) pregnane X and constitutive androstane receptor (bPXR and bCAR) with various CYP3A28 promoter constructs into hepatoma cell lines identified two main regions, the PP and the distal fragment F3 (-6899/-4937 bp), that were responsive to bPXR (both) and bCAR (F3 fragment only). Site-directed mutagenesis and deletion of NR motif ER6, hepatocyte nuclear factor 1 (HNF-1) and HNF-4 binding sites in the PP suggested either the involvement of ER6 element in bPXR-mediated activation or the cooperation between bPXR and liver-enriched transcription factors (LETFs) in PP transactivation. A putative DR5 element within the F3 fragment was involved in bCAR-mediated PP+F3 transactivation. Although DNA enrichment by anti-human NR antibodies was quite low, ChIP investigations in control and RU486-treated BFH12 cells, suggested that retinoid X receptor α (RXRα) bound to ER6 and DR5 motifs and its recruitment was enhanced by RU486 treatment. The DR5 element seemed to be recognized mainly by bCAR, while no clear-cut results were obtained for bPXR. Present results point to species-differences in CYP3A regulation and the complexity of bovine CYP3A28 regulatory elements, but further confirmatory studies are needed.

Validation of epigenetic mechanisms regulating gene expression in canine B-cell lymphoma: An in vitro and in vivo approach.

Despite canine B-cell Lymphoma (BCL) representing the most common haematological tumour, epigenetic events driving development and progression are scarcely known. Recently, canine Diffuse Large BCL (DLBCL) DNA methylome by genome-wide CpG microarray has identified genes and pathways associated to pathogenesis. To validate data previously obtained by array analysis, the CLBL-1 cell line was used and the HOXD10, FGFR2, ITIH5 and RASAL3 genes were selected. CLBL-1 cells were treated with two hypomethylating drugs (HDs; IC50, 50% inhibitory concentration), i.e. azacytidine and decitabine (DEC), either alone or in combination with three histone deacetylase inhibitors (HDACis; IC20), i.e. valproic acid, trichostatin and vorinostat. Following the incubation with both HDs, an overall decrease of promoter methylation was highlighted, thus confirming target genes hypermethylation. The highest mRNA restoration was observed following the exposure to HDs combined with HDACis, and mostly with valproic acid. Contrasting results were only obtained for RASAL3. An in vivo confirmation was finally attempted treating Nod-Scid mice engrafted with CLBL-1 cells with DEC. Although DEC did not arrest tumour growth, target genes promoter methylation was significantly reduced in DEC-treated mice vs controls. Overall, this work demonstrates that CLBL-1 cell line represents a reliable in vitro model to validate the methylation-dependent silencing of key genes for BCL; moreover, it may be useful for xenograft models in mice, despite its aggressive behaviour. In future, functional studies will be performed to deepen the role of selected genes on BCL pathogenesis and progression, and their methylation-dependent mechanism of regulation.

Targeting Canine KIT Promoter by Candidate DNA G-Quadruplex Ligands.

G-quadruplexes (G4) are nucleic acid secondary structures frequently assumed by G-rich sequences located mostly at telomeres and proto-oncogenes promoters. Recently, we identified, in canine KIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog) promoter, two G-rich sequences able to fold into G4: d_kit1 and d_kit2_A16. In this study, an anthraquinone (AQ1) and an anthracene derivative (AN6), known to stabilize the G4 structures of the corresponding human h_kit1 and h_kit2, were tested on the canine G4 and in two canine mast cell tumor (MCT) cell lines (C2 and NI-1) to verify their capability to down-regulate KIT expression. The cytotoxicity of AQ1 and AN6 was determined using the Alamar Blue test; also the constitutive expression of KIT and other proto-oncogenes containing G4 structures in their promoter (BCL2, VEGFα, VEGFR2, KRAS, and TERT) was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). Then the time- and dose-dependent effects of both ligands on target gene expression were assessed by qRT-PCR. All target genes were constitutively expressed up to 96 hours of culture. Both ligands decreased KIT mRNA levels and c-kit protein amount, and AN6 was comparatively fairly more effective. DNA interaction studies and a dual-luciferase gene reporter assay performed on a noncancerous canine cell line (Madin-Darby Canine Kidney cells) proved that this down-regulation was the result of the interaction of AN6 with KIT proximal promoter. Interestingly, our results only partially overlap with those previously obtained in human cell lines, where AQ1 was found as the most effective compound. These preliminary data might suggest AN6 as a promising candidate for the selective targeting of canine KIT-dependent tumors.

In Silico Identification of Piperidinyl-amine Derivatives as Novel Dual Binders of Oncogene c-myc/c-Kit G-quadruplexes.

In the last years, it has been shown that the DNA secondary structure known as G-quadruplex is also involved in the regulation of oncogenes transcription, such as c-myc, c-Kit, KRAS, Bcl-2, VEGF, and PDGF. DNA G-quadruplexes, formed in the promoter region of these proto-oncogenes, are considered alternative anticancer targets since their stabilization causes a reduction of the related oncoprotein overexpression. In this study, a structure-based virtual screening toward the experimental DNA G-quadruplex structures of c-myc and c-Kit was performed by using Glide for the docking analysis of a commercial library of approximately 693 000 compounds. The best hits were submitted to thermodynamic and biophysical studies, highlighting the effective stabilization of both G-quadruplex oncogene promoter structures for three N-(4-piperidinylmethyl)amine derivatives, thus proposed as a new class of dual G-quadruplex binders.

DNA methylation profiling reveals common signatures of tumorigenesis and defines epigenetic prognostic subtypes of canine Diffuse Large B-cell Lymphoma.

Epigenetic deregulation is a hallmark of cancer characterized by frequent acquisition of new DNA methylation in CpG islands. To gain insight into the methylation changes of canine DLBCL, we investigated the DNA methylome in primary DLBCLs in comparison with control lymph nodes by genome-wide CpG microarray. We identified 1,194 target loci showing different methylation levels in tumors compared with controls. The hypermethylated CpG loci included promoter, 5'-UTRs, upstream and exonic regions. Interestingly, targets of polycomb repressive complex in stem cells were mostly affected suggesting that DLBCL shares a stem cell-like epigenetic pattern. Functional analysis highlighted biological processes strongly related to embryonic development, tissue morphogenesis and cellular differentiation, including HOX, BMP and WNT. In addition, the analysis of epigenetic patterns and genome-wide methylation variability identified cDLBCL subgroups. Some of these epigenetic subtypes showed a concordance with the clinical outcome supporting the hypothesis that the accumulation of aberrant epigenetic changes results in a more aggressive behavior of the tumor. Collectively, our results suggest an important role of DNA methylation in DLBCL where aberrancies in transcription factors were frequently observed, suggesting an involvement during tumorigenesis. These findings warrant further investigation to improve cDLBCL prognostic classification and provide new insights on tumor aggressiveness.

Indenocinnoline derivatives as G-quadruplex binders, topoisomerase IIα inhibitors and antiproliferative agents.

DNA intercalating agents are a consolidated therapeutic option in the treatment of tumor diseases. Starting from previous findings in the antiproliferative efficacy of a series of indeno[1,2-c]cinnoline-11-one derivatives, we performed a suitable decoration of this scaffold by means of a simple and straightforward chemistry, aiming to a) enlarge the planar core to a pentacyclic benzo[h]indeno[1,2-c]cinnoline-13-one and b) introduce a basic head tethered through a simple polymethylene chain. In fluorescence melting and fluorescence intercalator displacement assays, these new compounds displayed fair to very good intercalating properties on different nucleic acid strands, with preference for G-quadruplex sequences. Inhibition of human topoisomerase IIα and antiproliferative assays on HeLa and MCF7 tumor cell lines outlined a multitarget antiproliferative profile for tetracyclic 6 and pentacyclic derivative 20, both bearing a N,N-dimethylamine as the protonatable moiety. Particularly, compound 6 displayed a very potent inhibition of tumor cell proliferation, while 20 returned the highest thermal stabilization in melting experiments. In summary, these results outlined a potential of such highly planar scaffolds for nucleic acid binding and antiproliferative effects.

Screening of candidate G-quadruplex ligands for the human c-KIT promotorial region and their effects in multiple in-vitro models.

Stabilization of G-quadruplex (G4) structures in promoters is a novel promising strategy to regulate gene expression at transcriptional and translational levels. c-KIT proto-oncogene encodes for a tyrosine kinase receptor. It is involved in several physiological processes, but it is also dysregulated in many diseases, including cancer. Two G-rich sequences able to fold into G4, have been identified in c-KIT proximal promoter, thus representing suitable targets for anticancer intervention. Herein, we screened an "in house" library of compounds for the recognition of these G4 elements and we identified three promising ligands. Their G4-binding properties were analyzed and related to their antiproliferative, transcriptional and post-transcriptional effects in MCF7 and HGC27 cell lines. Besides c-KIT, the transcriptional analysis covered a panel of oncogenes known to possess G4 in their promoters.From these studies, an anthraquinone derivative (AQ1) was found to efficiently downregulate c-KIT mRNA and protein in both cell lines. The targeted activity of AQ1 was confirmed using c-KIT-dependent cell lines that present either c-KIT mutations or promoter engineered (i.e., α155, HMC1.2 and ROSA cells).Present results indicate AQ1 as a promising compound for the target therapy of c-KIT-dependent tumors, worth of further and in depth molecular investigations.

Sequencing and G-quadruplex folding of the canine proto-oncogene KIT promoter region: might dog be used as a model for human disease?

Downregulation of gene expression by induction of non-canonical DNA structures at promotorial level is a novel attractive anticancer strategy. In human, two guanine-rich sequences (h_kit1 and h_kit2) were identified in the promotorial region of oncogene KIT. Their stabilization into G-quadruplex structures can find applications in the treatment of leukemias, mastocytosis, gastrointestinal stromal tumor, and lung carcinomas which are often associated to c-kit mis-regulation. Also the most common skin cancer in domestic dog, mast cell tumor, is linked to a mutation and/or to an over-expression of c-kit, thus supporting dog as an excellent animal model. In order to assess if the G-quadruplex mediated mechanism of regulation of c-kit expression is conserved among the two species, herein we cloned and sequenced the canine KIT promoter region and we compared it with the human one in terms of sequence and conformational equilibria in physiologically relevant conditions. Our results evidenced a general conserved promotorial sequence between the two species. As experimentally confirmed, this grants that the conformational features of the canine kit1 sequence are substantially shared with the human one. Conversely, two isoforms of the kit2 sequences were identified in the analyzed dog population. In comparison with the human counterpart, both of them showed an altered distribution among several folded conformations.

Atrial fibrillation and right bundle branch block complicating coil embolization of a huge pulmonary arterio-venous malformation.

Coil embolization is a safe therapy for pulmonary arterio-venous malformations (PAVMs). We report the case of a 72-year-old woman affected by hereditary hemorrhagic teleangectasia who experienced right bundle branch block and atrial fibrillation 36 hours after embolization of an extended PAVM. To our knowledge, this is the first case of such a complication of embolization of a pulmonary fistula, presumably due to an acute pressure overload on both the right atrium and ventricle. This case report suggests that, after embolization of extended pulmonary fistulas, patients should be carefully monitored for rhythm disturbance and new onset of intraventricular conduction defects.

Processos de criação em educadoras: uma experiência e suas implicações / Creation process in contexts of continued formation: one experience and its implications

Este trabalho busca analisar o processo de criação de uma atividade de produção literária, desenvolvida em um curso de formação para professores, intitulado 'Oficinas Estéticas: prática pedagógica e atividade criadora'. Seu objetivo foi proporcionar a construção de 'olhares estéticos' que redimensionassem as relações envolvidas na ação pedagógica por meio de vivências criadoras. Analisou-se o processo de criação, utilizando-se as filmagens das oficinas e o material produzido pelos participantes durante uma atividade de reconstrução de uma história infantil. Os resultados demonstraram que, por meio da imaginação, elementos de histórias conhecidas são combinados aos novos, re-significando e transformando os movimentos dos sujeitos.(AU)

This work analyzes the process of creation of an activity of literary production, developed in a course of formation, intitled 'Aesthetic Workshops: practical pedagogical and creative activity'. Its objective was to provide of 'aesthetic looks' that to review involved relations in the pedagogical action by means of creative experiences. The work was analyzed, using the filmings of the workshops and the material produced for the participants, during an activity of reconstruction of an infantile history. The results had demonstrated that, by means of the imagination, elements of known histories are combined to the news, re-meaning the movements of the individuals.(AU)

Processos de criação em educadoras: uma experiência e suas implicações / Creation process in contexts of continued formation: one experience and its implications

Este trabalho busca analisar o processo de criação de uma atividade de produção literária, desenvolvida em um curso de formação para professores, intitulado "Oficinas Estéticas: prática pedagógica e atividade criadora". Seu objetivo foi proporcionar a construção de "olhares estéticos" que redimensionassem as relações envolvidas na ação pedagógica por meio de vivências criadoras. Analisou-se o processo de criação, utilizando-se as filmagens das oficinas e o material produzido pelos participantes durante uma atividade de reconstrução de uma história infantil. Os resultados demonstraram que, por meio da imaginação, elementos de histórias conhecidas são combinados aos novos, re-significando e transformando os movimentos dos sujeitos.

This work analyzes the process of creation of an activity of literary production, developed in a course of formation, intitled "Aesthetic Workshops: practical pedagogical and creative activity". Its objective was to provide of "aesthetic looks" that to review involved relations in the pedagogical action by means of creative experiences. The work was analyzed, using the filmings of the workshops and the material produced for the participants, during an activity of reconstruction of an infantile history. The results had demonstrated that, by means of the imagination, elements of known histories are combined to the news, re-meaning the movements of the individuals.

Relações estéticas, atividade criadora e constituição do sujeito: algumas reflexões sobre a formação de professores(as) / Aesthetic relations, creative activity and subjectivity: some reflections on teachers education

Este trabalho tem por objetivo refletir sobre as temáticas constituição do sujeito, atividade criadora e relação estética, consideradas fundamentais para propostas de formação continuada de professores(as). Busca-se discutir na interface entre os campos de conhecimento da psicologia e da pedagogia e a partir do enfoque histórico-cultural a relevância destas temáticas para a prática pedagógica diante dos desafios dos processos de escolarização formal. Ressalta-se a importância da educação estética e, principalmente da formação estética dos professores(as), uma vez que se entende que todo processo de ensinar e aprender é constitutivo dos sujeitos, sendo a experiência estética uma possibilidade de vivenciar novas formas de relação. Relações pautadas na imaginação e no fazer criativo em contraposição a práticas e relações excessivamente técnicas e reprodutoras, que predominam na sociedade atual em que vivemos

This work has as objective to reflect about the thematic subjectivity, creative activity and aesthetic relation, considered basic for proposals of teacher’s education. It search to argue, in the interface between the knowledge fields of psychology and the pedagogy and from the historical-cultural approach, the relevance of these thematic for the pedagogical practice ahead the challenges of the processes of the school education. Stands out the importance of aesthetic education and, mainly of the aesthetic formation of the teachers, therefore it is understood that all process of teaching and learning is constituent of the subjectivity, being the aesthetic experience a possibility to live new forms of relation. Relations based in the imagination and creative making in contraposition to practices and to relations excessively techniques and reproductive, that are predominate in the current society where we live

Movimento de objetivação e subjetivação mediado pela criação artística / Movement of objectivity and subjectivity, mediated for the artistic creation

Este texto tem por objetivo refletir sobre o movimento de um sujeito e sua relação com a criação artística, apontando que neste processo articulam-se dialeticamente o saber, o fazer e o sentir. As análises sustentam-se nas falas desse sujeito, coletadas por meio de entrevistas e registros em vídeo de sua participação em uma disciplina de um curso de graduação em Psicologia. As reflexões decorrentes, por sua vez, pautam-se nos aportes teóricos da Psicologia Histórico-Cultural e revelam a complexidade do processo de constituição do sujeito que, via atividade criadora, parte da realidade e a transforma, transformando nesse movimento a si mesmo e ao modo como significa tanto a sua própria trajetória quanto o contexto do qual ativamente participa(AU)

Movimento de objetivação e subjetivação mediado pela criação artística / Movement of objectivity and subjectivity, mediated for the artistic creation

Este texto tem por objetivo refletir sobre o movimento de um sujeito e sua relação com a criação artística, apontando que neste processo articulam-se dialeticamente o saber, o fazer e o sentir. As análises sustentam-se nas falas desse sujeito, coletadas por meio de entrevistas e registros em vídeo de sua participação em uma disciplina de um curso de graduação em Psicologia. As reflexões decorrentes, por sua vez, pautam-se nos aportes teóricos da Psicologia Histórico-Cultural e revelam a complexidade do processo de constituição do sujeito que, via atividade criadora, parte da realidade e a transforma, transformando nesse movimento a si mesmo e ao modo como significa tanto a sua própria trajetória quanto o contexto do qual ativamente participa

Doce, pirâmide ou flor?: o processo de produção de sentidos em um contexto de ensinar e aprender / Candy, pyramid or flower?: the process of production of meaning in a context of teaching and learning

Este texto discute o processo de produção de sentidos sobre o produto de uma atividade estética em uma situação formal de ensinar e aprender. Tal atividade foi proposta a acadêmicos matriculados em uma disciplina do curso de psicologia da Universidade Federal de Santa Catarina. Os procedimentos escolhidos para coleta e análise de dados foram a videografia e a análise microgenética. A atenção centrou-se no diálogo estabelecido entre professoras e alunos no decorrer da atividade e na produção de uma das participantes, enfatizando as mediações engendradas e seus resultados. As reflexões fundamentaram-se nas contribuições de Vigotski, especificamente nas discussões sobre o processo de produção/apropriação de sentidos e a constituição do sujeito. Como resultado foram identificados elementos indicadores das mediações ali objetivadas e do processo vivido pela protagonista da situação, que resultou no reconhecimento de si como capaz de produzir criativamente algo que engendra múltiplos sentidos para os sujeitos em relação (AU)

Doce, pirâmide ou flor?: o processo de produção de sentidos em um contexto de ensinar e aprender / Candy, pyramid or flower?: the process of production of meaning in a context of teaching and learning

Este texto discute o processo de produção de sentidos sobre o produto de uma atividade estética em uma situação formal de ensinar e aprender. Tal atividade foi proposta a acadêmicos matriculados em uma disciplina do curso de psicologia da Universidade Federal de Santa Catarina. Os procedimentos escolhidos para coleta e análise de dados foram a videografia e a análise microgenética. A atenção centrou-se no diálogo estabelecido entre professoras e alunos no decorrer da atividade e na produção de uma das participantes, enfatizando as mediações engendradas e seus resultados. As reflexões fundamentaram-se nas contribuições de Vigotski, especificamente nas discussões sobre o processo de produção/apropriação de sentidos e a constituição do sujeito. Como resultado foram identificados elementos indicadores das mediações ali objetivadas e do processo vivido pela protagonista da situação, que resultou no reconhecimento de si como capaz de produzir criativamente algo que engendra múltiplos sentidos para os sujeitos em relação

Mediação pedagógica: reflexões sobre o olhar estético em contexto de escolarização formal / Pedagogical mediation: reflections about the aesthetics look

Neste texto é analisada a mediação pedagógica no processo de constituição do olhar estético, a partir de dois momentos de reflexão coletiva sobre o processo vivido por um sujeito numa oficina de pintura. A atividade estética relatada foi proposta a acadêmicos de psicologia da Universidade Federal de Santa Catarina regularmente matriculados em uma disciplina optativa intitulada 'Psicologia da Criatividade'. Para a coleta de dados, optou-se pela videografia. A análise fundamentou-se no arcabouço teórico da psicologia histórico-cultural, priorizando discussões sobre a percepção enquanto atividade semioticamente mediada e as tramas que entrelaçam a produção e apropriação de significações num contexto de ensinar e aprender. Finalmente, através do discurso da protagonista da ação, foram identificados elementos que apontam para o reconhecimento de que a obra por ela produzida, ao engendrar múltiplos sentidos para os sujeitos em relação, pôde ser percebida através de outros ângulos, inusitados e imprevisíveis, possibilitando um novo olhar(AU)