Identification of new genetic alterations in pancreatic ductal adenocarcinoma and precursor lesions by Whole Genome Sequencing and Oncoscan Array technology

L’Adenocarcinoma pancreatico (PDAC) è uno dei tumori più letali con un tasso di sopravvivenza a cinque anni di circa il 5%. Un certo miglioramento nell’outcome di questa neoplasia si è ottenuto con lo sviluppo delle terapie target e con l’introduzione di innovativi schemi chemioterapici, quali il FOLFIRINOX e l’associazione gemcitabina più Nab-Paclitaxel, ma la percentuale delle persone che ne beneficia è bassissima.Attualmente il cancro del pancreas non è ascrivibile al gruppo dei tumori meglio caratterizzati sotto il profilo biologico e, a dispetto delle numerose informazioni fornite dagli studi di caratterizzazione genetica della neoplasia condotti negli ultimi anni, non è ancora ad oggi ipotizzabile un risvolto clinico di tali conoscenze. Sulla base di queste premesse lo scopo di questo progetto è stato quello di analizzare alterazioni genetiche che caratterizzano l’eterogenità dell’adenocarcinoma pancreatico tramite sequenziamento massivo dell’esoma (WES) e analisi mediante tecnologia l’Oncoscan FFPE Assay, di campioni bioptici, tessuto fresco, e paraffinato provenienti da tumori pancreatici a diversi stadi di malattia (resecabili, localmente avanzati e metastatici). Quindi si è verificato o meno la correlazione di profili mutazionali con il diverso andamento clinico dei pazienti sottoposti al trattamento medico, chemioterapico o radio-chemioterapico integrato. In particolare questo progetto è stato condotto su circa 20 campioni in paraffina con metodologia Oncoscan Affymetrix FFPE per l’identificazione di copy number variation e su 30 campioni di tessuto fresco o bioptico con tecnologia WES per la ricerca di mutazioni puntiformi. Questo approccio integrato potrebbe permettere di avere un quadro biologico completo di tutte le alterazioni genetiche della malattia dalla lesione precancerosa al carcinoma metastatico per poter permettere di capire quale o quali sono gli eventi neoplastici che lo generano e poter quindi permettere uno studio farmacologico più mirato e più efficace.PDA is the fourth leading cause of cancer death, with a 5-years survival rate of 5% . Surgery remains the most effective treatment, but only 20% of patients are suitable for radical resection. Advances in chemotherapy, represented by FOLFIRINOX and by gemcitabine plus nab-paclitaxel regimens, have resulted in a modest outcome improvement..A thorough understanding of the genetic changes that will drive pancreatic carcinogenesis can lead to identification of biomarkers for early detection and targets for therapy. We used an approach with high resolution cytogenetic analysis Oncoscan Array and WES a bioinformatic, clinically-oriented interpretation of data to understand what are the most relevant pathways altered in precursor lesions and overt cancers to identify new therapeutic options for patients affected by PDA. In this study a total of 20 formalin fixed paraffin embedded samples from IPMN, profiled by Oncoscan and were analysed 30 fresh-frozen biopsies by WES . We identified in IPMN multiple copy number alterations and interestingly, differences were seen in the lesions at different stages, with 7 IPMN with low-intermediate dysplasia carrying a nearly normal karyotype and 13 IPMN with complex Karyotype (> 4 alterations), showing high grade dysplasia. A specific gain of chromosome arm 3q was found in IPMN with complex Karyotype (92%). This gain of 3q is particularly interesting for the presence of oncogenes such as PIK3CA, GATA2 and TERC that are part of pathways that deregulate cell growth and promote disease progression. In 30 sample analyzed with WES our results confirmed the high prevalence of KRAS, CDKN2A, TP53 and SMAD4 mutations. In particular, 93.7% of tumor samples exhibited somatic mutations acti¬vating KRAS and gene amplifications .The identification of these markers at an early stage of disease onset helps to identify patients at risk for cancer progression and new candidates for a more specific targeted therap

Hábitat popular e organização comunitária em bairros periféricos de Gran La Plata - Argentina perante o COVID 19

En este artículo se presentan avances del proyecto de investigación “Atención primaria del hábitat en barrios populares del Gran La Plata”, cuyo objetivo es elaborar un diagnóstico de las condiciones de criticidad del hábitat popular durante la pandemia por Covid 19, fortaleciendo el nexo entre necesidades sociales y políticas públicas. Se utiliza una metodología basada en herramientas cualitativas y cuantitativas con estrategias de recolección de datos múltiples —datos oficiales, entrevistas y focus group— en dos barrios del Gran La Plata (Argentina): Las Palmeras y Villa Progreso, como casos ejemplificadores respecto de las dificultades y lógicas de organización que experimentaron ciertos sectores sociales signados por la precariedad habitacional y la vulnerabilidad socioeconómica. Los resultados del trabajo exponen cómo estas estrategias desplegadas interpelan las formas en que se planifican las ciudades desde el urbanismo tradicional, mostrando por medio de la organización territorial y comunitaria de estos barrios otras formas de habitar las periferias.This article presents advances of the research project “Primary attention to habitat in popular neighborhoods of Gran La Plata”, whose objective is to elaborate a diagnosis of the critical conditions of the popular habitat during the Covid 19 pandemic, strengthening the nexus between social needs and public policies. A methodology based on qualitative and quantitative tools is used with multiple data collection strategies official data, interviews and focus groups in two neighborhoods of Gran La Plata (Argentina): Las Palmeras and Villa Progreso, as exemplary cases regarding the difficulties and organiza - tional logics experienced by certain social sectors marked by housing precariousness and socioeconomic vulnerability. The results of the work show how these strategies question the ways in which cities are planned from traditional urbanism, showing through the territorial and community organization of these neighborhoods other ways of inhabiting the peripheries.Neste artigo apresenta-se o andamento do projeto de pesquisa “Atenção primária do hábitat em bairros populares do Gran La Plata”, cujo objetivo é elaborar um diagnóstico das condições de criticidade do hábitat popular durante a pandemia pelo Covid 19, fortalecendo o nexo entre necessidades sociais e políticas públicas. Foi utilizada uma metodologia baseada em ferramentas qualitativas e quantitativas com estratégias de coleta de dados múltiplos —dados oficiais, entrevistas e focus group— em dois bairros do Gran La Plata (Argentina): Las Palmeras e Villa Progreso, como casos exemplares quanto as dificuldades e lógicas de organização vivenciados por certos setores sociais marcados pela precariedade habitacional e pela vulnerabilidade socioeconômica. Os resultados do traba - lho expõem como essas estratégias desfraldadas questionam as formas em que as cidades são planejadas desde o urbanismo tradicional, mostrando através da organização territorial e comunitária destes bairros, outras formas de habitar as periferias.Fil: Ursino, Sandra Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Arquitectura y Urbanismo. Centro Interdisciplinario de Estudios Complejos; ArgentinaFil: Vila, Mariana Paola. Universidad Nacional de La Plata. Facultad de Arquitectura y Urbanismo. Centro Interdisciplinario de Estudios Complejos; ArgentinaFil: Durante, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Arquitectura y Urbanismo. Centro Interdisciplinario de Estudios Complejos; Argentin

Osteogenic differentiation of MG63 cells in biodegradable scaffolds based on gelatin and genipin

Gelatin is a denatured collagen and commercially available as a biodegradable polymer. It has been extensively utilized for pharmaceutical and medical purposes, and its biosafety has been proven through long clinical applications. It showed an high cell attachment and proliferation, thus it has been widely considered as one of the best material to be used in medicine regenerative. Scaffolds based on pure gelatin showed a low mechanical and biological stability, so that a chemical crosslinking is necessary to improve its mechanical proprieties and success in clinical application. The aim of this study was to test the influence of a new porous composite scaffold based on gelatin/genipin composition on cell adhesion, proliferation and osteogenic potential. Genipin is a natural non toxic crosslinking agent which significantly improve the mechanical stability of the scaffold1. Osteoblast like cell (MG63) were seeded in collagen/genipin scaffolds for 24h, 7, 14, 21 and 28 days. Cell proliferation assay, light and electron microscopy analysis were performed to evaluate cell growth and morphological changes induced by cell/ scaffold interactions. Real Time PCR were carried out to evaluate the expression of the osteogenic markers such as collagen type I, osteonectin, osteopontin, ostecalcin and alkaline phosphatase proteins in MG63 seeded on gelatin/genipin scaffolds. Our results showed that gelatin/genipin scaffold is an excellent substrate for the growth and cell proliferation. Microscopy analysis showed an high cell adhesion on scaffold surface and an deep penetration in the macropores of the sponge. The Real Time data reveal a significant difference in the expression of the ostegenic markers in MG63 grown on gelatin/genipin scaffold compared to control samples. In conclusion, our data demonstrated that gelatin/genipin scaffold showed an high biocompatibility with human cells and an high osteogenic potential and it could be a potential tool in regenerative medicine

Changes of microenvironment in an in vitro co-culture system

For the enhanced understanding of fundamental cancer biology and improving molecular diagnostics and therapeutics, the role of the microenvironment during the initiation and progression of carcinogenesis is thought to be of critical importance (1, 2). The aim of this study was to establish an in vitro model based on a co-culture of healthy human fibroblasts (HFs) and human osteosarcoma cells (MG-63s) to simulate the microenvironment including tumor and healthy cells. The HFs and MG-63s were in vitro co-cultured for a period of time ranging from 24h to 7 days. Cell morphology and organization were studied by phase contrast microscopy while the expression of Human Cartilage Glycoprotein 39 (YKL-40), Vascular Endothelial Growth Factor (VEGF), Matrix Metalloprotease 1 (MMP1) and IL1 alpha was investigated by Real Time PCR and Western Blotting. To better correlate the role of YKL-40, VEGF, MMP1 and IL1 alpha, siRNA knockdown of YKL-40 in MG-63 cells was performed, and changes in protein expression in the co-cultures were analyzed. Results showed a characteristic disposition of tumor and healthy co-cultured cells in columns which are not visible in tumor and healthy cells grown singularly. The expression of YKL-40, VEGF and MMP1 significantly changed in co-cultured cells compared to HFs and MG-63s separately cultured. siRNA knockdown of YKL-40 was responsible of a down-regulation of VEGF expression on HFs, and an increase of IL1 alpha expression in MG-63s. These results suggest that the tumor microenvironment has an influence on the protein expression of the healthy surrounding tissues and the process of tumorigenicity

INIBITÓRIO COGNITIVO E SUA INFLUÊNCIA NA APRENDIZAGEM: um estudo de caso

Esse artigo é resultado do estágio clínico supervisionado durante a Pós-Graduação em Psicopedagogia Clínica e Institucional, que teve como eixo norteador o processo de ensino e aprendizagem e fatores que possam inibi-lo. Um dos fatores que pode exercer influência sobre esse processo denomi-na-se inibitório cognitivo, que está relacionado com a capacidade de inibir e selecionar pensamentos e memórias específicas e faz parte do controle inibi-tório, que envolve processos de controle do comportamento, da atenção e dos pensamentos/emoções. Assim, o objetivo geral do estudo foi diagnosti-car qual o tipo de problema ocorre com um menino de 7 anos de idade. Con-siderando o método que indica os meios técnicos da investigação, apropriou-se do método clínico, que apoia-se numa relação profunda entre pesquisador e pesquisado. Esse método indica que o sujeito possui um vínculo negativo com a aprendizagem, o qual reside em questões emocionais. Para aprender na escola a criança tem de exibir dois sistemas operativos: cognitivo e emo-cional, bem como as alterações na trajetória de desenvolvimento podem pro-vocar consequências e impactos graves na vida escolar, social, emocional e comportamental da criança

Tres nuevas especies del género Aphis Linnaeus (Hemiptera, Aphididae) viviendo sobre verbenáceas (Verbenaceae) en la Argentina

Se describen tres especies del género Aphis Linnaeus (Hemiptera, Aphididae, Aphidinae) a partir de hembras vivíparas ápteras y aladas recogidas sobre especies de Junellia, Lippia y Mulguraea (Verbenaceae) en localidades de la Argentina, respectivamente: Aphis junelliae González Rodríguez & Nieto Nafría sp. n., Aphis lippiae Ortego & Nieto Nafría sp. n., Aphis mulguraeae Nieto Nafría & Mier Durante sp. n. Se exponen las características que permiten diferenciar las nuevas especies entre sí y de las restantes especies de Aphis conocidas en América del Sur.Three new species belonging to the genus Aphis Linnaeus (Hemiptera, Aphididae, Aphidinae) are described from apterous and alate viviparous females collected on species of Junellia, Lippia and Mulguraea (Verbenaceae) in localities of Argentina; they respectively are: Aphis junelliae González Rodríguez & Nieto Nafría sp. n., Aphis lippiae Ortego & Nieto Nafría sp. n., Aphis mulguraeae Nieto Nafría & Mier Durante sp. n. Features that distinguish the new species between them and from other species of Aphis known in South America are exposed.EEA MendozaFil: González Rodríguez, Sandra. Universidad de León. Departamento de Biodiversidad y Gestión Ambiental (área de Zoología); EspañaFil: Mier Durante, Milagros Pilar. Universidad de León. Departamento de Biodiversidad y Gestión Ambiental (área de Zoología); EspañaFil: Ortego, Jaime. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Nieto Nafría, Juan Manuel. Universidad de León. Departamento de Biodiversidad y Gestión Ambiental (área de Zoología); Españ

Trypanosoma Cruzi: Parasite Persistence In Tissues In Chronic Chagasic Brazilian Patients.

Chagas disease in the chronic phase may develop into cardiac and/or digestive forms. The pathogenesis of the disease is not yet clear and studies have been carried out to elucidate the role of parasite persistence in affected organs. The aim of this study was to detect and quantify Trypanosoma cruzi in paraffin-embedded tissue samples from chronic patients using NPCR (nested polymerase chain reaction) and QPCR (quantitative polymerase chain reaction) methods. These results were correlated to anatomopathological alterations in the heart and gastrointestinal tract (GIT). Of the 23 patients studied, 18 presented the cardiac form and five presented the cardiodigestive form of Chagas disease. DNA samples were randomly isolated from formalin-fixed paraffin-embedded sections of heart and GIT tissue of 23 necropsies and were analyzed through NPCR amplification. T. cruzi DNA was detected by NPCR in 48/56 (85.7%) heart and 35/42 (83.3%) GIT samples from patients with the cardiac form. For patients with the cardiodigestive form, NPCR was positive in 12/14 (85.7%) heart and in 14/14 (100%) GIT samples. QPCR, with an efficiency of 97.6%, was performed in 13 samples (11 from cardiac and 2 from cardiodigestive form) identified previously as positive by NPCR. The number of T. cruzi copies was compared to heart weight and no statistical significance was observed. Additionally, we compared the number of copies in different tissues (both heart and GIT) in six samples from the cardiac form and two samples from the cardiodigestive form. The parasite load observed was proportionally higher in heart tissues from patients with the cardiac form. These results show that the presence of the parasite in tissues is essential to Chagas disease pathogenesis.10685-9

Ultrastructural changes in human gingival fibroblasts after exposure to 2-hydroxy-ethyl methacrylate

Polymerized resin-based materials are successfully utilized in medical applications. One drawback is the release of monomers from the matrix due to an incomplete polymerization or degradation processes. Released monomers can diffuse in the systemic circulation and induce adverse effects to biological tissues. Although there are many hypotheses about the induction of cell death by resin monomers, the underlying mechanisms are still under discussion. The aim of the study was to investigate the morphological modifications in human gingival fibroblasts exposed to 2-hydroxy-ethyl methacrylate (HEMA) to better elucidate the mechanism of cell death induced by resin monomers. Primary cultures of gingival fibroblasts were exposed to 3mM HEMA for 24 h, 72 h, 96 h. Morphological investigations were performed by scanning and transmission electron microscopy, while western blot for caspase-3 was carried out to verify apoptosis. Electron microscopy images showed deep changes in the cell surface and cytoplasm after 72 h and 96 h of HEMA treatment. Autophagic vesicles were easily observed just after 24 h. Cleaved caspase-3 was detected after 72 h of treatment. These findings suggest that resin based materials induced cell death by the cooperation of apoptosis and autophagy mechanisms. The understanding of these mechanisms will lead to the development of smart biomaterials without or with low adverse effects

Influence of a biomimetic gelatin porous scaffold in chondrogenic and osteogenic differentiation of mesenchymal stem cells

Recently, tissue engineering has merged with stem cell technology to develop new sources of transplantable material for injury or disease treatment. Eminently interesting, are bone and joint injuries/disorders because of the low self-regenerating capacity of the matrix secreting cells, particularly chondrocytes (1). Gelatin based scaffolds are considered to be a highly suitable 3D material for tissue regeneration, due to high biocompatibility and adaptation to native tissues. In the present study, the chondrogenic and osteogenic potential of a porous gelatin based scaffold (2), alone or in combination with hydroxyapatite crystals, was investigated in human mesenchymal stem cells. Cells were culture up to 4 weeks on the scaffold and on monolayer. MTT assay was performed to evaluate cell viability, light and transmission electron microscopy were carried out to demonstrate cell colonization inside the porous architecture of the biomaterial and scaffold adhesion. The expression of chondrogenic markers such as SOX9, collagen type II, aggregan and versican and osteogenic markers such as Collagen type I, Runx -2, osteopontin and bone matrix protein, were investigated by Real Time PCR. Results showed an high cell viability, adhesion and colonization of the scaffold. Real Time PCR data demonstrated an up-regulation of all the chondrogenic and osteogenic markers analyzed. In conclusion, gelatin porous scaffold provides an improved environment for chondrogenic and osteogenic differentiation of stem cells compared to cell monolayer culture system

Simultaneous monitoring of CMV and human herpesvirus 6 infections and diseases in liver transplant patients: one-year follow-up

OBJECTIVE: The aim of this study was to simultaneously monitoring cytomegalovirus and human herpesvirus 6 active infections using nested-polymerase chain reaction and, together with clinical findings, follow the clinical status of patients undergoing liver transplant. INTRODUCTION: The human β-herpesviruses, including cytomegalovirus and human herpesvirus 6, are ubiquitous among human populations. Active infections of human herpesvirus 6 and cytomegalovirus are common after liver transplantation, possibly induced and facilitated by allograft rejection and immunosuppressive therapy. Both viruses affect the success of the transplant procedure. METHODS: Thirty patients submitted to liver transplant at the Liver Transplant Unit, at the Gastro Center, State University of Campinas, SP, Brazil, were studied prospectively from six months to one year, nested-polymerase chain reaction for cytomegalovirus and human herpesvirus 6 DNA detections. Two or more consecutive positive nested-polymerase chain reaction were considered indicative of active infection. RESULTS: Active infection by cytomegalovirus was detected in 13/30 (43.3%) patients, median time to first cytomegalovirus detection was 29 days after transplantation (range: 0-99 days). Active infection by human herpesvirus 6 was detected in 12/30 (40%) patients, median time to first human herpesvirus 6 detection was 23.5 days after transplantation (range: 0-273 days). The time-related appearance of each virus was not statistically different (p = 0.49). Rejection of the transplanted liver was observed in 16.7% (5/30) of the patients. The present analysis showed that human herpesvirus 6 and/or cytomegalovirus active infections were frequent in liver transplant recipients at our center. CONCLUSIONS: Few patients remain free of betaherpesviruses after liver transplantation. Most patients presenting active infection with more than one virus were infected sequentially and not concurrently. Nested-polymerase chain reaction can be considered of limited value for clinically monitoring cytomegalovirus and human herpesvirus 6