SHAPES Marketplace: Transparency, Trust and Fair Competition in the Healthy Ageing Market.

Access to digital health and care solutions and services that promote healthy ageing, independent living, and ageing in place is limited due to significant market barriers and challenges. The SHAPES project addresses the challenge of ageing populations by developing a sociotechnical ecosystem comprising a variety of health and care digital solutions, tools and services to enable and facilitate active, independent, and healthy ageing at home. Within the SHAPES project, the SHAPES Marketplace serves as a one-stop-shop for digital solutions and services designed for the Silver Economy that target the smart and healthy ageing and independent living markets. Delivering a dynamic catalogue of health and care digital solutions and services, the Marketplace promotes a transparent expansion of a trusted market offer on digital solutions and services for healthy ageing and independent living on a pan-European scale, thereby preventing vendor lock-in and enhancing the agile and fair competitiveness of the health and care industry, particularly in Europe. This paper introduces the SHAPES Marketplace and considers its function as a market driver to raise awareness on the benefits and impact of health and care digital solutions and services, as well as to shape the healthy ageing market, upholding the Systems-Market for Assistive and Related Technologies (SMART) Thinking Matrix to stimulate transparency, trust and fair competition.

Essential role of CK2α for the interaction and stability of replication fork factors during DNA synthesis and activation of the S-phase checkpoint.

The ataxia telangiectasia mutated and Rad3-related (ATR)-CHK1 pathway is the major signalling cascade activated in response to DNA replication stress. This pathway is associated with the core of the DNA replication machinery comprising CDC45, the replicative MCM2-7 hexamer, GINS (altogether forming the CMG complex), primase-polymerase (POLε, -α, and -δ) complex, and additional fork protection factors such as AND-1, CLASPIN (CLSPN), and TIMELESS/TIPIN. In this study, we report that functional protein kinase CK2α is critical for preserving replisome integrity and for mounting S-phase checkpoint signalling. We find that CDC45, CLSPN and MCM7 are novel CK2α interacting partners and these interactions are particularly important for maintenance of stable MCM7-CDC45, ATRIP-ATR-MCM7, and ATR-CLSPN protein complexes. Consistently, cells depleted of CK2α and treated with hydroxyurea display compromised replisome integrity, reduced chromatin binding of checkpoint mediator CLSPN, attenuated ATR-mediated S-phase checkpoint and delayed recovery of stalled forks. In further support of this, differential gene expression analysis by RNA-sequencing revealed that down-regulation of CK2α accompanies global shutdown of genes that are implicated in the S-phase checkpoint. These findings add to our understanding of the molecular mechanisms involved in DNA replication by showing that the protein kinase CK2α is essential for maintaining the stability of the replisome machinery and for optimizing ATR-CHK1 signalling activation upon replication stress.

Implementation of a pan-European ecosystem and an interoperable platform for Smart and Healthy Ageing in Europe: An Innovation Action research protocol.

As life expectancy continues to increase in most EU Member States, smart technologies can help enable older people to continue living at home, despite the challenges accompanying the ageing process. The Innovation Action (IA) SHAPES 'Smart and Healthy Ageing through People Engaging in Supportive Systems' funded by the EU under the Horizon 2020 Research and Innovation Programme (grant agreement number 857159) attends to these topics to support active and healthy ageing and the wellbeing of older adults. This protocol article outlines the SHAPES project's objectives and aims, methods, structure, and expected outcomes. SHAPES seeks to build, pilot, and deploy a large-scale, EU-standardised interoperable, and scalable open platform. The platform will facilitate the integration of a broad range of technological, organisational, clinical, educational, and social solutions. SHAPES emphasises that the home is much more than a house-space; it entails a sense of belonging, a place and a purpose in the community. SHAPES creates an ecosystem - a network of relevant users and stakeholders - who will work together to scale-up smart solutions. Furthermore, SHAPES will create a marketplace seeking to connect demand and supply across the home, health and care services. Finally, SHAPES will produce a set of recommendations to support key stakeholders seeking to integrate smart technologies in their care systems to mediate care delivery. Throughout, SHAPES adopts a multidisciplinary research approach to establish an empirical basis to guide the development of the platform. This includes long-term ethnographic research and a large-scale pan-European campaign to pilot the platform and its digital solutions within the context of seven distinct pilot themes. The project will thereby address the challenges of ageing societies in Europe and facilitate the integration of community-based health and social care. SHAPES will thus be a key driver for the transformation of healthcare and social care services across Europe.

Phytochemicals in cancer and their effect on the PI3K/AKT-mediated cellular signalling.

Protein kinases belong to the largest family of enzymes controlling every aspect of cellular activity including gene expression, cell division, differentiation and metabolism. They are part of major intracellular signalling pathways. Hence, it is not surprising that they are involved in the development of major diseases such as cardiovascular disorders, diabetes, dementia and, most importantly, cancer when they undergo mutations, modifications and unbalanced expression. This review will explore the possibility to draw a connection between the application of natural phytochemicals and the treatment of cancer. We have chosen to focus on the PI3K/AKT cellular signalling pathway which has been shown to be a major target by natural compounds in cell cultures and animal models.

Shaping the Future of Digitally Enabled Health and Care.

People generally need more support as they grow older to maintain healthy and active lifestyles. Older people living with chronic conditions are particularly dependent on healthcare services. Yet, in an increasingly digital society, there is a danger that efforts to drive innovations in eHealth will neglect the needs of those who depend on healthcare the most-our ageing population. The SHAPES (Smart and Healthy Ageing through People Engaging in Supportive Systems) Innovation Action aims to create an open European digital platform that facilitates the provision of meaningful, holistic support to older people living independently. A pan-European pilot campaign will evaluate a catalogue of digital solutions hosted on the platform that have been specifically adapted for older people. 'Medicines control and optimisation' is one of seven themes being explored in the campaign and will investigate the impact of digital solutions that aim to optimise medicines use by way of fostering effective self-management, while facilitating timely intervention by clinicians based on remote monitoring and individualised risk assessments powered by artificial intelligence. If successful, the SHAPES Innovation Action will lead to a greater sense of self-sufficiency and empowerment in people living with chronic conditions as they grow older.

Down-Regulation of CK2α Leads toUp-Regulation of the Cyclin-Dependent Kinase Inhibitor p27KIP1 in Conditions Unfavorable for the Growth of Myoblast Cells.

BACKGROUND/AIMS: Compelling evidence indicates that CK2α, which is one of the two catalytic isoforms of protein kinase CK2, is required for cell viability and plays an important role in cell proliferation and differentiation. While much is known on CK2 in the context of disease states, particularly cancer, its critical role in non-cancerous cell growth has not been extensively investigated. METHODS: In the present study, we have employed a cell line derived from rat heart with inducible down-regulation of CK2α and CK2α-knockout mouse tissue to identify CK2-mediated molecular mechanisms regulating cell growth. For this, we have performed Incucyte® live-cell analysis and applied flow cytometry, western blot, immunoprecipitation, immunohistochemistry, RT-qPCR and luciferase-based methods. RESULTS: Here, we show that lack of CK2α results in significantly delayed cell cycle progression through G1, inhibition of cyclin E-CDK2 complex, decreased phosphorylation of Rb protein at S795, and inactivation of E2F transcription factor. These events are accompanied by nuclear accumulation and up-regulation of the cyclin-dependent kinase inhibitor p27KIP1 in cells and CK2α-knockout mouse tissues. We found that increased levels of p27KIP1 are mainly attributable to post-translational modifications, namely phosphorylation at S10 and T197 amino acid residues catalyzed by Dyrk1B and AMPK, respectively, as silencing of FoxO3A transcription factor, which activates CDKN1B the gene coding for p27KIP1, does not result in markedly decreased expression levels of the corresponding protein. Interestingly, simultaneous silencing of CK2α and p27KIP1 significantly impairs cell cycle progression without increasing cell death. CONCLUSION: Taken together, our study sheds light on the molecular mechanisms controlling cell cycle progression through G1 phase when myoblasts proliferation potential is impaired by CK2α depletion. Our results suggest that elevated levels of p27KIP1, which follows CK2α depletion, contribute to delay the G1-to-S phase transition. Effects seen when p27KIP1 is down-regulated are independent of CK2α and reflect the protective role exerted by p27KIP1 under unfavorable cell growth conditions.

Role of Protein Kinase CK2 in Aberrant Lipid Metabolism in Cancer.

Uncontrolled proliferation is a feature defining cancer and it is linked to the ability of cancer cells to effectively adapt their metabolic needs in response to a harsh tumor environment. Metabolic reprogramming is considered a hallmark of cancer and includes increased glucose uptake and processing, and increased glutamine utilization, but also the deregulation of lipid and cholesterol-associated signal transduction, as highlighted in recent years. In the first part of the review, we will i) provide an overview of the major types of lipids found in eukaryotic cells and their importance as mediators of intracellular signaling pathways ii) analyze the main metabolic changes occurring in cancer development and the role of oncogenic signaling in supporting aberrant lipid metabolism and iii) discuss combination strategies as powerful new approaches to cancer treatment. The second part of the review will address the emerging role of CK2, a conserved serine/threonine protein kinase, in lipid homeostasis with an emphasis regarding its function in lipogenesis and adipogenesis. Evidence will be provided that CK2 regulates these processes at multiple levels. This suggests that its pharmacological inhibition combined with dietary restrictions and/or inhibitors of metabolic targets could represent an effective way to undermine the dependency of cancer cells on lipids to interfere with tumor progression.

Ensino de comportamento verbal elementar por exemplares múltiplos em crianças com autismo / Teaching elementary verbal behavior by multiple exemplar in children with autism / Enseñanza del comportamiento verbal elemental mediante múltiples copias en niños con autismo

No escopo da investigação de sob quais condições ocorre a aprendizagem e a emergência de operantes verbais, diferentes condições de ensino têm sido planejadas. Considerando que a população com Transtorno do Espectro Autista (TEA) apresenta frequentemente um repertório de operantes verbais ausente ou fracamente estabelecido, é necessário o estudo do planejamento de intervenções sistemáticas desses repertórios com condições de favorecer não só a aquisição de vocabulário, mas também o seu potencial gerativo de novas funções verbais. O Multiple Exemplar Instruction (MEI) é uma estrutura de ensino que tem demonstrado resultados promissores pela sua capacidade de estabelecer relações entre comportamentos de ouvinte e de falante e gerar novas respostas verbais. O objetivo deste estudo foi verificar os efeitos do MEI sobre o estabelecimento e integração entre os repertórios de ouvinte e de falante (ecoico, tato e mando). Participaram duas crianças com TEA, com idades de 7 e 8 anos e cuja comunicação era muito restrita e baseada em trocas de figuras. O ensino adotou três conjuntos com três estímulos cada. O ensino com cada conjunto foi realizado separadamente. Ora o ensino consistia no treino de ouvinte baseado em seleção, ora tentativas de ouvinte, ecoico, tato e mando, eram apresentadas de forma rotativa. Sondas múltiplas intercalaram os ensinos e verificaram os efeitos destes sobre o repertório de falante com os demais conjuntos. Os resultados demonstraram um aumento na emissão de respostas de ouvir e falar após o ensino por MEI para os dois participantes, ambos com repertório verbal restrito, mas o procedimento foi mais efetivo para uma das crianças...(AU)

In the scope of research about under what conditions the learning and emergency of verbal operants occurs, different teaching conditions have been planned. Considering that the population with Autistic Spectrum Disorder (ASD) often has a repertoire of verbal operants absent or poorly established, it is necessary to study the planning of systematic interventions in these repertoires with conditions to favor not only vocabulary acquisition, but also their generative potential of new verbal functions. The Multiple Exemplar Instruction (MEI) is a teaching structure that has shown promising results for its ability to establish relationships between listening and speaker behaviors and to generate new verbal responses. The objective of this study was to verify the effects of the MEI on the establishment and integration between the listener repertoires and the speaker (echoic, tact and mand). Two children with ASD, aged between 7 and 8 years old, participated in the study, whose communication was very restricted and based on exchanges of figures. The teaching adopted three sets with three stimuli each. Teaching with each set was carried out separately. The teaching consisted of the training of listener based on selection, sometimes attempts of listener, echoic, tact and mand, were presented in a rotating way. Multiple probes intercalated the teachings and verified the effects of these on the repertoire of speaker with the other sets. The results showed an increase in the emission of listening and speaking responses after MEI teaching for the two participants, both with restricted verbal repertoire, but the procedure was more effective for one of the children...(AU)

El el ámbito de la investigación de en qué condiciones se produce el aprendizaje y la aparición de operantes verbales, se han planificado diferentes condiciones de enseñanza. Teniendo en cuenta que la población con Trastorno del Espectro Autista (TEA) con frecuencia presenta un repertorio de operantes verbales ausentes o débilmente establecidos, es necesario estudiar la planificación de intervenciones sistemáticas en estos repertorios con condiciones para favorecer no solo la adquisición de vocabulario, sino también su potencial generativo de nuevas funciones verbales. La Instrucción Ejemplar Múltiple (MEI) es una estructura de enseñanza que ha mostrado resultados prometedores por su capacidad para establecer relaciones entre los comportamientos del oyente y el hablante y generar nuevas respuestas verbales. El objetivo de este estudio fue verificar los efectos del MEI en el establecimiento e integración entre los repertorios de oyentes y hablantes (eco, tacto y comando). Participaron dos niños con TEA, de 7 y 8 años, cuya comunicación era muy restringida y basada en intercambios de figuras. La enseñanza adoptó tres conjuntos con tres estímulos cada uno. La enseñanza con cada conjunto se llevó a cabo por separado. Algunas veces la enseñanza consistía en entrenar al oyente en base a la selección, a veces los intentos de escuchar, eco, tacto y comando, se presentaban de manera rotativa. Múltiples sondas intercalan las enseñanzas y verifican sus efectos en el repertorio de los hablantes con los otros grupos. Los resultados mostraron un aumento en la emisión de respuestas para escuchar y hablar después de la enseñanza de MEI para ambos participantes, ambos con repertorio verbal restringido, pero el procedimiento fue más efectivo para uno de los niños...(AU)

Ensino de Ecoico em Pessoas com Transtorno do Espectro Autista: Revisão Sistemática de Literatura / Teaching of Echoic in People with Autism Spectrum Disorders: Systematic Review of Literature

RESUMO: A literatura tem demonstrado uma grande lacuna em estudos com o objetivo de estabelecer ecoico em pessoas com Transtorno do Espectro Autista (TEA). Assim sendo, este trabalho teve por objetivo identificar o acervo de pesquisas direcionadas para o ensino de ecoico em indivíduos com TEA. A revisão foi pautada no modelo Preferred Reporting Items for Systematics Review and Meta-Analysis (PRISMA). A busca, de todas as ocorrências, sem limite de tempo, até o ano de 2018, foi realizada nas bases de dados Web of Science, Pubmed, Scopus, ERIC e PsicINFO. Adotaram-se as palavras-chave e marcadores boleanos autism [or] autist [or] autistic [and] echoic [and] verbal behavior. Os critérios de inclusão foram artigos em português, inglês e espanhol que estabeleceram ecoico como variável dependente em intervenções com pessoas com TEA, apresentar delineamento experimental de sujeito único e demonstrar os dados da aquisição do ecoico no decorrer das sessões de ensino. Como resultados, foram encontrados 338 artigos e, com a aplicação dos critérios de inclusão, foram selecionados três. Os resultados corroboraram os dados da literatura que apontam o déficit de estudos com objetivo de estabelecer ecoico como alvo principal de ensino em pessoas com TEA. A maior parte dos estudos adota o ecoico como prompt para o estabelecimento de outro comportamento verbal. Ainda que escassos, os artigos apontam os efeitos de procedimentos distintos sobre a aquisição do comportamento ecoico em pessoas com TEA e com repertórios iniciais diversificados.

ABSTRACT: Literature has shown a large gap in studies aiming to establish an echoic in people with Autism Spectrum Disorder (ASD). Thus, the objective of this study was to identify the collection of research aimed at teaching echoic in individuals with ASD. The review was based on the Preferred Reporting Items for Systematics Review and Meta-Analysis (PRISMA) model. The search, of all occurrences, without time limit, until the year 2018, was performed in the Web of Science, Pubmed, Scopus, ERIC and PsichINFO database. The keywords and boolean operators autism [or] autist [or] autistic [and] echoic [and] verbal behavior were adopted. The inclusion criteria were articles in Portuguese, English and Spanish that established echoic as a dependent variable in interventions with people with ASD, present a single subject experimental design and demonstrate the data of the echoic acquisition during the teaching sessions. As a result, 338 articles were found and, with the application of the inclusion criteria, three were selected. The results corroborate the data in the literature that point out the deficit of studies to establish echoic as the main target of teaching in people with ASD. Most studies adopt echoic as a prompt for establishing other verbal behavior. Even if scarce, the articles indicate the effects of different procedures under the acquisition of the echoic behavior in people with ASD and with diversified initial repertories.

Down-regulation of CK2α correlates with decreased expression levels of DNA replication minichromosome maintenance protein complex (MCM) genes.

Protein kinase CK2 is a serine/threonine kinase composed of two catalytic subunits (CK2α and/or CK2α') and two regulatory subunits (CK2ß). It is implicated in every stage of the cell cycle and in the regulation of various intracellular pathways associated with health and disease states. The catalytic subunits have similar biochemical activity, however, their functions may differ significantly in cells and in vivo. In this regard, homozygous deletion of CK2α leads to embryonic lethality in mid-gestation potentially due to severely impaired cell proliferation. To determine the CK2α-dependent molecular mechanisms that control cell proliferation, we established a myoblast-derived cell line with inducible silencing of CK2α and carried out a comprehensive RNA-Seq analysis of gene expression. We report evidence that CK2α depletion causes delayed cell cycle progression through the S-phase and defective response to replication stress. Differential gene expression analysis revealed that the down-regulated genes were enriched in pathways implicated in cell cycle regulation, DNA replication and DNA damage repair. Interestingly, the genes coding for the minichromosome maintenance proteins (MCMs), which constitute the core of the replication origin recognition complex, were among the most significantly down-regulated genes. These findings were validated in cells and whole mouse embryos. Taken together, our study provides new evidence for a critical role of protein kinase CK2 in controlling DNA replication initiation and the expression levels of replicative DNA helicases, which ensure maintenance of proliferative potential and genome integrity in eukaryotic cells.

Natural Compounds and Derivatives as Ser/Thr Protein Kinase Modulators and Inhibitors.

The need for new drugs is compelling, irrespective of the disease. Focusing on medical problems in the Western countries, heart disease and cancer are at the moment predominant illnesses. Owing to the fact that ~90% of all 21,000 cellular proteins in humans are regulated by phosphorylation/dephosphorylation it is not surprising that the enzymes catalysing these reactions (i.e., protein kinases and phosphatases, respectively) have attracted considerable attention in the recent past. Protein kinases are major team players in cell signalling. In tumours, these enzymes are found to be mutated disturbing the proper function of signalling pathways and leading to uncontrolled cellular growth and sustained malignant behaviour. Hence, the search for small-molecule inhibitors targeting the altered protein kinase molecules in tumour cells has become a major research focus in the academia and pharmaceutical companies.

Delivery of proteins encapsulated in chitosan-tripolyphosphate nanoparticles to human skin melanoma cells.

We have successfully encapsulated two proteins, bovine serum albumin (BSA) and p53, in chitosan-tripolyphosphate (TPP) nanoparticles at various pH values from 5.5 to 6.5 and delivered the particles to human melanoma cells. The particles have diameters ranging from 180 nm to 280 nm and a zeta potential of +15 to + 40 mV. Cellular uptake of the particles by human skin melanoma cells was evaluated by: (i) fluorescence microscopy and (ii) gel electrophoresis showing that FITC-labeled BSA and p53 could be recovered in the soluble cell fraction after lysis of the cells. Our data also show that the highest cellular uptake takes place at the lowest pH as the particles have the highest positive charge under these conditions. The method we describe appears to be a general method for delivery of proteins to cells using chitosan-TPP nanoparticles as a drug delivery system, since structurally unrelated proteins such as BSA and p53 with different isoelectrical points can be encapsulated in the chitosan-TPP nanoparticles and be effectively internalized by the cells.

Ensino de repertórios requisitos e os efeitos sobre comportamentos incompatíveis com aprendizagem em crianças com Transtorno do Espectro Autista / The teaching of repertoires and its effects on behavior incompatible with learning in children with on the Autism Spectrum Disorder / Enseñanza de repertorios requisitos y los efectos sobre comportamientos incompatibles con el aprendizaje en niños con Trastorno del Espectro Autista

Pessoas com Transtorno do Espectro Autista (TEA) podem apresentar déficits significativos no repertório básico requisito para receber programas de ensino e se engajar com frequência em comportamentos repetitivos e inadequados. Considerando-se que tais comportamentos são incompatíveis com o ensino e resultam em consequências negativas, sejam sociais e/ou acadêmicas, esta pesquisa teve por objetivo ensinar sistematicamente operantes requisitos, como sentar, permanecer sentado, olhar, imitar, imitação generalizada e rastreamento visual para duas crianças com TEA, e verificar o efeito do procedimento sobre os comportamentos inadequados e estereotipias. Os resultados demonstraram que um participante aprendeu todos os comportamentos alvo de ensino enquanto o outro não atingiu o critério de aprendizagem em imitação. Houve redução dos comportamentos incompatíveis com a aprendizagem, logo após a introdução das contingências para os comportamentos de ensino, para ambos. Os resultados demonstram a aquisição de repertórios requisitos importantes em crianças com TEA severo e demonstram que a redução de comportamentos incompatíveis com aprendizagem pode ocorrer mediante ensino de comportamentos desejados.

People with Autism Spectrum Disorder (ASD) may have significant deficits in basic repertoire to accept teaching programs and may engage frequently in repetitive and inappropriate behaviors. Considering that such behavior is incompatible with learning and results in negative consequences, whether social and/or academic, this study aimed to systematically teach operational requirements such as sit, stay seated, look, imitation, generalized imitation and visual tracking to two children with ASD, and check the effect of the procedure on the inappropriate and stereotypical behavior. The results demonstrated that one participant learned all the target behavior that was taught whilst the other participant did not reach the learning criterion in imitation. There was a reduction of behavior incompatible with learning after the introduction of the contingencies for teaching behaviors for both. The results demonstrate the acquisition of important repertoire requirements in children with severe ASD and show that the rebate of incompatible behavior with learning is possible through the teaching of desired behaviors.

Las personas con Trastorno del Espectro Autista (TEA) pueden presentar déficit significativo en el repertorio básico necesario para recibir programas de aprendizaje formal, presentando con frecuencia comportamientos repetitivos e inadecuados. Considerando que tales comportamientos son incompatibles con el sistema formal de educación, trayendo consecuencias negativas, ya sean sociales e/o académicas. La presente investigación tuvo como objetivo enseñar sistemáticamente operantes requisitos, como sentarse, permanecer sentado, mirar, imitar, imitación generalizada y rastreo visual para dos niños con TEA, y verificar el efecto del procedimiento sobre los comportamientos inadecuados y estereotipias. Los resultados demostraron que uno de los participantes aprendió todos los comportamientos propuestos mientras que el otro no alcanzó el criterio de aprendizaje en el quesito imitación. En ambos participantes hubo una reducción de los comportamientos incompatibles con el aprendizaje después de la introducción de las contingencias para los comportamientos enseñados. Los resultados demuestran la adquisición de repertorios necesarios en niños con TEA grave y demuestran que la reducción de comportamientos incompatibles con el aprendizaje puede ocurrir mediante la enseñanza de comportamientos deseados.

Blocking of an intronic splicing silencer completely rescues IKBKAP exon 20 splicing in familial dysautonomia patient cells.

Familial dysautonomia (FD) is a severe genetic disorder causing sensory and autonomic dysfunction. It is predominantly caused by a c.2204+6T>C mutation in the IKBKAP gene. This mutation decreases the 5' splice site strength of IKBKAP exon 20 leading to exon 20 skipping and decreased amounts of full-length IKAP protein. We identified a binding site for the splicing regulatory protein hnRNP A1 downstream of the IKBKAP exon 20 5'-splice site. We show that hnRNP A1 binds to this splicing regulatory element (SRE) and that two previously described inhibitory SREs inside IKBKAP exon 20 are also bound by hnRNP A1. Knockdown of hnRNP A1 in FD patient fibroblasts increases IKBKAP exon 20 inclusion demonstrating that hnRNP A1 is a negative regulator of IKBKAP exon 20 splicing. Furthermore, by mutating the SREs in an IKBKAP minigene we show that all three SREs cause hnRNP A1-mediated exon repression. We designed splice switching oligonucleotides (SSO) that blocks the intronic hnRNP A1 binding site, and demonstrate that this completely rescues splicing of IKBKAP exon 20 in FD patient fibroblasts and increases the amounts of IKAP protein. We propose that this may be developed into a potential new specific treatment of FD.

Bone-Marrow-Derived Mesenchymal Stromal Cells (MSC) from Diabetic and Nondiabetic Rats Have Similar Therapeutic Potentials / Células Estromais Mesenquimais (CEM) Derivadas de Medula Óssea de Ratos com e sem Diabetes têm Potencial Terapêutico Similar

Abstract Background: Diabetes mellitus is a severe chronic disease leading to systemic complications, including cardiovascular dysfunction. Previous cell therapy studies have obtained promising results with the use bone marrow mesenchymal stromal cells derived from healthy animals (MSCc) in diabetes animal models. However, the ability of MSC derived from diabetic rats to improve functional cardiac parameters is still unknown. Objectives: To investigate whether bone-marrow-derived MSC from diabetic rats (MSCd) would contribute to recover metabolic and cardiac electrical properties in other diabetic rats. Methods: Diabetes was induced in Wistar rats with streptozotocin. MSCs were characterized by flow cytometry, morphological analysis, and immunohistochemistry. Cardiac electrical function was analyzed using recordings of ventricular action potential. Differences between variables were considered significant when p < 0.05. Results: In vitro properties of MSCc and MSCd were evaluated. Both cell types presented similar morphology, growth kinetics, and mesenchymal profile, and could differentiate into adipogenic and osteogenic lineages. However, in an assay for fibroblast colony-forming units (CFU-F), MSCd formed more colonies than MSCc when cultured in expansion medium with or without hydrocortisone (1 µM). In order to compare the therapeutic potential of the cells, the animals were divided into four experimental groups: nondiabetic (CTRL), diabetic (DM), diabetic treated with MSCc (DM + MSCc), and diabetic treated with MSCd (DM + MSCd). The treated groups received a single injection of MSC 4 weeks after the development of diabetes. MSCc and MSCd controlled hyperglycemia and body weight loss and improved cardiac electrical remodeling in diabetic rats. Conclusions: MSCd and MSCc have similar in vitro properties and therapeutic potential in a rat model of diabetes induced with streptozotocin.

Resumo Fundamentos: O diabetes mellitus é uma doença crônica grave que leva a complicações sistêmicas, como a disfunção cardiovascular. Estudos anteriores de terapia celular obtiveram resultados promissores com utilização de células estromais mesenquimais (CEM) derivadas de medula óssea de animais saudáveis (CEMc) em modelos de animais diabéticos. No entanto, a capacidade das CEM derivadas de ratos diabéticos em melhorar parâmetros cardíacos funcionais é ainda desconhecida. Objetivos: Investigar se CEM derivadas de medula óssea de ratos diabéticos (CEMd) poderiam contribuir para a recuperação metabólica e de propriedades elétricas cardíacas em outros ratos também com diabetes. Métodos: O diabetes foi induzido em ratos Wistar com estreptozotocina. As CEM foram caracterizadas por citometria de fluxo, análise morfológica e imunohistoquímica. A função elétrica cardíaca foi analisada através de registro do potencial de ação ventricular. As diferenças entre as variáveis foram consideradas significativas quando p < 0,05. Resultados: As propriedades in vitro das CEMc e CEMd foram avaliadas. Ambos os tipos celulares apresentaram morfologia, cinética de crescimento e perfil mesenquimal semelhante, e puderam ser diferenciadas em linhagens adipogênica e osteogênica. No entanto, em ensaios para unidades formadoras de colônias de fibroblastos (UFC-F), as CEMd formaram mais colônias em comparação às CEMc quando cultivadas em meio com ou sem hidrocortisona (1 µM). Para comparar o potencial terapêutico das células, os animais foram divididos em quatro grupos experimentais: não diabéticos (CTRL), diabéticos (DM), diabéticos tratados com CEMc (DM + CEMc) e diabéticos tratados com CEMd (DM + CEMd). Os grupos tratados receberam uma única injeção de CEM 4 semanas após o estabelecimento do diabetes. Ambas CEMc e CEMd controlaram a hiperglicemia e a perda de peso corporal e melhoraram o remodelamento elétrico cardíaco em ratos com diabetes. Conclusão: As CEMd e CEMc possuem propriedades in vitro e potencial terapêutico semelhante em um modelo de rato com diabetes induzido por estreptozotocina. (Arq Bras Cardiol. 2017; 109(6):579-589)

Protein kinase CK2 regulates redox homeostasis through NF-κB and Bcl-xL in cardiomyoblasts.

Oxygen consumption is particularly elevated in cardiac cells as they are equipped with a large number of mitochondria and high levels of respiratory chain components. Consequently, production of reactive oxygen species (ROS) is tightly controlled as an imbalance in redox reactions can lead to irreversible cellular damage. siRNA-mediated down-regulation of protein kinase CK2 has been implicated in the accumulation of ROS in cells. The present study was undertaken in order to investigate the role of CK2 in redox homeostasis in cardiomyoblasts. We found that inhibition or silencing of CK2 causes elevated levels of ROS, notably superoxide radical, and this is accompanied by suppression of NF-κB transcriptional activity and mitochondrial dysfunction. We show that CK2 regulates the expression of manganese superoxide dismutase, the enzyme catalyzing the dismutation of superoxide, in cancer cells but not in cardiomyoblasts. Furthermore, we report evidence that impaired expression of CK2 results in destabilization of the Bcl-2 mammalian homolog Bcl-xL, which is known to stabilize the mitochondrial membrane potential, through a mechanism involving disruption of the chaperone function of heat shock protein 90. Analysis of differential mRNA expression related to oxidative stress revealed that CK2 silencing caused a statistically significant deregulation of four genes associated with the oxidative damage, i.e., Fmo2, Ptgs1, Dhcr24, and Ptgs2. Overall, the results reported here are consistent with the notion that CK2 plays a role in conferring protection against oxidative stress by positively regulating pro-survival signaling molecules and the protein folding machinery in cardiomyoblasts.

The small-molecule kinase inhibitor D11 counteracts 17-AAG-mediated up-regulation of HSP70 in brain cancer cells.

Many types of cancer express high levels of heat shock proteins (HSPs) that are molecular chaperones regulating protein folding and stability ensuring protection of cells from potentially lethal stress. HSPs in cancer cells promote survival, growth and spreading even in situations of growth factors deprivation by associating with oncogenic proteins responsible for cell transformation. Hence, it is not surprising that the identification of potent inhibitors of HSPs, notably HSP90, has been the primary research focus, in recent years. Exposure of cancer cells to HSP90 inhibitors, including 17-AAG, has been shown to cause resistance to chemotherapeutic treatment mostly attributable to induction of the heat shock response and increased cellular levels of pro-survival chaperones. In this study, we show that treatment of glioblastoma cells with 17-AAG leads to HSP90 inhibition indicated by loss of stability of the EGFR client protein, and significant increase in HSP70 expression. Conversely, co-treatment with the small-molecule kinase inhibitor D11 leads to suppression of the heat shock response and inhibition of HSF1 transcriptional activity. Beside HSP70, Western blot and differential mRNA expression analysis reveal that combination treatment causes strong down-regulation of the small chaperone protein HSP27. Finally, we demonstrate that incubation of cells with both agents leads to enhanced cytotoxicity and significantly high levels of LC3-II suggesting autophagy induction. Taken together, results reported here support the notion that including D11 in future treatment regimens based on HSP90 inhibition can potentially overcome acquired resistance induced by the heat shock response in brain cancer cells.

Unraveling Chagas disease transmission through the oral route: Gateways to Trypanosoma cruzi infection and target tissues.

Oral transmission of Trypanosoma cruzi, the causative agent of Chagas disease, is the most important route of infection in Brazilian Amazon and Venezuela. Other South American countries have also reported outbreaks associated with food consumption. A recent study showed the importance of parasite contact with oral cavity to induce a highly severe acute disease in mice. However, it remains uncertain the primary site of parasite entry and multiplication due to an oral infection. Here, we evaluated the presence of T. cruzi Dm28c luciferase (Dm28c-luc) parasites in orally infected mice, by bioluminescence and quantitative real-time PCR. In vivo bioluminescent images indicated the nasomaxillary region as the site of parasite invasion in the host, becoming consistently infected throughout the acute phase. At later moments, 7 and 21 days post-infection (dpi), luminescent signal is denser in the thorax, abdomen and genital region, because of parasite dissemination in different tissues. Ex vivo analysis demonstrated that the nasomaxillary region, heart, mandibular lymph nodes, liver, spleen, brain, epididymal fat associated to male sex organs, salivary glands, cheek muscle, mesenteric fat and lymph nodes, stomach, esophagus, small and large intestine are target tissues at latter moments of infection. In the same line, amastigote nests of Dm28c GFP T. cruzi were detected in the nasal cavity of 6 dpi mice. Parasite quantification by real-time qPCR at 7 and 21 dpi showed predominant T. cruzi detection and expansion in mouse nasal cavity. Moreover, T. cruzi DNA was also observed in the mandibular lymph nodes, pituitary gland, heart, liver, small intestine and spleen at 7 dpi, and further, disseminated to other tissues, such as the brain, stomach, esophagus and large intestine at 21 dpi. Our results clearly demonstrated that oral cavity and adjacent compartments is the main target region in oral T. cruzi infection leading to parasite multiplication at the nasal cavity.

Ensino de comportamento verbal por múltiplos exemplares em uma criança com desordem do espectro da neuropatia auditiva: estudo de caso / Verbal behavior teaching by multiple exemplars in a child with auditory neuropathy spectrum disorder: a case study

RESUMO A Desordem do Espectro da Neuropatia Auditiva (DENA) é caracterizada pela dificuldade no estabelecimento do comportamento verbal nas modalidades de ouvinte e de falante, por uma dessincronia na condução nervosa da estimulação sonora recebida, ocasionando perdas auditivas. O implante coclear tem sido uma alternativa que estabiliza a detecção sonora, porém, discriminar e reconhecer o que se ouve e estabelecer relações com a fala irá requerer aprendizagem. Um procedimento de reconhecida eficácia no estabelecimento de repertórios verbais em crianças com pouca ou nenhuma linguagem é o Multiple Exemplar Instruction (MEI). O objetivo deste estudo foi verificar a replicabilidade dos resultados obtidos com o MEI com outras populações no ensino e integração entre os repertórios de ouvir e de falar em uma criança de seis anos com DENA e implante coclear bilateral. O ensino foi conduzido com três conjuntos de estímulos, e consistiu na apresentação rotativa de respostas de ouvinte, baseadas na seleção de figuras mediante palavra ditada e, de falante, após uma palavra ditada (ecoico) e mediante a nomeação de uma figura (tato). Repetidas sondas avaliaram o desempenho nos conjuntos de estímulos. Ainda que com alguma variabilidade no responder, as respostas de seleção foram estabelecidas primeiro e as respostas de falante, gradativamente, ao longo de sucessivas sessões. As condições sob as quais o repertório de falante pode ser refinado e a generalização dos resultados para outros participantes com DENA devem ser investigadas.

ABSTRACT Auditory Neuropathy Spectrum Disorder (ANSD) is characterized by a difficulty in establishing verbal behavior in the speaker and listener modalities due to an asynchrony in the nervous conduction of sound stimuli, leading to hearing loss. Cochlear implants are an alternative that stabilizes sound detection; however, discriminating and recognizing what is heard and establishing relations to speech requires learning. A procedure with recognized efficacy in establishing verbal repertoires in children with little or no language is Multiple Exemplar Instruction (MEI). The objective of this study was to verify replicability of results obtained from MEI with other populations for teaching and integrating listener and speaker behaviors on a six year-old child with ANSD and bilateral cochlear implants. The study was conducted with three sets of stimuli and consisted in a rotated presentation of listener responses, based on figure selection from dictated words, and of speaker responses, after a dictated word (echoic) and figure naming (tact). Repeated probes evaluated performance on the set of stimuli. Even though there is some variability in responding, selection responses were established first and speaker responses occurred gradually throughout successive sessions. The conditions under which a speaker’s repertoire may be refined, and generalization of these results to other participants with ANSD should be further investigated.

D11-Mediated Inhibition of Protein Kinase CK2 Impairs HIF-1α-Mediated Signaling in Human Glioblastoma Cells.

Compelling evidence indicates that protein kinase CK2 plays an important role in many steps of cancer initiation and progression, therefore, the development of effective and cell-permeable inhibitors targeting this kinase has become an important objective for the treatment of a variety of cancer types including glioblastoma. We have recently identified 1,3-dichloro-6-[(E)-((4-methoxyphenyl)imino)methyl]dibenzo(b,d)furan-2,7-diol (D11) as a potent and selective inhibitor of protein kinase CK2. In this study, we have further characterized this compound and demonstrated that it suppresses CK2 kinase activity by mixed type inhibition (KI 7.7 nM, KI' 42 nM). Incubation of glioblastoma cells with D11 induces cell death and upon hypoxia the compound leads to HIF-1α destabilization. The analysis of differential mRNA expression related to human hypoxia signaling pathway revealed that D11-mediated inhibition of CK2 caused strong down-regulation of genes associated with the hypoxia response including ANGPTL4, CA9, IGFBP3, MMP9, SLC2A1 and VEGFA. Taken together, the results reported here support the notion that including D11 in future treatment regimens might turn out to be a promising strategy to target tumor hypoxia to overcome resistance to radio- and chemotherapy.