Transcriptional Dysregulation and Impaired Neuronal Activity in FMR1 Knock-Out and Fragile X Patients' iPSC-Derived Models.

Fragile X syndrome (FXS) is caused by a repression of the FMR1 gene that codes the Fragile X mental retardation protein (FMRP), an RNA binding protein involved in processes that are crucial for proper brain development. To better understand the consequences of the absence of FMRP, we analyzed gene expression profiles and activities of cortical neural progenitor cells (NPCs) and neurons obtained from FXS patients' induced pluripotent stem cells (IPSCs) and IPSC-derived cells from FMR1 knock-out engineered using CRISPR-CAS9 technology. Multielectrode array recordings revealed in FMR1 KO and FXS patient cells, decreased mean firing rates; activities blocked by tetrodotoxin application. Increased expression of presynaptic mRNA and transcription factors involved in the forebrain specification and decreased levels of mRNA coding AMPA and NMDA subunits were observed using RNA sequencing on FMR1 KO neurons and validated using quantitative PCR in both models. Intriguingly, 40% of the differentially expressed genes were commonly deregulated between NPCs and differentiating neurons with significant enrichments in FMRP targets and autism-related genes found amongst downregulated genes. Our findings suggest that the absence of FMRP affects transcriptional profiles since the NPC stage, and leads to impaired activity and neuronal differentiation over time, which illustrates the critical role of FMRP protein in neuronal development.

CNS-selective noncompetitive cholinesterase inhibitors derived from the natural piperidine alkaloid (-)-spectaline.

LASSBio-767 [(-)-3-O-acetyl-spectaline] and LASSBio-822 [(-)-3-O-tert-Boc-spectaline] were recently described as cholinesterase inhibitors derived from the natural piperidine alkaloid (-)-spectaline, obtained from the flowers of Senna spectabilis (Fabaceae). We investigated their mechanism of inhibition of acetylcholinesterase and their efficacy in reversing scopolamine-induced amnesia. Competition assays with the substrate acetylthiocholine showed a concentration-dependent reduction in rat brain cholinesterase Vmax without changes in apparent Km. The kinetic data for LASSBio-767 and LASSBio-822 were best fit by a model of simple linear noncompetitive inhibition with Ki of 6.1 microM and 7.5 microM, respectively. A dilution assay showed a fast and complete reversal of inhibition, independent of incubation time. Simulated docking of the compounds into the catalytic gorge of Torpedo acetylcholinesterase showed interactions with the peripheral anionic site, but not with the catalytic triad. Anti-amnestic effects in mice were assessed in a step-down passive avoidance test and in the Morris water maze 30 min after injection of scopolamine (1 mg/kg i.p.). Saline, LASSBio-767, or LASSBio-822 was administered 15 min before scopolamine. Both compounds reversed the scopolamine-induced reduction in step-down latency at 0.1 mg/kg i.p. LASSBio-767 reversed scopolamine-induced changes in water maze escape latency at 1 mg/kg i.p. or p.o., while its cholinergic side effects were absent or mild up to 30 mg/kg i.p. (LD50 above 100 mg/kg i.p.). Thus, the (-)-spectaline derivatives are potent cholinergic agents in vivo, with a unique profile combining noncompetitive cholinesterase inhibition and CNS selectivity, with few peripheral side effects.

Improvement in the production of the human recombinant enzyme N-acetylgalactosamine-6-sulfatase (rhGALNS) in Escherichia coli using synthetic biology approaches.

Previously, we demonstrated production of an active recombinant human N-acetylgalactosamine-6-sulfatase (rhGALNS) enzyme in Escherichia coli as a potential therapeutic alternative for mucopolysaccharidosis IVA. However, most of the rhGALNS produced was present as protein aggregates. Here, several methods were investigated to improve production and activity of rhGALNS. These methods involved the use of physiologically-regulated promoters and alternatives to improve protein folding including global stress responses (osmotic shock), overexpression of native chaperones, and enhancement of cytoplasmic disulfide bond formation. Increase of rhGALNS activity was obtained when a promoter regulated under σ s was implemented. Additionally, improvements were observed when osmotic shock was applied. Noteworthy, overexpression of chaperones did not have any effect on rhGALNS activity, suggesting that the effect of osmotic shock was probably due to a general stress response and not to the action of an individual chaperone. Finally, it was observed that high concentrations of sucrose in conjunction with the physiological-regulated promoter proU mod significantly increased the rhGALNS production and activity. Together, these results describe advances in the current knowledge on the production of human recombinant enzymes in a prokaryotic system such as E. coli, and could have a significant impact on the development of enzyme replacement therapies for lysosomal storage diseases.

Anaerobic sulfatase maturase AslB from Escherichia coli activates human recombinant iduronate-2-sulfate sulfatase (IDS) and N-acetylgalactosamine-6-sulfate sulfatase (GALNS).

Maturation of type I sulfatases requires the conversion of the cysteine (Cys) or serine (Ser) present in the active site to formylglycine (FGly). This activation represents a limiting step during the production of recombinant sulfatases in bacteria and eukaryotic hosts. AslB, YdeM and YidF have been proposed to participate in the activation of sulfatases in Escherichia coli. In this study, we combined in-silico and experimental approaches to study the interaction between Escherichia coli BL21(DE3) AslB and human sulfatases, more specifically iduronate-2-sulfate sulfatase (IDS) and N-acetylgalactosamine-6-sulfate sulfatase (GALNS). In-silico results show that AslB has a higher affinity for the residual motif of GALNS (-9.4kcalmol-1), Cys- and Ser-type, than for the one of IDS (-8.0kcalmol-1). However, the distance between the AslB active residue and the target motif favors the interaction with IDS (4.4Å) more than with GALNS (5.5Å). Experimental observations supported in-silico results where the co-expression of AslB with GALNS Cys- and Ser-type presented an activity increment of 2.0- and 1.5-fold compared to the control cultures, lacking overexpressed AslB. Similarly, IDS activity was increased in 4.6-fold when co-expressed with AslB. The higher sulfatase activity of AslB-IDS suggests that the distance between the AslB active residue and the motif target is a key parameter for the in-silico search of potential sulfatase activators. In conclusion, our results suggest that AslB is involve in the maturation of heterologous human sulfatases in E. coli BL21(DE3), and that it can have important implications in the production of recombinant sulfatases for therapeutic purposes and research.

Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris.

Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A.

De seniores para seniores, Proalfa: um projeto de alfabetização com dinâmicas de animação / From seniors to seniors Proalfa: a literacy project with socio-educational animation dynamics

Num contexto de transformações demográficas, de incentivo à educação ao longo da vida, de necessidade de valorizar o papel dos seniores na sociedade e de promover a convivência entre gerações, foi criado o projeto ProAlfa, para utentes de uma estrutura residencial para pessoas idosas de Leiria ­ Portugal. O objetivo é potenciar a aprendizagem da leitura e da escrita, a partir de estratégias de animação socioeducativa, e promover a interação social. As oficinas decorrem uma vez por semana, no espaço da instituição, sendo dinamizadas, voluntariamente, por quatro estudantes seniores do Programa 60+ do Politécnico de Leiria. O presente artigo apresenta parte da investigação realizada no âmbito do ProAlfa, com os seguintes objetivos: conhecer os motivos que levam os seniores a participar no projeto, identificar as atividades que realizam e as que mais valorizam, além de conhecer as expetativas dos participantes sobre o mesmo. Em nível metodológico, a pesquisa é um estudo de caso, de abordagem mista concretizado a partir de um inquérito por entrevista, de cariz estruturado, aplicado a dezesseis participantes no ProAlfa. Estes são maioritariamente mulheres, têm idades compreendidas entre os 58 e os 102 anos, e são pouco escolarizados. Os resultados evidenciam que a oportunidade para a realização de aprendizagens valiosas e para conviver são os motivos apontados para a participação no projeto. Realizam semanalmente atividades de leitura, escrita e pintura, sendo esta última a mais valorizada. Apre- sentam expectativas reduzidas quanto à sua participação no projeto, nomeadamente em relação à aquisição de competências de leitura.(AU)

In a context of demographic changes, of encouraging lifelong education, of valuing the role of seniors in society and of promoting coexistence between generations, the ProAlfa project was created for users of a residential structure for elderly people in Leiria ­ Portugal. The objective is to enhance the learning of reading and writing, based on socio-educational animation strategies, and to promote social interaction. The workshops take place once a week, in the institution's space, being voluntarily promoted by four senior students of the Programa 60+ of the Polytechnic of Leiria. This article presents part of the research carried out within the scope of ProAlfa, with the following objectives: know the reasons that lead seniors to participate in the project, identify the activities they carry out and those that they value most, and to know the expectations of the participants about the project. At a methodological level, the research is a case study, with a mixed approach, obtained through an interview survey, of a structured nature, applied to 16 participants in ProAlfa. These are mostly women, ages between 58 and 102 years old, and with low level of education. The results show that the opportunity to learn valuable skills and to enjoy their time together are the reasons given for participating in the project. They carry out weekly reading, writing and painting sessions, the latter being the most valued. They have low expectations regarding their participation in the project, namely in relation to the acquisition of reading skills.(AU)

The Multi-Target Drug M30 Shows Pro-Cognitive and Anti-Inflammatory Effects in a Rat Model of Alzheimer's Disease.

Current therapies for Alzheimer's disease (AD) offer partial symptomatic relief and do not modify disease progression. There is substantial evidence indicating a disease onset years before clinical diagnosis, at which point no effective therapy has been found. In this study, we investigated the efficacy of a new multi-target drug, M30, at relatively early stages of the AD-like amyloid pathology in a robust rat transgenic model. McGill-R-Thy1-APP transgenic rats develop the full AD-like amyloid pathology in a progressive fashion, and have a minimal genetic burden. McGill rats were given 5 mg/kg M30 or vehicle per os, every 2 days for 4 months, starting at a stage where the transgenic animals suffer detectable cognitive impairments. At the completion of the treatment, cognitive functions were assessed with Novel Object Location and Novel Object Recognition tests. The brains were then analyzed to assess amyloid-ß (Aß) burden and the levels of key inflammatory markers. Long-term treatment with M30 was associated with both the prevention and the reversal of transgene-related cognitive decline. The effects on cognition were accompanied by a shift of the Aß-immunoreactive material toward an amyloid plaque aggregated molecular form, diminished molecular signs of CNS inflammation and a change in microglia morphology toward a surveying phenotype. This study is the first to demonstrate the therapeutic potential of M30 in a rat model of the AD amyloid pathology. It provides a rationale for further investigations with M30 and with potential multi-target approaches to delay, prevent or reverse the progression the AD pathology at early disease-stages.

Projeto en-red-versad@s-clube de poesia on- line- relato de uma experiência de utilização de um blogue por seniores portugueses e espanhóis / En-red- versad@s project-online poetry club- report of an experience of ising a blog by portuguese and spanish seniors

O presente relato é parte de um projeto mais vasto de intervenção e investigação em curso no âmbito do Projeto En­Red­Versad@s.Clube de poesia on line, dinamizado pela Universidade Sénior da Universidade da Corunha e pelo seu parceiro Programa 60+ do Politécnico de Leiria. Este clube visa, através da dinamização de um blogue, a divulgar a língua e a poesia portuguesas, bem como desenvolver competências linguísticas, culturais e digitais, através do domínio e uso das Tecnologias da Informação e Comunicação (TIC). O projeto desenvolve-se no quadro de uma educação plurilinguística, poética e digital, ao longo da vida, em ordem a um envelhecimento ativo e saudável, bem como à construção de uma cidadania consciente e comprometida por parte dos estudantes seniores. Através deste relato, pretende-se apresentar os objetivos, participantes, metodologias de ação e os primeiros dados de participação dos seniores, decorrentes do registo de acesso ao blogue e aos conteúdos partilhados, cuja análise se insere num paradigma quantitativo. Os resultados obtidos, através de estatístisca descritiva, sugerem que o projeto tem potencialidades formativas que impulsionam o envolvimento dos participantes seniores no clube de poesia on line, abrindo a discussão sobre as vantagens de um alargamento da sua participação e sobre as estratégias pedagógicas para a promover. Afirma-se como uma resposta socioeducativa válida, em ordem à valorização pessoal e intergeracional dos participantes.(AU)

This report is part of a broader intervention and research project integrated within the scope of the En­Red­Versad@s Project. Online poetry club. Such project is developed by the Senior University of the University of A Coruña and its partner Program 60+ at the Polytechnic of Leiria. Through a blog, this club aims to promote the Portuguese language and poetry, as well as develop linguistic, cultural and digital competences, increasing proficiency with technologies and computers. The project is developed within the framework of a multilingual, poetic and digital education, through the life course, in order to achieve an active and healthy aging, as well as construct a conscious and committed citizenship by the senior students. This report intends to present the aims, participants and methods of action, as well as the first data of participation from the seniors, including their access records to the blog and the contents shared. The analysis is based on a quantitative paradigm. The results, obtained through, descriptive statisticssuggest that the project has training opportunities that can enhance the involvement of senior participants in the online poetry club. This opens the discussion about the advantages of widening their participation and the pedagogical ways of promoting it. This arises as a valid socio-educational response, in order to enhance the personal and intergenerational value of the participants.(AU)

Neuronal driven pre-plaque inflammation in a transgenic rat model of Alzheimer's disease.

Chronic brain inflammation is associated with Alzheimer's disease (AD) and is classically attributed to amyloid plaque deposition. However, whether the amyloid pathology can trigger early inflammatory processes before plaque deposition remains a matter of debate. To address the possibility that a pre-plaque inflammatory process occurs, we investigated the status of neuronal, astrocytic, and microglial markers in pre- and post-amyloid plaque stages in a novel transgenic rat model of an AD-like amyloid pathology (McGill-R-Thy1-APP). In this model, we found a marked upregulation of several classical inflammatory markers such as COX-2, IL-1ß, TNF-α, and fractalkine (CX3CL1) in the cerebral cortex and hippocampus. Interestingly, many of these markers were highly expressed in amyloid beta-burdened neurons. Activated astrocytes and microglia were associated with these Aß-burdened neurons. These findings confirm the occurrence of a proinflammatory process preceding amyloid plaque deposition and suggest that Aß-burdened neurons play a crucial role in initiating inflammation in AD.

Evaluation of risk factors that contribute to falls among the elderly / Avaliação dos fatores de risco que contribuem para queda em idosos

Abstract Objective: to determine the incidence of falls among the elderly population of the city of Barbacena in the state of Minas Gerais, together with causal factors, circumstances and major consequences. Methods: a cross-sectional study was performed through questionnaires applied to 206 patients over the age of 60, from November 2014 to February 2015 in the city of Barbacena, in the state of Minas Gerais. Risk factors related to falls were analyzed, as well as the incidence of falls and the consequences for the lives of elderly persons. The existence of a relationship between the reporting of falls and possible risk factors was determined by the Chi-squared and Fischer's exact tests as indicated. Results: an incidence of falls of 36.41% was observed among the elderly, 45.95% of which occurred outside the home. A total of 85.71% of respondents had previously suffered strokes and 39.78% were taking medication. Among elderly persons who have fallen and suffered fractures (18.67%), 50% had suffered strokes, 50% were suffering from chronic kidney disease, and 61.54% could not perform their activities of daily living after the fall. Conclusion: it was concluded that the incidence of falls among the elderly was 36.41%, while the most correlated factors were drug use, stroke victims and people with chronic kidney disease. Among those who suffered fractures, 61.54% failed to perform activities of daily living. Preventing falls is a public health concern, and simple changes can reduce its prevalence. AU

Resumo Objetivo: descrever incidência de quedas em idosos no município de Barbacena, MG, com seus fatores causais, circunstâncias e consequências. Método: estudo de corte transversal, realizado mediante aplicação de questionários em 206 pacientes acima de 60 anos de idade, de novembro 2014 a fevereiro de 2015. Foram analisados fatores de risco, incidência e consequência das quedas na vida dos idosos. Foram construídas distribuições de frequência e calculadas as médias, desvios-padrão e percentagens. A existência de relação entre o relato de quedas e seus possíveis fatores de risco foi determinada por Teste qui-quadrado e exato de Fischer conforme a indicação. Resultados: observou-se a incidência de 36,41% de queda em idoso, sendo que 45,95% ocorreram fora de casa, 85,71% dos pesquisados já haviam sofrido acidente vascular encefálico (AVE) e 39,78% faziam uso de medicamento. Dos idosos que caíram e sofreram fratura (18,67%), 50% já tinham sofrido episódio de AVE e 50% eram portadores de doença renal crônica, sendo que 61,54% deixaram de realizar suas atividades diárias após a queda. Conclusão: a incidência de queda em idosos foi de 36,41%. Os fatores mais correlacionados foram uso de medicamentos, vítimas de AVE, portadores de doença renal crônica e, dos que sofreram fratura, 61,54% deixaram de realizar atividades diárias. Desta forma, a prevenção das quedas é uma preocupação de saúde pública e mudanças relativamente simples podem reduzi-las. AU

New selective acetylcholinesterase inhibitors designed from natural piperidine alkaloids.

Five new piperidine alkaloids were designed from natural (-)-3-O-acetyl-spectaline and (-)-spectaline that were obtained from the flowers of Senna spectabilis (sin. Cassia spectabilis, Leguminosae). Two semi-synthetic analogues (7 and 9) inhibited rat brain acetylcholinesterase, showing IC50 of 7.32 and 15.1 microM, and were 21 and 9.5 times less potent against rat brain butyrylcholinesterase, respectively. Compound 9 (1mg/kg, i.p.) was fully efficacious in reverting scopolamine-induced amnesia in mice. The two active compounds (7 and 9) did not show overt toxic effects at the doses tested in vivo.