Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosineDNA glycosylase to a sequence-specific DNA binding domain

DNA methylation is a crucial epigenetic mark associated to gene silencing, and its targeted removal is amajor goal of epigenetic editing. In animal cells, DNA demethylation involves iterative 5mC oxidation byTET enzymes followed by replication-dependent dilution and/or replication-independent DNA repair of itsoxidized derivatives. In contrast, plants use specific DNA glycosylases that directly excise 5mC and initiateits substitution for unmethylated C in a base excision repair process. In this work, we have fused thecatalytic domain ofArabidopsisROS1 5mC DNA glycosylase (ROS1_CD) to the DNA binding domain ofyeast GAL4 (GBD). We show that the resultant GBD-ROS1_CD fusion protein binds specifically a GBD-targeted DNA sequencein vitro. We also found that transientin vivoexpression of GBD-ROS1_CD inhuman cells specifically reactivates transcription of a methylation-silenced reporter gene, and that suchreactivation requires both ROS1_CD catalytic activity and GBD binding capacity. Finally, we show thatreactivation induced by GBD-ROS1_CD is accompanied by decreased methylation levels at several CpGsites of the targeted promoter. All together, these results show that plant 5mC DNA glycosylases can beused for targeted active DNA demethylation in human cells

Active DNA demethylation in plants

Methylation of cytosine (5-meC) is a critical epigenetic modification in many eukaryotes, and genomic DNA methylation landscapes are dynamically regulated by opposed methylation and demethylation processes. Plants are unique in possessing a mechanism for active DNA demethylation involving DNA glycosylases that excise 5-meC and initiate its replacement with unmodified C through a base excision repair (BER) pathway. Plant BER-mediated DNA demethylation is a complex process involving numerous proteins, as well as additional regulatory factors that avoid accumulation of potentially harmful intermediates and coordinate demethylation and methylation to maintain balanced yet flexible DNA methylation patterns. Active DNA demethylation counteracts excessive methylation at transposable elements (TEs), mainly in euchromatic regions, and one of its major functions is to avoid methylation spreading to nearby genes. It is also involved in transcriptional activation of TEs and TE-derived sequences in companion cells of male and female gametophytes, which reinforces transposon silencing in gametes and also contributes to gene imprinting in the endosperm. Plant 5-meC DNA glycosylases are additionally involved in many other physiological processes, including seed development and germination, fruit ripening, and plant responses to a variety of biotic and abiotic environmental stimuli

New insights into the exploitation of vitis vinifera l. Cv. aglianico leaf extracts for nutraceutical purposes

The leaves of Vitis vinifera L. have been used for a long time in traditional medicine for the treatment of many ailments. Grape polyphenols, indeed, have been demonstrated to be able to defend against oxidative stress, responsible for various disorders such as cancer, diabetes and neurodegenerative diseases. The effects of different extraction techniques, Soxhlet (SOX), Accelerated Solvent (ASE 40, ASE 50) and Ultrasound Assisted Extraction (UAE) were studied in this work to evaluate their impact on the chemical profile and bioactive potential of Vitis vinifera L. (cv. Aglianico) leaf extracts. The phytochemical profile was investigated by HPLC-DAD and 9 phenolic compounds were identified and quantified in the extract. Moreover, the antioxidant, anticholinesterase and antityrosinase activities were evaluated. In detail, the total polyphenol content and antioxidant activity (2,2-diphenyl-1-picrylhydrazyl, Oxygen Radical Absorbance Capacities and β-Carotene Bleaching assays) were evaluated and compared to assess the Relative Antioxidant Capacity Index (RACI). To test the inhibitory activity of extracts towards cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition assays were performed. SOX and ASE 50 have shown the highest value of RACI, 0.76 and 0.65, respectively. Regarding enzymatic inhibitory activity, ASE 50 (IC50 = 107.16 ± 8.12 µg/mL) and SOX (IC50 = 171.34 ± 12.12 µg/mL) extracts exhibited the highest AChE and BChE inhibitory activity, respectively, while UAE (IC50 = 293.2 ± 25.6 µg/mL, followed by SOX (IC50 = 302.5 ± 38.3 µg/mL) showed the highest tyrosinase inhibition value. Our results demonstrated for the first time that Aglianico leaves are important sources of phenols that could be used to prevent oxidative stress and be potentially helpful in diseases treatable with tyrosinase and cholinesterase inhibitors, like myasthenia gravis or Alzheimer’s

Identification and location of hot and cold spots of treated prevalence of depression in Catalonia (Spain)

Abstract Background Spatial analysis is a relevant set of tools for studying the geographical distribution of diseases, although its methods and techniques for analysis may yield very different results. A new hybrid approach has been applied to the spatial analysis of treated prevalence of depression in Catalonia (Spain) according to the following descriptive hypotheses: 1) spatial clusters of treated prevalence of depression (hot and cold spots) exist and, 2) these clusters are related to the administrative divisions of mental health care (catchment areas) in this region. Methods In this ecological study, morbidity data per municipality have been extracted from the regional outpatient mental health database (CMBD-SMA) for the year 2009. The second level of analysis mapped small mental health catchment areas or groups of municipalities covered by a single mental health community centre. Spatial analysis has been performed using a Multi-Objective Evolutionary Algorithm (MOEA) which identified geographical clusters (hot spots and cold spots) of depression through the optimization of its treated prevalence. Catchment areas, where hot and cold spots are located, have been described by four domains: urbanicity, availability, accessibility and adequacy of provision of mental health care. Results MOEA has identified 6 hot spots and 4 cold spots of depression in Catalonia. Our results show a clear spatial pattern where one cold spot contributed to define the exact location, shape and borders of three hot spots. Analysing the corresponding domain values for the identified hot and cold spots no common pattern has been detected. Conclusions MOEA has effectively identified hot/cold spots of depression in Catalonia. However these hot/cold spots comprised municipalities from different catchment areas and we could not relate them to the administrative distribution of mental care in the region. By combining the analysis of hot/cold spots, a better statistical and operational-based visual representation of the geographical distribution is obtained. This technology may be incorporated into Decision Support Systems to enhance local evidence-informed policy in health system research.</p

Intervenção sistêmica estruturada numa empresa do setor aeronáutico

The main objective of this research work is to present and detail the application of a systemic intervention method in a medium-sized company in the aeronautical sector to outline actions to address complex problems. It is a hybrid approach obtained from the synergy of foundations and methods of systemic and business management epistemologies, which aims to explore three aspects: team training for systemic intervention, structured dialogue between stakeholders and active leadership. The proposed method is structured in stages and steps, supported by pillars and critical heuristics to deepen the analysis of the problem situation. As a result, from the perspective of the problem, the proposal of viable actions was obtained and, from the strategic point of view, growth, and perceived learning regarding critical and systemic sensitivity on the part of those involved. The structured and systematic realisation of dialogues for exploration and mutual understanding of the problem situation proved to be efficient in promoting a more cordial and tolerant work environment, as well as it was observed that a process of awareness and metacognitive growth of people in critical-systemic thinking was initiated, leading to an increase in the organisation\u27s capacity to solve complex problems. It is hoped that the method can be successfully replicated in other companies.O principal objetivo deste trabalho de pesquisa é apresentar e detalhar a aplicação de um método de intervenção sistêmica numa empresa de médio porte do setor aeronáutico visando traçar ações para tratar problemas complexos. Trata-se de uma abordagem híbrida obtida da sinergia de fundamentos e métodos das epistemologias sistêmica e de gestão de negócios, que visa explorar três aspectos: a capacitação da equipe para intervenção sistêmica, o diálogo estruturado entre as partes interessadas e a liderança ativa. O método proposto é estruturado em etapas e passos, suportados por pilares e heurísticas críticas para aprofundamento na análise da situação-problema. Como resultados, obteve-se, na perspectiva do problema, a proposição de ações viáveis e, sob o ponto de vista estratégico, crescimento e aprendizado percebido quanto à sensibilidade crítica e sistêmica por parte dos envolvidos. A realização estruturada e sistemática de diálogos para exploração e entendimento mútuo da situação-problema se mostrou eficiente na promoção de um ambiente de trabalho mais cordial e tolerante, bem como observou-se que um processo de conscientização e crescimento metacognitivo das pessoas em pensamento crítico-sistêmico foi iniciado, levando ao aumento da capacidade da organização em solucionar problemas complexos. Espera-se que o método possa ser replicado em outras empresas com sucesso. ARK CAICYT: http://id.caicyt.gov.ar/ark:/s18539777/gr9kz41r3

AAV-mediated expression of secreted and transmembrane αKlotho isoforms rescues relevant aging hallmarks in senescent SAMP8 mice

Senescence represents a stage in life associated with elevated incidence of morbidity and increased risk of mortality due to the accumulation of molecular alterations and tissue dysfunction, promoting a decrease in the organism's protective systems. Thus, aging presents molecular and biological hallmarks, which include chronic inflammation, epigenetic alterations, neuronal dysfunction, and worsening of physical status. In this context, we explored the AAV9-mediated expression of the two main isoforms of the aging-protective factor Klotho (KL) as a strategy to prevent these general age-related features using the senescence-accelerated mouse prone 8 (SAMP8) model. Both secreted and transmembrane KL isoforms improved cognitive performance, physical state parameters, and different molecular variables associated with aging. Epigenetic landscape was recovered for the analyzed global markers DNA methylation (5-mC), hydroxymethylation (5-hmC), and restoration occurred in the acetylation levels of H3 and H4. Gene expression of pro- and anti-inflammatory mediators in central nervous system such as TNF-α and IL-10, respectively, had improved levels, which were comparable to the senescence-accelerated-mouse resistant 1 (SAMR1) healthy control. Additionally, this improvement in neuroinflammation was supported by changes in the histological markers Iba1, GFAP, and SA β-gal. Furthermore, bone tissue structural variables, especially altered during senescence, recovered in SAMP8 mice to SAMR1 control values after treatment with both KL isoforms. This work presents evidence of the beneficial pleiotropic role of Klotho as an anti-aging therapy as well as new specific functions of the KL isoforms for the epigenetic regulation and aged bone structure alteration in an aging mouse model