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

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

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

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

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. Intraventricular administration of AAV vectors expressing secreted and transmembrane Klotho isoforms, rescued accelerated aging phenotype of SAMP8 mice. An improvement in cognitive and physical performance, recovery of epigenetic, inflammatory and senescence markers, as well as structural changes in long bones of these mice was detected