Publicaciones Nanomedicina

Hello world! Please change me in Site Preferences -> This Category/Section -> Lower Description Bar

07

Dec 2018

Quantification of Global DNA Methylation Levels by Mass Spectrometry

Posted by / in Publicaciones 2018, Publicaciones Destacadas, Publicaciones Nanomedicina, Publications Nanomedicine 2018, Últimas Publicaciones CINN / No comments yet

Global DNA methylation was classically considered the relative percentage of 5-methylcysine (5mC) with respect to total cytosine (C). Early approaches were based on the use of high-performance separation technologies and UV detection. However, the recent development of protocols using mass spectrometry for the detection has increased sensibility and permitted the precise identification of peak compounds based on their molecular masses. This allows work to be conducted with much less genomic DNA starting material and also to quantify 5-hydroxymethyl-cytosine (5hmC), a recently identified form of methylated cytosine that could play an important role in active DNA demethylation. Here, we describe the protocol that we currently use in our laboratory to analyze 5mC and 5hmC by mass spectrometry. The protocol, which is based on the method originally developed by Le and colleagues using Ultra Performance Liquid Chromatography (UPLC) and mass spectrometry (triple Quadrupole (QqQ)) detection, allows for the rapid and accurate quantification of relative global 5mC and 5hmC levels starting from just 1 μg of genomic DNA, which allows for the rapid and accurate quantification of relative global 5mC and 5hmC levels.

Please select the social network you want to share this page with:

22

Nov 2018

Deregulation of the imprinted DLK1-DIO3 locus ncRNAs is associated with replicative senescence of human adipose-derived stem cells

Posted by / in Publicaciones 2018, Publicaciones Nanomedicina, Publications Nanomedicine 2018, Últimas Publicaciones CINN / No comments yet

Background

Human adult adipose-derived stem cells (hADSCs) have become the most promising cell source for regenerative medicine. However the prolonged ex vivo expansion periods required to obtain the necessary therapeutic dose promotes progressive senescence, with the concomitant reduction of their therapeutic potential.

Aim and scope

A better understanding of the determinants of hADSC senescence is needed to improve biosafety while preserving therapeutic efficiency. Here, we investigated the association between deregulation of the imprinted DLK1-DIO3 region and replicative senescence in hADSC cultures.

Methods

We compared hADSC cultures at short (PS) and prolonged (PL) passages, both in standard and low [O2] (21 and 3%, respectively), in relation to replicative senescence. hADSCs were evaluated for expression alterations in the DLK1-DIO3 region on chromosome 14q32, and particularly in its main miRNA cluster.

Results

Comparison of hADSCs cultured at PL or PS surprisingly showed a quite significant fraction (69%) of upregulated miRNAs in PL cultures mapping to the imprinted 14q32 locus, the largest miRNA cluster described in the genome. In agreement, expression of the lncRNA MEG3(Maternally Expressed 3; Meg3/Gtl2), cultured at 21 and 3% [O2], was also significantly higher in PL than in PS passages. During hADSC replicative senescence the AcK16H4 activating mark was found to be significantly associated with the deregulation of the entire DLK1-DIO3 locus, with a secondary regulatory role for the methylation of DMR regions.

Conclusion

A direct relationship between DLK1-DIO3 deregulation and replicative senescence of hADSCs is reported, involving upregulation of a very significant fraction of its largest miRNA cluster (14q32.31), paralleled by the progressive overexpression of the lncRNA MEG3, which plays a central role in the regulation of Dlk1/Dio3 activation status in mice.

Please select the social network you want to share this page with:

22

Nov 2018

Alzheimer’s disease DNA methylome of pyramidal layers in frontal cortex: Laser-assisted microdissection study

Posted by / in Publicaciones 2018, Publicaciones Destacadas, Publicaciones Nanomedicina, Publications Nanomedicine 2018, Últimas Publicaciones CINN / No comments yet

Objective: To study DNA methylation patterns of cortical pyramidal layers susceptible to late-onset Alzheimer’s disease (LOAD) neurodegeneration.

Methods: Laser-assisted microdissection to select pyramidal layers’ cells in frontal cortex of 32 human brains (18 LOAD) and Infinium DNA Methylation 450K analysis were performed to find differential methylated positions and regions, in addition to the corresponding gene set functional enrichment analyses. Results:Differential hypermethylation in several genomic regions and genes mainly in HOXA3, GSTP1, CXXC1-3 and BIN1. The functional enrichment analysis revealed genes significantly related to oxidative-stress and synapsis.

Conclusion: The present results indicate the differentially methylated genes related to neural projections, synapsis, oxidative stress and epigenetic regulator genes and represent the first epigenome of cortical pyramidal layers in LOAD.

Please select the social network you want to share this page with: