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Ravina M.,University of Santiago de Compostela | Cubillo E.,Instituto Nacional Of Toxicologia Y Ciencias Forenses Intcf | Novoa-Carballal R.,University of Santiago de Compostela | Fernandez-Megia E.,University of Santiago de Compostela | And 4 more authors.
Pharmaceutical Research | Year: 2010

Purpose To design hyaluronic acid (HA) and chitosan-g-poly (ethylene glycol) (CS-g-PEG) nanoparticles intended for a broad range of gene delivery applications. Methods Nanoparticles formulated at different HA/CS-g-PEG mass ratios were developed to associate either pDNA or siRNA. The physico-chemical characteristics, morphology, association efficiency and nuclease protection ability of the nanocarriers were compared for these two molecules. Their biological performance, including transfection effciency, nanoparticle cellular uptake and citotoxicity, was assesed. Results The resulting nanoparticles showed an adequate size (between 130 and 180 nm), and their surface charge could be modulated according to the nanoparticle composition (from +30 mV to -20 mV). All prototypes exhibited a greater association efficiency and nuclease protection for pDNA than for siRNA. However, cell culture experiments evidenced that HA/CS-g-PEG nanoparticles were effective carriers for the delivery of both, siRNA and pDNA, eliciting a biological response with minimal cytotoxicity. Moreover, experiments performed in the HEK-EGFP-Snail1 cell line showed the potential of the HA/CS-g-PEG nanoparticles to silence the expression of the Snail1 transcription factor, an important mediator in tumor progression. Conclusions HA/CS-g-PEG nanoparticles can be easily modulated for the delivery of different types of gene molecules, offering great potential for gene therapy applications, as evidenced by their biological performance. © Springer Science+Business Media, LLC 2010.


Vullo C.M.,Forensic Genetics Laboratory | Romero M.,Forensic Genetics Laboratory | Catelli L.,Forensic Genetics Laboratory | Sakic M.,International Commission for Missing Persons ICMP | And 13 more authors.
Forensic Science International: Genetics | Year: 2016

The GHEP-ISFG Working Group has recognized the importance of assisting DNA laboratories to gain expertise in handling DVI or missing persons identification (MPI) projects which involve the need for large-scale genetic profile comparisons. Eleven laboratories participated in a DNA matching exercise to identify victims from a hypothetical conflict with 193 missing persons. The post mortem database was comprised of 87 skeletal remain profiles from a secondary mass grave displaying a minimal number of 58 individuals with evidence of commingling. The reference database was represented by 286 family reference profiles with diverse pedigrees. The goal of the exercise was to correctly discover re-associations and family matches. The results of direct matching for commingled remains re-associations were correct and fully concordant among all laboratories. However, the kinship analysis for missing persons identifications showed variable results among the participants. There was a group of laboratories with correct, concordant results but nearly half of the others showed discrepant results exhibiting likelihood ratio differences of several degrees of magnitude in some cases. Three main errors were detected: (a) some laboratories did not use the complete reference family genetic data to report the match with the remains, (b) the identity and/or non-identity hypotheses were sometimes wrongly expressed in the likelihood ratio calculations, and (c) many laboratories did not properly evaluate the prior odds for the event. The results suggest that large-scale profile comparisons for DVI or MPI is a challenge for forensic genetics laboratories and the statistical treatment of DNA matching and the Bayesian framework should be better standardized among laboratories. © 2015 Elsevier Ireland Ltd. All rights reserved.


PubMed | Analisis ADN Forense, University of Santiago de Compostela, University of the Basque Country, Instituto Nacional Of Medicina Legal E Ciencias Forenses and 7 more.
Type: | Journal: Forensic science international. Genetics | Year: 2016

The GHEP-ISFG Working Group has recognized the importance of assisting DNA laboratories to gain expertise in handling DVI or missing persons identification (MPI) projects which involve the need for large-scale genetic profile comparisons. Eleven laboratories participated in a DNA matching exercise to identify victims from a hypothetical conflict with 193 missing persons. The post mortem database was comprised of 87 skeletal remain profiles from a secondary mass grave displaying a minimal number of 58 individuals with evidence of commingling. The reference database was represented by 286 family reference profiles with diverse pedigrees. The goal of the exercise was to correctly discover re-associations and family matches. The results of direct matching for commingled remains re-associations were correct and fully concordant among all laboratories. However, the kinship analysis for missing persons identifications showed variable results among the participants. There was a group of laboratories with correct, concordant results but nearly half of the others showed discrepant results exhibiting likelihood ratio differences of several degrees of magnitude in some cases. Three main errors were detected: (a) some laboratories did not use the complete reference family genetic data to report the match with the remains, (b) the identity and/or non-identity hypotheses were sometimes wrongly expressed in the likelihood ratio calculations, and (c) many laboratories did not properly evaluate the prior odds for the event. The results suggest that large-scale profile comparisons for DVI or MPI is a challenge for forensic genetics laboratories and the statistical treatment of DNA matching and the Bayesian framework should be better standardized among laboratories.


Cubillo E.,Autonomous University of Madrid | Cubillo E.,Instituto Nacional Of Toxicologia Y Ciencias Forenses Intcf | Diaz-Lopez A.,Autonomous University of Madrid | Cuevas E.P.,Autonomous University of Madrid | And 8 more authors.
PLoS ONE | Year: 2013

E12/E47 proteins (encoded by E2A gene) are members of the class I basic helix-loop-helix (bHLH) transcription factors (also known as E proteins). E47 has been described as repressor of E-cadherin and inducer of epithelial-mesenchymal transition (EMT). We reported previously that EMT mediated by E47 in MDCK cells occurs with a concomitant overexpression of Id1 and Id3 proteins. Id proteins belong to class V of HLH factors that lack the basic domain; they dimerise with E proteins and prevent their DNA interaction, thus, acting as dominant negative of E proteins. Here, we show that E47 interacts with Id1 in E47 overexpressing MDCK cells that underwent a full EMT as well as in mesenchymal breast carcinoma and melanoma cell lines. By conducting chromatin immunoprecipitation assays we demonstrate that E47 binds directly to the endogenous E-cadherin promoter of mesenchymal MDCK-E47 cells in a complex devoid of Id1. Importantly, our data suggest that both E47 and Id1 are required to maintain the mesenchymal phenotype of MDCK-E47 cells. These data support the collaboration between E47 and Id1 in the maintenance of EMT by mechanisms independent of the dominant negative action of Id1 on E47 binding to E-cadherin promoter. Finally, the analysis of several N0 breast tumour series indicates that the expression of E47 and ID1 is significantly associated with the basal-like phenotype supporting the biological significance of the present findings. © 2013 Cubillo et al.


PubMed | Gendiag SL, University of Barcelona, Instituto Nacional Of Toxicologia Y Ciencias Forenses Intcf, Institute Of Medicina Legal I Ciencies Forenses Of Catalonia Imlcfc and University of Girona
Type: | Journal: Forensic science international | Year: 2017

Sudden cardiac arrest is a leading cause of death worldwide. Most cardiac arrests happen in patients who have previously suffered a myocardial infarct. The risk of sudden death after infarction may increase in people who carry a pathogenic genetic alteration in cardiac ion channels. We hypothesized that micro-ischemia could trigger lethal arrhythmogenesis, thus we sought to identify genetic alterations in cardiac ion channels in patients with micro-ischemic disease. We studied a cohort of 56 post-mortem samples. Autopsy studies identified myocardial infarction as the cause of death in each case. We used both Sanger sequencing and next-generation sequencing to screen candidate genes associated with sudden cardiac death. We identified six rare missense genetic variations in five unrelated patients. Two variants have been previously reported; one is associated with atrial fibrillation (SCN5A_p.H445D), and the other is predicted to be benign (ANK2_p.T2059M). The novel variants were predicted in silico as benign, except for one (RyR2_p.M4019T), which was classified as deleterious. Our post-mortem, micro-infarction cohort displayed a rate of nearly 10% non-common genetic variants. However, the clinical significance of most of the identified variants remains unknown due to lack of family assessment. Further analyses should be performed in large cohorts to clarify the role of ion-channel gene analysis in samples showing microscopic ischemic alterations.

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