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

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 (P S ) and prolonged (P L ) passages, both in standard and low [O 2 ] (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 P L or P S surprisingly showed a quite significant fraction (69%) of upregulated miRNAs in P L 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% [O 2 ], was also significantly higher in P L than in P S 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.This work was supported by grants to AB from the Spanish Ministry of Economy, Industry (SAF2015-70882-R; AEI/FEDER, UE), Comunidad Autónoma de Madrid (S2010/BMD-2420), Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0018) and the European Commission (FP7-HEALTH- 2009/CARE-MI). AMS was supported by grants from the MINECO (SAF2010–17167) and Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0013), and MFF and RGU by grants from the Plan Nacional de I+D+I 2013-2016/FEDER (PI15/ 00892), the Asturias Regional Government (GRUPIN14-052), the IUOPA (Obra Social Cajastur) and the Fundación Científica de la AECC. SGL held a predoctoral fellowship from the Spanish Programa de Formación del Profesorado Universitari

DNA polymerase lambda (Pol λ), a novel eukaryotic DNA polymerase with a potential role in meiosis

A new gene (POLL) encoding a novel DNA polymerase (Pol λ) has been identified at mouse chromosome 19. Murine Pol λ, consisting of 573 amino acid residues, has a 32 % identity to Pol β, involved in nuclear DNA repair in eukaryotic cells. It is interesting that Pol λ contains all the critical residues involved in DNA binding, nucleotide binding and selection, and catalysis of DNA polymerization, that are conserved in Pol β and other DNA polymerases belonging to family X. Murine Pol λ, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity when assessed by in situ gel analysis. Pol λ also conserves the critical residues of Pol β required for its intrinsic deoxyribose phosphate lyase (dRPase) activity. The first 230 amino acid residues of Pol λ, that have no counterpart in Pol β, contain a BRCT domain, present in a variety of cell-cycle check-point control proteins responsive to DNA damage and proteins involved in DNA repair. Northern blotting, in situ hybridization analysis and immunostaining showed high levels of Pol λ specifically expressed in testis, being developmentally regulated and mainly associated to pachytene spermatocytes. These first evidences, although indirect, suggest a potential role of Pol λ in DNA repair synthesis associated with meiosis.This work has been granted by DGES (PB97-1192) and CAM (08.1/0044/98) to LB; CAM(08.1/0044.2/98) to AB; DGICYT (PB 95-0119), EC PL96-0183 and CAM (07/0022) to JM, and by an institutional grant from Fundación Ramón Areces

Global hyperactivation of enhancers stabilizes human and mouse naïve pluripotency through inhibition of CDK8/19 Mediator kinases

Pluripotent stem cells (PSCs) transition between cell states in vitro and reflect developmental changes in the early embryo. PSCs can be stabilized in the naïve state by blocking extracellular differentiation stimuli, particularly FGF-MEK signaling. Here, we report that multiple features of the naïve state in human and mouse PSCs can be recapitulated without affecting FGF-MEK-signaling or global DNA methylation. Mechanistically, chemical inhibition of CDK8 and CDK19 kinases removes their ability to repress the Mediator complex at enhancers. Thus CDK8/19 inhibition increases Mediator-driven recruitment of RNA Pol II to promoters and enhancers. This efficiently stabilizes the naïve transcriptional program and confers resistance to enhancer perturbation by BRD4 inhibition. Moreover, naïve pluripotency during embryonic development coincides with a reduction in CDK8/19. We conclude that global hyperactivation of enhancers drives naïve pluripotency, and this can be achieved in vitro by inhibiting CDK8/19 kinase activity. These principles may apply to other contexts of cellular plasticity

Properties of Graphene: A Theoretical Perspective

In this review, we provide an in-depth description of the physics of monolayer and bilayer graphene from a theorist's perspective. We discuss the physical properties of graphene in an external magnetic field, reflecting the chiral nature of the quasiparticles near the Dirac point with a Landau level at zero energy. We address the unique integer quantum Hall effects, the role of electron correlations, and the recent observation of the fractional quantum Hall effect in the monolayer graphene. The quantum Hall effect in bilayer graphene is fundamentally different from that of a monolayer, reflecting the unique band structure of this system. The theory of transport in the absence of an external magnetic field is discussed in detail, along with the role of disorder studied in various theoretical models. We highlight the differences and similarities between monolayer and bilayer graphene, and focus on thermodynamic properties such as the compressibility, the plasmon spectra, the weak localization correction, quantum Hall effect, and optical properties. Confinement of electrons in graphene is nontrivial due to Klein tunneling. We review various theoretical and experimental studies of quantum confined structures made from graphene. The band structure of graphene nanoribbons and the role of the sublattice symmetry, edge geometry and the size of the nanoribbon on the electronic and magnetic properties are very active areas of research, and a detailed review of these topics is presented. Also, the effects of substrate interactions, adsorbed atoms, lattice defects and doping on the band structure of finite-sized graphene systems are discussed. We also include a brief description of graphane -- gapped material obtained from graphene by attaching hydrogen atoms to each carbon atom in the lattice.Comment: 189 pages. submitted in Advances in Physic

7th Drug hypersensitivity meeting: part two

No abstract availabl

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified