Cis-regulatory elements are harbored in Intron5 of the RUNX1 gene

BACKGROUND: Human RUNX1 gene is one of the most frequent target for chromosomal translocations associated with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). The highest prevalence in AML is noted with (8; 21) translocation; which represents 12 to 15% of all AML cases. Interestingly, all the breakpoints mapped to date in t(8;21) are clustered in intron 5 of the RUNX1 gene and intron 1 of the ETO gene. No homologous sequences have been found at the recombination regions; but DNase I hypersensitive sites (DHS) have been mapped to the areas of the genes involved in t(8;21). Presence of DHS sites is commonly associated with regulatory elements such as promoters, enhancers and silencers, among others. RESULTS: In this study we used a combination of comparative genomics, cloning and transfection assays to evaluate potential regulatory elements located in intron 5 of the RUNX1 gene. Our genomic analysis identified nine conserved non-coding sequences that are evolutionarily conserved among rat, mouse and human. We cloned two of these regions in pGL-3 Promoter plasmid in order to analyze their transcriptional regulatory activity. Our results demonstrate that the identified regions can indeed regulate transcription of a reporter gene in a distance and position independent manner; moreover, their transcriptional effect is cell type specific. CONCLUSIONS: We have identified nine conserved non coding sequence that are harbored in intron 5 of the RUNX1 gene. We have also demonstrated that two of these regions can regulate transcriptional activity in vitro. Taken together our results suggest that intron 5 of the RUNX1 gene contains multiple potential cis-regulatory elements

CHK1 expression in gastric cancer is modulated by p53 and RB1/E2F1: Implications in chemo/radiotherapy response

Radiation has a limited but relevant role in the adjuvant therapy of gastric cancer (GC) patients. Since Chk1 plays a critical function in cellular response to genotoxic agents, we aimed to analyze the role of Chk1 in GC as a biomarker for radiotherapy resistance. We analyzed Chk1 expression in AGS and MKN45 human GC cell lines by RT-QPCR and WB and in a small cohort of human patient’s samples. We demonstrated that Chk1 overexpression specifically increases resistance to radiation in GC cells. Accordingly, abrogation of Chk1 activity with UCN-01 and its expression with shChk1 increased sensitivity to bleomycin and radiation. Furthermore, when we assessed Chk1 expression in human samples, we found a correlation between nuclear Chk1 accumulation and a decrease in progression free survival. Moreover, using a luciferase assay we found that Chk1’s expression is controlled by p53 and RB/E2F1 at the transcriptional level. Additionally, we present preliminary data suggesting a posttranscriptional regulation mechanism, involving miR-195 and miR-503, which are inversely correlated with expression of Chk1 in radioresistant cells. In conclusion, Chk1/microRNA axis is involved in resistance to radiation in GC, and suggests Chk1 as a potential tool for optimal stratification of patients susceptible to receive adjuvant radiotherapy after surgeryThis work was supported by Instituto de Salud Carlos III–Fondo de Investigación Sanitaria (PS09/1988 to ISP; PI11-00949, pI014-1495 and Feder Funds to RP); Comunidad Autónoma de Madrid-Universidad Autónoma de Madrid (CCG10-UAM/BIO-5871 to ISP); Fundación Leticia Castillejo Castillo and Ministerio de Ciencia e Innovación (SAF2012-30862 to RSP), Spain

Astrometric calibration and performance of the Dark Energy Camera

We characterize the ability of the Dark Energy Camera (DECam) to perform relative astrometry across its 500~Mpix, 3 deg^2 science field of view, and across 4 years of operation. This is done using internal comparisons of ~4x10^7 measurements of high-S/N stellar images obtained in repeat visits to fields of moderate stellar density, with the telescope dithered to move the sources around the array. An empirical astrometric model includes terms for: optical distortions; stray electric fields in the CCD detectors; chromatic terms in the instrumental and atmospheric optics; shifts in CCD relative positions of up to ~10 um when the DECam temperature cycles; and low-order distortions to each exposure from changes in atmospheric refraction and telescope alignment. Errors in this astrometric model are dominated by stochastic variations with typical amplitudes of 10-30 mas (in a 30 s exposure) and 5-10 arcmin coherence length, plausibly attributed to Kolmogorov-spectrum atmospheric turbulence. The size of these atmospheric distortions is not closely related to the seeing. Given an astrometric reference catalog at density ~0.7 arcmin^{-2}, e.g. from Gaia, the typical atmospheric distortions can be interpolated to 7 mas RMS accuracy (for 30 s exposures) with 1 arcmin coherence length for residual errors. Remaining detectable error contributors are 2-4 mas RMS from unmodelled stray electric fields in the devices, and another 2-4 mas RMS from focal plane shifts between camera thermal cycles. Thus the astrometric solution for a single DECam exposure is accurate to 3-6 mas (0.02 pixels, or 300 nm) on the focal plane, plus the stochastic atmospheric distortion.Comment: Submitted to PAS

Regulation of membrane ruffling by polarized STIM1 and ORAI1in cortactin-rich domains

La movilidad celular y la migración requieren la reorganización del citoesqueleto cortical en el borde principal de las células y la entrada de Ca2 + extracelular es esencial para esta reorganización. Sin embargo, la naturaleza molecular de los reguladores de esta vía es desconocida. Este trabajo contribuye a comprender el papel de STIM1 y ORAI1 en la promoción de la ondulación de la membrana al mostrar que la fosfo-STIM1 se localiza en el borde principal de las células, y que tanto phospho-STIM1 como ORAI1 se localizan conjuntamente con la cortactina (CTTN), un regulador del citoesqueleto en las zonas de rizo de la membrana. Las líneas celulares STIM1-KO y ORAI1-KO se generaron mediante la edición del genoma CRISPR / Cas9 en células U2OS. En ambos casos, las células KO presentaron una reducción notable de la entrada de Ca2 + operada por el almacén (SOCE) que se rescató mediante la expresión de STIM1-mCherry y ORAI1-mCherry. Estos resultados demostraron que SOCE regula la deformación de la membrana en el borde anterior de las células. Por otra parte, ORAI1 endógeno y ORAI1-GFP sobreexpresado coinmuno precipitado con CTTN endógeno. Este último resultado, además del fenotipo de las células KO, la preservación de la co-localización de ORAI1-CTTN durante el fruncido, y la inhibición de la rizo de la membrana por parte del inhibidor del canal de Ca2 + SKF96365, apoya aún más un vínculo funcional entre el SOCE y el fruncido de la membrana.Cell motility and migration requires the reorganization of the cortical cytoskeleton at the leading edge of cells and extracellular Ca2+ entry is essential for this reorganization. However the molecular nature of the regulators of this pathway is unknown. This work contributes to understanding the role of STIM1 and ORAI1 in the promotion of membrane ruffling by showing that phospho-STIM1 localizes at the leading edge of cells, and that both phospho-STIM1 and ORAI1 co-localize with cortactin (CTTN), a regulator of the cytoskeleton at membrane ruffling areas. STIM1-KO and ORAI1-KO cell lines were generated by CRISPR/Cas9 genome editing in U2OS cells. In both cases, KO cells presented a notable reduction of store-operated Ca2+ entry (SOCE) that was rescued by expression of STIM1-mCherry and ORAI1-mCherry. These results demonstrated that SOCE regulates membrane ruffling at the leading edge of cells. Moreover, endogenous ORAI1 and overexpressed ORAI1-GFP co-immuno precipitated with endogenous CTTN. This latter result, in addition to the KO cells’ phenotype, the preservation of ORAI1-CTTN co-localization during ruffling, and the inhibition of membrane ruffling g by the Ca2+- channel inhibitor SKF96365, further supports a functional link between SOCE and membrane ruffling.• Ministerio de Economía y Competitividad y Fondo Social Europeo. Becas BFU2011-22798 y BFU2014-52401-P, para Francisco Javier Martín Romero • Consejo de Investigación Médica. Beca MC_UU_12016 / 2, para Darío R. Alessi • Ministerio de Economía y Competitividad. Beca BES-2012-052061, para Aida María López Guerrero • Gobierno de Extremadura. Ayuda PD10081, para Patricia Tomás Martín • Ministerio de Educación, Cultura y Deporte. Beca FPU13 / 03430, para Carlos Pascual Caro • Consejo de Investigación Médica. Ayuda MR / K015869 / 1, para Graeme Ball • EMBO. Beca ASTF-311-2014, para Eulalia Pozo Guisado • Ministerio de Educación, Cultura Española y Deporte. Beca PRX14 / 00176, para Francisco Javier Martín RomeropeerReviewe

Observation of the decay Bc→J/ψK+K−π+B_c \rightarrow J/\psi K^+ K^- \pi^+

The decay $B_c\rightarrow J/\psi K^+ K^- \pi^+$ is observed for the first time, using proton-proton collisions collected with the LHCb detector corresponding to an integrated luminosity of 3fb$^{-1}$. A signal yield of $78\pm14$ decays is reported with a significance of 6.2 standard deviations. The ratio of the branching fraction of \B_c \rightarrow J/\psi K^+ K^- \pi^+ decays to that of $B_c \rightarrow J/\psi \pi^+$ decays is measured to be $0.53\pm 0.10\pm0.05$, where the first uncertainty is statistical and the second is systematic.Comment: 18 pages, 2 figure

Measurements of the branching fractions of B+→ppK+ decays

The branching fractions of the decay B+ → pp̄K+ for different intermediate states are measured using data, corresponding to an integrated luminosity of 1.0 fb-1, collected by the LHCb experiment. The total branching fraction, its charmless component Mpp̄ < 2.85 GeV/c2 and the branching fractions via the resonant cc̄ states η c(1S) and ψ(2S) relative to the decay via a J/ψ intermediate state are [Equation not available: see fulltext.] Upper limits on the B + branching fractions into the η c(2S) meson and into the charmonium-like states X(3872) and X(3915) are also obtained

Observation of the decay B+c→Bºsπ+

The result of a search for the decay B+c→Bºsπ+ is presented, using the Bºs→Ds-π+ and Bºs→J/ψϕ channels. The analysis is based on a data sample of pp collisions collected with the LHCb detector, corresponding to an integrated luminosity of 1  fb-1 taken at a center-of-mass energy of 7 TeV, and 2  fb-1 taken at 8 TeV. The decay B+c→Bºsπ+ is observed with significance in excess of 5 standard deviations independently in both decay channels. The measured product of the ratio of cross sections and branching fraction is [σ(Bc+)/σ(Bºs)]×B(Bc+→Bºsπ+)=[2.37±0.31 (stat)±0.11 (syst)-0.13+0.17(τBc+)]×10-3, in the pseudorapidity range 2<η(B)<5, where the first uncertainty is statistical, the second is systematic, and the third is due to the uncertainty on the Bc+ lifetime. This is the first observation of a B meson decaying to another B meson via the weak interaction