Marek's disease virus-encoded miR-155 ortholog critical for the induction of lymphomas is not essential for the proliferation of transformed cell lines

MicroRNAs (miRNAs) are small noncoding RNAs with profound regulatory roles in many areas of biology, including cancer. MicroRNA 155 (miR-155), one of the extensively studied multifunctional miRNAs, is important in several human malignancies such as diffuse large B cell lymphoma and chronic lymphocytic leukemia. Moreover, miR-155 orthologs KSHV-miR-K12-11 and MDV-miR-M4, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) and Marek's disease virus (MDV), respectively, are also involved in oncogenesis. In MDV-induced T-cell lymphomas and in lymphoblastoid cell lines derived from them, MDV-miR-M4 is highly expressed. Using excellent disease models of infection in natural avian hosts, we showed previously that MDV-miR-M4 is critical for the induction of T-cell lymphomas as mutant viruses with precise deletions were significantly compromised in their oncogenicity. However, those studies did not elucidate whether continued expression of MDV-miR-M4 is essential for maintaining the transformed phenotype of tumor cells. Here using an in situ CRISPR/Cas9 editing approach, we deleted MDV-miR-M4 from the MDV-induced lymphoma-derived lymphoblastoid cell line MDCC-HP8. Precise deletion of MDV-miR-M4 was confirmed by PCR, sequencing, quantitative reverse transcription-PCR (qRT-PCR), and functional analysis. Continued proliferation of the MDV-miR-M4-deleted cell lines demonstrated that MDV-miR-M4 expression is not essential for maintaining the transformed phenotype, despite its initial critical role in the induction of lymphomas. Ability to examine the direct role of oncogenic miRNAs in situ in tumor cell lines is valuable in delineating distinct determinants and pathways associated with the induction or maintenance of transformation in cancer cells and will also contribute significantly to gaining further insights into the biology of oncogenic herpesviruses.IMPORTANCE Marek's disease virus (MDV) is an alphaherpesvirus associated with Marek's disease (MD), a highly contagious neoplastic disease of chickens. MD serves as an excellent model for studying virus-induced T-cell lymphomas in the natural chicken hosts. Among the limited set of genes associated with MD oncogenicity, MDV-miR-M4, a highly expressed viral ortholog of the oncogenic miR-155, has received extensive attention due to its direct role in the induction of lymphomas. Using a targeted CRISPR-Cas9-based gene editing approach in MDV-transformed lymphoblastoid cell lines, we show that MDV-miR-M4, despite its critical role in the induction of tumors, is not essential for maintaining the transformed phenotype and continuous proliferation. As far as we know, this was the first study in which precise editing of an oncogenic miRNA was carried out in situ in MD lymphoma-derived cell lines to demonstrate that it is not essential in maintaining the transformed phenotype

A VSA search for the extended Sunyaev-Zel'dovich Effect in the Corona Borealis Supercluster

We present interferometric imaging at 33 GHz of the Corona Borealis supercluster, using the extended configuration of the Very Small Array. A total area of 24 deg^2 has been imaged, with an angular resolution of 11 arcmin and a sensitivity of 12 mJy/beam. The aim of these observations is to search for Sunyaev-Zel'dovich (SZ) detections from known clusters of galaxies in this supercluster and for a possible extended SZ decrement due to diffuse warm/hot gas in the intercluster medium. We measure negative flux values in the positions of the ten richest clusters in the region. Collectively, this implies a 3.0-sigma detection of the SZ effect. In the clusters A2061 and A2065 we find decrements of approximately 2-sigma. Our main result is the detection of two strong and resolved negative features at -70+-12 mJy/beam (-157+-27 microK) and -103+-10 mJy/beam (-230+-23 microK), respectively, located in a region with no known clusters, near the centre of the supercluster. We discuss their possible origins in terms of primordial CMB anisotropies and/or SZ signals related to either unknown clusters or to a diffuse extended warm/hot gas distribution. Our analyses have revealed that a primordial CMB fluctuation is a plausible explanation for the weaker feature (probability of 37.82%). For the stronger one, neither primordial CMB (probability of 0.33%) nor SZ can account alone for its size and total intensity. The most reasonable explanation, then, is a combination of both primordial CMB and SZ signal. Finally, we explore what characteristics would be required for a filamentary structure consisting of warm/hot diffuse gas in order to produce a significant contribution to such a spot taking into account the constraints set by X-ray data.Comment: 16 pages, 10 figures. Accepted in MNRA

Radio source calibration for the VSA and other CMB instruments at around 30 GHz

Accurate calibration of data is essential for the current generation of CMB experiments. Using data from the Very Small Array (VSA), we describe procedures which will lead to an accuracy of 1 percent or better for experiments such as the VSA and CBI. Particular attention is paid to the stability of the receiver systems, the quality of the site and frequent observations of reference sources. At 30 GHz the careful correction for atmospheric emission and absorption is shown to be essential for achieving 1 percent precision. The sources for which a 1 percent relative flux density calibration was achieved included Cas A, Cyg A, Tau A and NGC7027 and the planets Venus, Jupiter and Saturn. A flux density, or brightness temperature in the case of the planets, was derived at 33 GHz relative to Jupiter which was adopted as the fundamental calibrator. A spectral index at ~30 GHz is given for each. Cas A,Tau A, NGC7027 and Venus were examined for variability. Cas A was found to be decreasing at $0.394 \pm 0.019$ percent per year over the period March 2001 to August 2004. In the same period Tau A was decreasing at $0.22\pm 0.07$ percent per year. A survey of the published data showed that the planetary nebula NGC7027 decreased at $0.16\pm 0.04$ percent per year over the period 1967 to 2003. Venus showed an insignificant ($1.5 \pm 1.3$ percent) variation with Venusian illumination. The integrated polarization of Tau A at 33 GHz was found to be $7.8\pm 0.6$ percent at pa $= 148^\circ \pm 3^\circ$.}Comment: 13 pages, 15 figures, submitted to MNRA

Very Small Array observations of the Sunyaev-Zel'dovich effect in nearby galaxy clusters

We present VSA observations (~34GHz) on scales ~20 arcmin towards a complete, X-ray-flux-limited sample of seven clusters at redshift z<0.1. Four have significant SZ detections in the presence of CMB primordial anisotropy. We use a bayesian MCMC method for inference from the VSA data, with X-ray priors on cluster positions and temperatures, and radio priors on sources. We make assumptions of beta-model gas distributions and of hydrostatic equilibrium, to evaluate probability densities for the gas mass and total mass out to r_200. Our combined estimate of the gas fraction is 0.08^{+0.06}_{-0.04}h^{-1} The random errors are poor (note that the errors are higher than would have been obtained with the usual chi-squared method) but the control of bias is good. We have described the MCMC analysis method specifically in terms of SZ but hope the description will be of more general use. We find that the effects of primordial CMB contamination tend to be similar in the estimates of both the gas mass and total mass over our narrow range of angular scales, so that there is little effect of primordials on the gas fraction determination. Using our total mass estimates we find a normalisation of the mass-temperature relation based on the profiles from the VSA cluster pressure maps that is in good agreement with recent M-T determinations from X-ray cluster measurements.Comment: Replaces earlier version. 16 pages, 5 figures, LaTeX. Minor revisions to content, accepted by MNRAS for publicatio

Searching for non-Gaussianity in the VSA data

We have tested Very Small Array (VSA) observations of three regions of sky for the presence of non-Gaussianity, using high-order cumulants, Minkowski functionals, a wavelet-based test and a Bayesian joint power spectrum/non-Gaussianity analysis. We find the data from two regions to be consistent with Gaussianity. In the third region, we obtain a 96.7% detection of non-Gaussianity using the wavelet test. We perform simulations to characterise the tests, and conclude that this is consistent with expected residual point source contamination. There is therefore no evidence that this detection is of cosmological origin. Our simulations show that the tests would be sensitive to any residual point sources above the data's source subtraction level of 20 mJy. The tests are also sensitive to cosmic string networks at an rms fluctuation level of $105 \mu K$ (i.e. equivalent to the best-fit observed value). They are not sensitive to string-induced fluctuations if an equal rms of Gaussian CDM fluctuations is added, thereby reducing the fluctuations due to the strings network to $74 \mu K$ rms . We especially highlight the usefulness of non-Gaussianity testing in eliminating systematic effects from our data.Comment: Minor corrections; accepted for publication to MNRA

Estimating the bispectrum of the Very Small Array data

We estimate the bispectrum of the Very Small Array data from the compact and extended configuration observations released in December 2002, and compare our results to those obtained from Gaussian simulations. There is a slight excess of large bispectrum values for two individual fields, but this does not appear when the fields are combined. Given our expected level of residual point sources, we do not expect these to be the source of the discrepancy. Using the compact configuration data, we put an upper limit of 5400 on the value of f_NL, the non-linear coupling parameter, at 95 per cent confidence. We test our bispectrum estimator using non-Gaussian simulations with a known bispectrum, and recover the input values.Comment: 17 pages, 16 figures, replaced with version accepted by MNRAS. Primordial bispectrum recalculated and figure 11 change

CMB observations from the CBI and VSA: A comparison of coincident maps and parameter estimation methods

We present coincident observations of the Cosmic Microwave Background (CMB) from the Very Small Array (VSA) and Cosmic Background Imager (CBI) telescopes. The consistency of the full datasets is tested in the map plane and the Fourier plane, prior to the usual compression of CMB data into flat bandpowers. Of the three mosaics observed by each group, two are found to be in excellent agreement. In the third mosaic, there is a 2 sigma discrepancy between the correlation of the data and the level expected from Monte Carlo simulations. This is shown to be consistent with increased phase calibration errors on VSA data during summer observations. We also consider the parameter estimation method of each group. The key difference is the use of the variance window function in place of the bandpower window function, an approximation used by the VSA group. A re-evaluation of the VSA parameter estimates, using bandpower windows, shows that the two methods yield consistent results.Comment: 10 pages, 6 figures. Final version. Accepted for publication in MNRA

High sensitivity measurements of the CMB power spectrum with the extended Very Small Array

We present deep Ka-band ($\nu \approx 33$ GHz) observations of the CMB made with the extended Very Small Array (VSA). This configuration produces a naturally weighted synthesized FWHM beamwidth of $\sim 11$ arcmin which covers an $\ell$-range of 300 to 1500. On these scales, foreground extragalactic sources can be a major source of contamination to the CMB anisotropy. This problem has been alleviated by identifying sources at 15 GHz with the Ryle Telescope and then monitoring these sources at 33 GHz using a single baseline interferometer co-located with the VSA. Sources with flux densities \gtsim 20 mJy at 33 GHz are subtracted from the data. In addition, we calculate a statistical correction for the small residual contribution from weaker sources that are below the detection limit of the survey. The CMB power spectrum corrected for Galactic foregrounds and extragalactic point sources is presented. A total $\ell$-range of 150-1500 is achieved by combining the complete extended array data with earlier VSA data in a compact configuration. Our resolution of $\Delta \ell \approx 60$ allows the first 3 acoustic peaks to be clearly delineated. The is achieved by using mosaiced observations in 7 regions covering a total area of 82 sq. degrees. There is good agreement with WMAP data up to $\ell=700$ where WMAP data run out of resolution. For higher $\ell$-values out to $\ell = 1500$, the agreement in power spectrum amplitudes with other experiments is also very good despite differences in frequency and observing technique.Comment: 16 pages. Accepted in MNRAS (minor revisions