Regional gene repression by DNA double-strand breaks in G1 phase cells

DNA damage responses (DDR) to double-strand breaks (DSBs) alter cellular transcription programs at the genome-wide level. Through processes that are less well understood, DSBs also alter transcriptional responses locally, which may be important for efficient DSB repair. Here, we developed an approach to elucidate th

Inhibition of the PI3K/AKT/mTOR pathway activates autophagy and compensatory Ras/Raf/MEK/ERK signalling in prostate cancer

The PI3K/AKT/mTOR pathway is frequently activated in advanced prostate cancer, due to loss of the tumour suppressor PTEN, and is an important axis for drug development. We have assessed the molecular and functional consequences of pathway blockade by inhibiting AKT and mTOR kinases either in combination or as individual drug treatments. In established prostate cancer cell lines, a decrease in cell viability and in phospho-biomarker expression was observed. Although apoptosis was not induced, a G1 growth arrest was observed in PTEN null LNCaP cells, but not in BPH1 or PC3 cells. In contrast, when the AKT inhibitor AZD7328 was applied to patient-derived prostate cultures that retained expression of PTEN, activation of a compensatory Ras/MEK/ERK pathway was observed. Moreover, whilst autophagy was induced following treatment with AZD7328, cell viability was less affected in the patient-derived cultures than in cell lines. Surprisingly, treatment with a combination of both AZD7328 and two separate MEK1/2 inhibitors further enhanced phosphorylation of ERK1/2 in primary prostate cultures. However, it also induced irreversible growth arrest and senescence.Ex vivo treatment of a patient-derived xenograft (PDX) of prostate cancer with a combination of AZD7328 and the mTOR inhibitor KU-0063794, significantly reduced tumour frequency upon re-engraftment of tumour cells.The results demonstrate that single agent targeting of the PI3K/AKT/mTOR pathway triggers activation of the Ras/MEK/ERK compensatory pathway in near-patient samples. Therefore, blockade of one pathway is insufficient to treat prostate cancer in man

High-throughput, quantitative analyses of genetic interactions in E. coli.

Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor-driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli

Ultraviolet Signposts of Resonant Dynamics in the Starburst-Ringed Sab Galaxy, M94 (NGC 4736)

M94 (NGC 4736) is investigated using images from the Ultraviolet Imaging Telescope (FUV-band), Hubble Space Telescope (NUV-band), Kitt Peak 0.9-m telescope (H-alpha, R, and I bands), and Palomar 5-m telescope (B-band), along with spectra from the International Ultraviolet Explorer and Lick 1-m telescopes. The wide-field UIT image shows FUV emission from (a) an elongated nucleus, (b) a diffuse inner disk, where H-alpha is observed in absorption, (c) a bright inner ring of H II regions at the perimeter of the inner disk (R = 48 arcsec. = 1.1 kpc), and (d) two 500-pc size knots of hot stars exterior to the ring on diametrically opposite sides of the nucleus (R= 130 arcsec. = 2.9 kpc). The HST/FOC image resolves the NUV emission from the nuclear region into a bright core and a faint 20 arcsec. long ``mini-bar'' at a position angle of 30 deg. Optical and IUE spectroscopy of the nucleus and diffuse inner disk indicates an approximately 10^7 or 10^8 yr-old stellar population from low-level starbirth activity blended with some LINER activity. Analysis of the H-alpha, FUV, NUV, B, R, and I-band emission along with other observed tracers of stars and gas in M94 indicates that most of the star formation is being orchestrated via ring-bar dynamics involving the nuclear mini-bar, inner ring, oval disk, and outer ring. The inner starburst ring and bi-symmetric knots at intermediate radius, in particular, argue for bar-mediated resonances as the primary drivers of evolution in M94 at the present epoch. Similar processes may be governing the evolution of the ``core-dominated'' galaxies that have been observed at high redshift. The gravitationally-lensed ``Pretzel Galaxy'' (0024+1654) at a redshift of approximately 1.5 provides an important precedent in this regard.Comment: revised figure 1 (corrected coordinate labels on declination axis); 19 pages of text + 19 figures (jpg files); accepted for publication in A

Using screen video capture software to aide and inform cognitive interviewing

Web-based surveys are a salient tool in the repertoire of social and behavioral scientists. The increase in web-based surveys is understandable considering the distinct advantages offered, including: (a) lower costs and reduced labor time, (b) ability to directly transfer data into statistical packages (reducing coding errors), (c) customization options enabling more attractive presentation, (d) ability to reduce respondent burden by embedding skip patterns, and (e) access to larger sample sizes in different geographic regions. It is important to note, however, that administering web-based surveys also introduces distinct sources of error (e.g., coverage, sampling and non-response). Regardless of format (e.g., paper-and-pencil or web-based), specific, prescribed steps must be followed when constructing an instrument in order to reduce survey error and lend credence to the data collected before subsequent analysis is performed. One of those crucial stages integral to the pretesting process is cognitive interviewing. Cognitive interviewing is a qualitative process, encompassing two main techniques: think aloud interviewing and verbal probing. Collectively, these two methods seek to (a) produce information on what the respondent is thinking while answering the questions, (b) the cognitive processes used to answer the questions, and (c) how the respondent answers the questions. The purpose of this article is to provide a practical guide outlining how Camtasia, a screen video capture software, can aide and inform the cognitive interview process

The impact of BCG strains and repeat vaccinations on immunodiagnostic tests in Eurasian badgers (Meles meles)

Publication history: Accepted - 29 June 2022; Published online - 9July 2022.Bacille Calmette-Guerin (BCG) is a potential tool in the control of Mycobacterium bovis in European badgers (Meles meles). A five year Test and Vaccinate or Remove (TVR) research intervention project commenced in 2014 using two BCG strains (BCG Copenhagen 1331 (Years 1–3/ BadgerBCG) and BCG Sofia SL2222 (Years 4–5). Badgers were recaptured around 9 weeks after the Year 5 vaccination and then again a year later. The Dual-Path Platform (DPP) Vet TB assay was used to detect serological evidence of M. bovis infection. Of the 48 badgers, 47 had increased Line 1 readings (MPB83 antigen) between the Year 5 vaccination and subsequent recapture. The number of BCG Sofia vaccinations influenced whether a badger tested positive to the recapture DPP VetTB assay Line 1 (p < 0.001) while the number of BadgerBCG vaccinations did not significantly affect recapture Line 1 results (p = 0.59). Line 1 relative light units (RLU) were more pronounced in tests run with sera than whole blood. The results from an in_house MPB83 ELISA results indicated that the WB DPP VetTB assay may not detect lower MPB83 IgG levels as well as the serum DPP VetTB assay. Changes in interferon gamma assay (IFN-γ) results were seen in 2019 with significantly increased CFP-10 and PPDB readings. Unlike BadgerBCG, BCG Sofia induces an immune response to MPB83 (the immune dominant antigen in M. bovis badger infection) that then affects the use of immunodiagnostic tests. The use of the DPP VetTB assay in recaptured BCG Sofia vaccinated badgers within the same trapping season is precluded and caution should be used in badgers vaccinated with BCG Sofia in previous years. The results suggest that the DPP VetTB assay can be used with confidence in badgers vaccinated with BadgerBCG as a single or repeated doses.This work was funded by the Department of Agriculture, Environment and Rural Affairs, Northern Ireland (DAERA)