Nucleosome resection at a double-strand break during Non-Homologous Ends Joining in mammalian cells - implications from repressive chromatin organization and the role of ARTEMIS

<p>Abstract</p> <p>Background</p> <p>The <it>S. cerevisiae </it>mating type switch model of double-strand break (DSB) repair, utilizing the HO endonuclease, is one of the best studied systems for both Homologous Recombination Repair (HRR) and direct ends-joining repair (Non-Homologous Ends Joining - NHEJ). We have recently transposed that system to a mammalian cell culture model taking advantage of an adenovirus expressing HO and an integrated genomic target. This made it possible to compare directly the mechanism of repair between yeast and mammalian cells for the same type of induced DSB. Studies of DSB repair have emphasized commonality of features, proteins and machineries between organisms, and differences when conservation is not found. Two proteins that stand out that differ between yeast and mammalian cells are DNA-PK, a protein kinase that is activated by the presence of DSBs, and Artemis, a nuclease whose activity is modulated by DNA-PK and ATM. In this report we describe how these two proteins may be involved in a specific pattern of ends-processing at the DSB, particularly in the context of heterochromatin.</p> <p>Findings</p> <p>We previously published that the repair of the HO-induced DSB was generally accurate and occurred by simple rejoining of the cohesive 3'-overhangs generated by HO. During continuous passage of those cells in the absence of puromycin selection, the locus appears to have become more heterochromatic and silenced by displaying several features. 1) The site had become less accessible to cleavage by the HO endonuclease; 2) the expression of the puro mRNA, which confers resistance to puromycin, had become reduced; 3) occupancy of nucleosomes at the site (ChIP for histone H3) was increased, an indicator for more condensed chromatin. After reselection of these cells by addition of puromycin, many of these features were reversed. However, even the reselected cells were not identical in the pattern of cleavage and repair as the cells when originally created. Specifically, the pattern of repair revealed discrete deletions at the DSB that indicated unit losses of nucleosomes (or other protein complexes) before religation, represented by a ladder of PCR products reminiscent of an internucleosomal cleavage that is typically observed during apoptosis. This pattern of cleavage suggested to us that perhaps, Artemis, a protein that is believed to generate the internucleosomal fragments during apoptosis and in DSB repair, was involved in that specific pattern of ends-processing. Preliminary evidence indicates that this may be the case, since knock-down of Artemis with siRNA eliminated the laddering pattern and revealed instead an extensive exonucleolytic processing of the ends before religation.</p> <p>Conclusions</p> <p>e have generated a system in mammalian cells where the absence of positive selection resulted in chromatin remodeling at the target locus that recapitulates many of the features of the mating-type switching system in yeast. Specifically, just as for yeast HML and HMR, the locus had become transcriptionally repressed; accessibility to cleavage by the HO endonuclease was reduced; and processing of the ends was drastically changed. The switch was from high-fidelity religation of the cohesive ends, to a pattern of release of internucleosomal fragments, perhaps in search of micro-homology stretches for ligation. This is consistent with reports that the involvement of ATM, DNA-PK and Artemis in DSB repair is largely focused to heterochromatic regions, and not required for the majority of IR-induced DSB repair foci in euchromatin.</p

The OH Megamaser galaxy IRAS11506−3851 : an AGN and star formation revealed by multiwavelength observations

We present Gemini Multi-Object Spectrograph (GMOS) Integral Field Unit (IFU), Hubble Space Telescope (HST), and Very Large Array (VLA) observations of the OH Megamaser (OHM) galaxy IRAS 11506−3851. The HST images reveal an isolated spiral galaxy and the combination with the GMOS-IFU flux distributions and VLA data allow us to identify a partial ring of star-forming regions surrounding the nucleus with a radius of ≈ 500 pc. While this ring shows starburst excitation and low velocity dispersion, the region internal to the ring shows higher excitation and velocity dispersion values, with values increasing towards its borders at ≈ 240 pc from the nucleus, resembling a projected bubble. The enhanced excitation and velocity dispersion of this bubble surrounds a 8.5-GHz radio emission structure, supporting its origin in a faint active galactic nucleus (AGN) that is mostly shocking the surrounding gas via a plasma ejection seen in radio at the present stage. This is the fourth of the 5 OHM galaxies we have studied so far (from our sample of 15 OHM) for which GMOS-IFU data indicate the presence of a previously unknown faint AGN at the nucleus, consistent with the hypothesis that OHM galaxies harbour recently triggered AGN

The National Security Implications of HIV/AIDS

Feldbaum and colleagues look at evidence on the links between HIV and national security, and evaluate the risks and benefits of addressing HIV/AIDS as a national security issue

Theory of hot electrons: general discussion

Sylwester Gawinkowski opened discussion of the paper by Javier Aizpurua: How many electrons are transferred by one ultrashort pulse? If you are thinking about applications in electronics, then it seems that this number may be too small. Is simply increasing the pulse energy and therefore significantly increasing the number of such emitted electrons possible

Gemini IFU, VLA, and HST observations of the OH megamaser galaxy IRAS F23199 + 0123 : the hidden monster and its outflow

We present Gemini Multi-Object Spectrograph (GMOS) Integral field Unit (IFU), Very Large Array (VLA), and Hubble Space Telescope (HST) observations of the OH megamaser (OHM) galaxy IRAS F23199+0123. Our observations show that this system is an interacting pair, with two OHM sources associated with the eastern (IRAS 23199E) member. The two members of the pair present somewhat extended radio emission at 3 and 20 cm, with flux peaks at each nucleus. The GMOS-IFU observations cover the inner ∼6 kpc of IRAS 23199E at a spatial resolution of 2.3 kpc. The GMOS-IFU flux distributions in Hα and [N II] λ6583 are similar to that of an HST [N II]+Hα narrow-band image, being more extended along the north-east–southwest direction, as also observed in the continuum HST F814W image. The GMOS-IFU Hα flux map of IRAS 23199E shows three extranuclear knots attributed to star-forming complexes. We have discovered a Seyfert 1 nucleus in this galaxy, as its nuclear spectrum shows an unresolved broad (full width at half-maximum ≈2170 km s−1) double-peaked Hα component, from which we derive a black hole mass of MBH = 3.8+0.3−0.2× 106M . The gas kinematics shows low velocity dispersions (σ) and low [NII]/Hα ratios for the star-forming complexes and higher σ and [N II]/Hα surrounding the radio emission region, supporting interaction between the radio plasma and ambient gas. The two OH masers detected in IRAS F23199E are observed in the vicinity of these enhanced σ regions, supporting their association with the active nucleus and its interaction with the surrounding gas. The gas velocity field can be partially reproduced by rotation in a disc, with residuals along the north–south direction being tentatively attributed to emission from the front walls of a bipolar outflow

Gemini IFU, VLA, and HST observations of the OH Megamaser Galaxy IRAS17526 + 3253

We present a multiwavelength study of the OH megamaser galaxy IRAS17526 + 3253, based on new Gemini multi-object spectrograph integral field unit (GMOS/IFU) observations, Hubble Space Telescope F814W, and H α + [N II] images, and archival 2MASS and 1.49 GHz VLA data. The Hubble Space Telescope (HST) images clearly reveal a mid-to-advanced stage major merger whose northwestern and southeastern nuclei have a projected separation of ∼8.5 kpc. Our HST/H α + [N II] image shows regions of ongoing star formation across the envelope on∼10 kpc scales, which are aligned with radio features, supporting the interpretation that the radio emission originates from star-forming regions. The measured H α luminosities imply that the unobscured star formation rate (SFR) is ∼10–30 M yr−1. The GMOS/IFU data reveal two structures in northwestern separated by 850 pc and by a discontinuity in the velocity field of ∼ 200 km s−1. We associate the blueshifted and redshifted components with, respectively, the distorted disc of northwestern and tidal debris, possibly a tail originating in southeastern. Star formation is the main ionization source in both components, which have SFRs of ∼2.6–7.9 M yr−1 and ∼1.5–4.5 M yr−1, respectively. Fainter line emission bordering these main components is consistent with shock ionization at a velocity ∼200 km s−1 and may be the result of an interaction between the tidal tail and the northwestern galaxy’s disc. IRAS17526 + 3253 is one of only a few systems known to host both luminous OH and H2O masers. The velocities of the OH and H2O maser lines suggest that they are associated with the northwestern and southeastern galaxies, respectively (Martin et al.; Wagner)

Highly contractile 3D tissue engineered skeletal muscles from human iPSCs reveal similarities with primary myoblast-derived tissues

Skeletal muscle research is transitioning toward 3D tissue engineered in vitro models reproducing muscle's native architecture and supporting measurement of functionality. Human induced pluripotent stem cells (hiPSCs) offer high yields of cells for differentiation. It has been difficult to differentiate high-quality, pure 3D muscle tissues from hiPSCs that show contractile properties comparable to primary myoblast-derived tissues. Here, we present a transgene-free method for the generation of purified, expandable myogenic progenitors (MPs) from hiPSCs grown under feeder-free conditions. We defined a protocol with optimal hydrogel and medium conditions that allowed production of highly contractile 3D tissue engineered skeletal muscles with forces similar to primary myoblast-derived tissues. Gene expression and proteomic analysis between hiPSC-derived and primary myoblast-derived 3D tissues revealed a similar expression profile of proteins involved in myogenic differentiation and sarcomere function. The protocol should be generally applicable for the study of personalized human skeletal muscle tissue in health and disease.</p

Low-smoke chulha in Indian slums: study protocol for a randomised controlled trial

Background Biomass fuel is used as a primary cooking source by more than half of the world’s population, contributing to a high burden of disease. Although cleaner fuels are available, some households continue using solid fuels because of financial constraints and absence of infrastructure, especially in non-notified slums. The present study documents a randomised controlled study investigating the efficacy of improved cookstove on the personal exposure to air pollution and the respiratory health of women and children in an Indian slum. The improved cookstove was based on co-creation of a low-smoke chulha with local communities in order to support adaption and sustained uptake. Methods The study will be conducted in a non-notified slum called Ashrayanagar in Bangalore, India. The study design will be a 1:1 randomised controlled intervention trial, including 250 households. The intervention group will receive an improved cookstove (low-smoke chulha) and the control group will continue using either the traditional cookstove (chulha) or a combination of the traditional stove and the kerosene/diesel stove. Follow-up time is 1 year. Outcomes include change in lung function (FEV1/FVC), incidence of pneumonia, change in personal PM2.5 and CO exposure, incidence of respiratory symptoms (cough, phlegm, wheeze and shortness of breath), prevalence of other related symptoms (headache and burning eyes), change in behaviour and adoption of the stove. Ethical clearance was obtained from the Institutional Ethics Committee of the Indian Institute of Public Health Hyderabad- Bengaluru Campus. Discussion The findings from this study aim to provide insight into the effects of improved cookstoves in urban slums. Results can give evidence for the decrease of indoor air pollution and the improvement of respiratory health for children and women. Trial registration The trial was registered with clinicaltrials.gov on 21 June 2016 with the identifier NCT02821650; A Study to Test the Impact of an Improved Chulha on the Respiratory Health of Women and Children in Indian Slums

Ladakh: Diverse, high-altitude extreme environments for off-earth analogue and astrobiology research

This paper highlights unique sites in Ladakh, India, investigated during our 2016 multidisciplinary pathfinding expedition to the region. We summarize our scientific findings and the site's potential to support science exploration, testing of new technologies and science protocols within the framework of astrobiology research. Ladakh has several accessible, diverse, pristine and extreme environments at very high altitudes (3000-5700 m above sea level). These sites include glacial passes, sand dunes, hot springs and saline lake shorelines with periglacial features. We report geological observations and environmental characteristics (of astrobiological significance) along with the development of regolith-landform maps for cold high passes. The effects of the diurnal water cycle on salt deliquescence were studied using the ExoMars Mission instrument mockup: HabitAbility: Brines, Irradiance and Temperature (HABIT). It recorded the existence of an interaction between the diurnal water cycle in the atmosphere and salts in the soil (which can serve as habitable liquid water reservoirs). Life detection assays were also tested to establish the best protocols for biomass measurements in brines, periglacial ice-mud and permafrost melt water environments in the Tso-Kar region. This campaign helped confirm the relevance of clays and brines as interest targets of research on Mars for biomarker preservation and life detection.The team would like to express its gratitude to BirbalSahni Institute of Palaeosciences, Department of Science and Technology,Office of Chief Wildlife Warden of Ladakh, Government of India for helpingarrange the requisite clearances and permits for the conducted work. Projectmentoring and guidance provided by Spaceward Bound members at NASAAmes Research Center. Financial and logistics support provided by TataMotors Ltd, Inspired Journeys Co, Pearl Travels Ltd and NationalGeographic Traveller India. Website and IT support provided by the BlueMarble Space Institute of Science. Audio-video documentation support pro-vided by Astroproject India and The H

Support for UNRWA's survival

The United Nations Relief and Works Agency for Palestine Refugees in the Near East (UNRWA) provides life-saving humanitarian aid for 5·4 million Palestine refugees now entering their eighth decade of statelessness and conflict. About a third of Palestine refugees still live in 58 recognised camps. UNRWA operates 702 schools and 144 health centres, some of which are affected by the ongoing humanitarian disasters in Syria and the Gaza Strip. It has dramatically reduced the prevalence of infectious diseases, mortality, and illiteracy. Its social services include rebuilding infrastructure and homes that have been destroyed by conflict and providing cash assistance and micro-finance loans for Palestinians whose rights are curtailed and who are denied the right of return to their homeland