Identification and Characterization of the DNA Damage Response Function of Human Rif1 and a Search For Human Rap1 Interacting Factors

Rifl, the ortholog of the yeast Rapl interacting factor 1, a telomere length regulator was identified. Immunofluorescence did not reveal an association of human Rifl with telomeres. Preliminary co-immunoprecipitation with known telomeric proteins and chromatin immunoprecipitation failed to show an interaction of human Rifl with telomeric protein complexes or with telomeric DNA. Unexpectedly, Rifl responded to ionizing radiation (IR), UV light, and clastogens, forming foci that co-localized with other DNA damage response factors such as 53BP1, ATM, BRCA1, Chkl, Nbsl, and Rad 17. Furthermore, Rifl localized to uncapped telomeres, as do other DNA damage response factors. Among DNA damage response proteins, Rifl showed a unique dependence on the ATM kinase. Whereas inhibition of ATR signaling did not suppress the Rifl response, ATM deficient cells treated with IR or UV lacked Rifl foci even after prolonged incubation or high radiation dose. Therefore, IR-induced Rifl foci constitute an assay for ATM status. The Rifl response also depended on the presence of 53BP1 but was not affected by reduced function of BRCA1, Chk2, Nbsl, and Mrell. RNAimediated Rifl inhibition resulted in increased radiosensitivity, indicating that Rifl contributes to ATM-mediated protection against exposure to ionizing radiation. In order to more fully understand telomere length regulation in mammalian cells and the evolutionary history of telomere-binding complexes, a search for novel hRapl -binding proteins and studies of the Rapl telomere complex at human telomeres were performed. Both genetic and biochemical methods were used in order to better understand the role of the Rapl complex, consisting of hRapl, TRF2, and the Mrell complex, in human telomere biology. A yeast two-hybrid screen was carried out using hRapl as a bait and two genes were identified. The first gene, called candidate 144 (C144), encodes a novel zinc-finger containing protein. Immunofluorescence and immunoprecipitation failed to reveal an association of C144 with telomeres. Deletion of the open-reading frame YDL175c in S. cerevisiae, the ortholog of C144, did not result in detectable changes in telomere length. The second gene, called FLASH, was previously identified to play a role as an adaptor in ligand-mediated apoptosis. In immunofluorescence experiments, FLASH did not co-localize with known telomeric proteins

The Coordination Chemistry of the Rhodizonate Dianion and Its Decomposition Products

The rhodizonate dianion is of importance for forensic testing, specifically with regard to the sodium rhodizonate test for lead in gunpowder residues. In an effort to understand this test from a chemical perspective, the coordination of rhodizonate and its decomposition products, croconate and oxalate, with several metals has been investigated with a variety of structural and spectroscopic methods. FT-IR data of the three sequential steps of the sodium rhodizonate test show coordination between Pb(II) atoms, tartrate anions, and rhodizonate dianions, with the tartrate being substituted for Cl- once a 5% HCl solution is added to produce the confirmatory blue-purple color for Pb. XPS data confirms the coordination of Cl- to Pb(II) with a shift from the major XPS peak of the Pb 4f7/2 peak from 138.6 eV to 139.0 eV. UV-Vis titrations show that Pb(II) and rhodizonate bind in a 1:1 ratio in water/ethanol (50/50 by volume). This solvent system was essential for generating these results, as in pure water, the formed Pb-rhodizonate complex immediately precipitates. Rhodizonate was also shown to complex with lanthanides. UV-Vis titrations show that rhodizonate coordinates to lanthanides in 1:1 ratio in water/ethanol (50/50 by volume). However, rhodizonate has higher affinity for Pb(II) over several Ln(III). Metal-rhodizonates with N-donating co-ligands such as 1,10-phenanthroline, 2,2’-bipyridine, and imidazole, were also shown to coordinate to toxic metals, such as Hg(II) in a 1:1 metal-rhodizonate/co-ligand ratio in water/ethanol (50/50 by volume), regardless of whether the co-ligand was added to a solution of metal-rhodizonate, or the metal was added to a solution of rhodizonate and co-ligand. Dilute solutions (~10-5 M) of sodium rhodizonate, which are suitable for UV-Vis spectroscopic work will typically decompose and lose their characteristic orange color in about 2 h, though full decomposition into croconate takes upwards of one week. We have now shown that by dissolving rhodizonate in water/ethanol (50/50 by volume), the stability of rhodizonate in solution is extended by several hours, allowing for accurate UV-Vis spectroscopic measurements, titration studies, and determination of binding constants. Crystals of the five-member croconate, a decomposition product of rhodizonate, with Pb(II) and Cd(II) show marked differences. Pb(II)-croconate crystals feature 3D-layers of alternating Pb(II) and croconate, while Cd(II)-croconate crystals feature 1D-layers of alternating Cd(II) and croconate held together through a hydrogen bonding network. Metal-rhodizonate solutions left over longer times will further decompose into oxalates, which can then form, 3D metal-oxalate frameworks. The 3D structures are unique among metal oxalates, forming large pores, in contrast to 1D and 2D metal-oxalates previously reported in the literature

A Highly Consistent Framework for the Evolution of the Star-Forming "Main Sequence" from z~0-6

Using a compilation of 25 studies from the literature, we investigate the evolution of the star-forming galaxy (SFG) Main Sequence (MS) in stellar mass and star formation rate (SFR) out to $z \sim 6$. After converting all observations to a common set of calibrations, we find a remarkable consensus among MS observations ($\sim 0.1$ dex 1$\sigma$ interpublication scatter). By fitting for time evolution of the MS in bins of constant mass, we deconvolve the observed scatter about the MS within each observed redshift bins. After accounting for observed scatter between different SFR indicators, we find the width of the MS distribution is $\sim 0.2$ dex and remains constant over cosmic time. Our best fits indicate the slope of the MS is likely time-dependent, with our best fit $\log\textrm{SFR}(M_*,t) = \left(0.84 \pm 0.02 - 0.026 \pm 0.003 \times t\right) \log M_* - \left(6.51 \pm 0.24 - 0.11 \pm 0.03 \times t\right)$, with $t$ the age of the Universe in Gyr. We use our fits to create empirical evolutionary tracks in order to constrain MS galaxy star formation histories (SFHs), finding that (1) the most accurate representations of MS SFHs are given by delayed-$\tau$ models, (2) the decline in fractional stellar mass growth for a "typical" MS galaxy today is approximately linear for most of its lifetime, and (3) scatter about the MS can be generated by galaxies evolving along identical evolutionary tracks assuming an initial $1\sigma$ spread in formation times of $\sim 1.4$ Gyr.Comment: 59 pages, 10 tables, 12 figures, accepted to ApJS; v2, slight changes to text, added new figure and fit

Gameplay, Interactive Drama, and Training: Authoring Edutainment Stories for Online Players (AESOP)

This paper describes initial efforts at providing some of the technological advances of the videogame genres in a coherent, accessible format to teams of educators. By providing these capabilities inside an interactive drama generator, we believe that the full potential of educational games may eventually be realized. Sections 1 and 2 postulate three goals for reaching that objective: a toolset for interactive drama authoring, ways to insulate authors from game engines, and reusable digital casts to facilitate composability. Sections 3 and 4 present progress on those tools and an in-depth case study that made use of the resulting toolset to create a large interactive drama. We close with lessons learned to date and a look at the remaining challenges: the unpleasant reality that state-of-the-art tools are not yet able to boost the productivity of edutainment authors

Authoring Edutainment Stories for Online Players (AESOP): Introducing Gameplay into Interactive Dramas

The video gaming industry has experienced extraordinary technological growth in the recent past, causing a boom in both the quality and revenue of these games. Educational games, on the other hand, have lagged behind this trend, as their creation presents major creative and pedagogical challenges in addition to technological ones. By providing the technological advances of the entertainment genres in a coherent, accessible format to teams of educators, and developing an interactive drama generator, we believe that the full potential of educational games can be realized. Section 1 postulates three goals for reaching that objective: a toolset for interactive drama authoring, ways to insulate authors from game engines, and reusable digital casts to facilitate composability. Sections 2 and 3 present progress on simple versions of those tools and a case study that made use of the resulting toolset to create an interactive drama

Lineage A betacoronavirus NS2 proteins and the homologous torovirus Berne pp1a carboxy-terminal domain are phosphodiesterases that antagonize activation of RNase L

Viruses in the family Coronaviridae, within the order Nidovirales, are etiologic agents of a range of human and animal diseases, including both mild and severe respiratory diseases in humans. These viruses encode conserved replicase and structural proteins as well as more diverse accessory proteins, encoded in the 3′ ends of their genomes, that often act as host cell antagonists. We previously showed that 2′,5′-phosphodiesterases (2′,5′-PDEs) encoded by the prototypical Betacoronavirus, mouse hepatitis virus (MHV), and by Middle East respiratory syndrome-associated coronavirus antagonize the oligoadenylate-RNase L (OAS-RNase L) pathway. Here we report that additional coronavirus superfamily members, including lineage A betacoronaviruses and toroviruses infecting both humans and animals, encode 2′,5′-PDEs capable of antagonizing RNase L. We used a chimeric MHV system (MHV(Mut)) in which exogenous PDEs were expressed from an MHV backbone lacking the gene for a functional NS2 protein, the endogenous RNase L antagonist. With this system, we found that 2′,5′-PDEs encoded by the human coronavirus HCoV-OC43 (OC43; an agent of the common cold), human enteric coronavirus (HECoV), equine coronavirus (ECoV), and equine torovirus Berne (BEV) are enzymatically active, rescue replication of MHV(Mut) in bone marrow-derived macrophages, and inhibit RNase L-mediated rRNA degradation in these cells. Additionally, PDEs encoded by OC43 and BEV rescue MHV(Mut) replication and restore pathogenesis in wild-type (WT) B6 mice. This finding expands the range of viruses known to encode antagonists of the potent OAS-RNase L antiviral pathway, highlighting its importance in a range of species as well as the selective pressures exerted on viruses to antagonize it. IMPORTANCE Viruses in the family Coronaviridae include important human and animal pathogens, including the recently emerged viruses severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and Middle East respiratory syndrome-associated coronavirus (MERS-CoV). We showed previously that two viruses within the genus Betacoronavirus, mouse hepatitis virus (MHV) and MERS-CoV, encode 2′,5′-phosphodiesterases (2′,5′-PDEs) that antagonize the OAS-RNase L pathway, and we report here that these proteins are furthermore conserved among additional coronavirus superfamily members, including lineage A betacoronaviruses and toroviruses, suggesting that they may play critical roles in pathogenesis. As there are no licensed vaccines or effective antivirals against human coronaviruses and few against those infecting animals, identifying viral proteins contributing to virulence can inform therapeutic development. Thus, this work demonstrates that a potent antagonist of host antiviral defenses is encoded by multiple and diverse viruses within the family Coronaviridae, presenting a possible broad-spectrum therapeutic target

Terpene Metabolic Engineering \u3cem\u3evia\u3c/em\u3e Nuclear or Chloroplast Genomes Profoundly and Globally Impacts Off-Target Pathways Through Metabolite Signalling

The impact of metabolic engineering on nontarget pathways and outcomes of metabolic engineering from different genomes are poorly understood questions. Therefore, squalene biosynthesis genes FARNESYL DIPHOSPHATE SYNTHASE (FPS) and SQUALENE SYNTHASE (SQS) were engineered via the Nicotiana tabacum chloroplast (C), nuclear (N) or both (CN) genome to promote squalene biosynthesis. SQS levels were ~4300-fold higher in C and CN lines than in N, but all accumulated ~150-fold higher squalene due to substrate or storage limitations. Abnormal leaf and flower phenotypes, including lower pollen production and reduced fertility, were observed regardless of the compartment or level of transgene expression. Substantial changes in metabolomes of all lines were observed: levels of 65-120 unrelated metabolites, including the toxic alkaloid nicotine, changed by as much as 32-fold. Profound effects of transgenesis on nontarget gene expression included changes in the abundance of 19 076 transcripts by up to 2000-fold in CN; 7784 transcripts by up to 1400-fold in N; and 5224 transcripts by as much as 2200-fold in C. Transporter-related transcripts were induced, and cell cycle-associated transcripts were disproportionately repressed in all three lines. Transcriptome changes were validated by qRT-PCR. The mechanism underlying these large changes likely involves matabolite-mediated anterograde and/or retrograde signalling irrespective of the level of transgene expression or end product, due to imbalance of metabolic pools, offering new insight into both anticipated and unanticipated consequences of metabolic engineering

A High-Resolution Spectroscopic Search for the Remaining Donor for Tycho's Supernova

In this paper, we report on our analysis using Hubble Space Telescope astrometry and Keck-I HIRES spectroscopy of the central six stars of Tycho's supernova remnant (SN 1572). With these data, we measured the proper motions, radial velocities, rotational velocities, and chemical abundances of these objects. Regarding the chemical abundances, we do not confirm the unusu- ally high [Ni/Fe] ratio previously reported for Tycho-G. Rather, we find that for all metrics in all stars, none exhibit the characteristics expected from traditional SN Ia single-degenerate-scenario calculations. The only possible exception is Tycho-B, a rare, metal-poor A-type star; however, we are unable to find a suitable scenario for it. Thus, we suggest that SN 1572 cannot be explained by the standard single-degenerate model.Comment: 34 pages, 11 Figures, revised and resubmitted to Ap

Heating Injection Drug Preparation Equipment Used for Opioid Injection May Reduce HIV Transmission Associated with Sharing Equipment

London, Canada, experienced an HIV outbreak among persons who inject drugs despite widespread distribution of harm reduction equipment. Hydromorphone controlled-release (HMC) is the local opioid of choice. Injection drug preparation equipment (IDPE; ie, cookers and filters) is often shared and reused because of the perception that there is residual HMC in the IDPE after use. The purpose of this study was to investigate the mechanisms of HIV transmission in this context.Methods:Residual hydromorphone, (controlled-release or immediate-release), remaining in the IDPE, was measured with liquid chromatography-tandem mass spectrometry, in conditions replicating persons who inject drug use. HIV was added to IDPE in the presence HMC, hydromorphone immediate-release, or microcrystalline cellulose (an HMC drug excipient). HIV viral persistence was measured by reverse transcriptase activity and infectivity of indicator Tzm-bl cells.Results:Forty-five percent of HMC remained in the IDPE after the first aspiration of solution, with no change after heating. HIV persistence and infectivity were preserved in the presence of HMC, and less so with microcrystalline cellulose. Heating the IDPE rapidly inactivated HIV.Conclusions:Sharing of IDPE is a potential means of HIV transmission. HMC encourages IDPE sharing because of the residual drug in the IDPE, and the HMC excipients preserve HIV viability. Heating IDPE before aspiration of the opioid may be a harm reduction strategy

The Massive Progenitor of the Type II-Linear Supernova 2009kr

We present early-time photometric and spectroscopic observations of supernova (SN) 2009kr in NGC 1832. We find that its properties to date support its classification as Type II-linear (SN II-L), a relatively rare subclass of core-collapse supernovae (SNe). We have also identified a candidate for the SN progenitor star through comparison of pre-explosion, archival images taken with WFPC2 on board the Hubble Space Telescope with SN images obtained using adaptive optics plus NIRC2 on the 10 m Keck-II telescope. Although the host galaxy's substantial distance (similar to 26 Mpc) results in large uncertainties in the relative astrometry, we find that if this candidate is indeed the progenitor, it is a highly luminous (M(V)(0) = -7.8 mag) yellow supergiant with initial mass similar to 18-24 M(circle dot). This would be the first time that an SN II-L progenitor has been directly identified. Its mass may be a bridge between the upper initial mass limit for the more common Type II-plateau SNe and the inferred initial mass estimate for one Type II-narrow SN.Hungarian OTKA K76816NSF AST-0707769, AST-0908886Sylvia & Jim Katzman FoundationTABASGO FoundationNASA through STScI AR-11248, GO-10877Harvard UniversityUC BerkeleyUniversity of VirginiaNASA/Swift NNX09AQ66GDOEAstronom