Isolation and Functional Mapping of Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein DNA-Damage Complexes

Human T-cell Leukemia Virus Type 1 (HTLV-1) is a transforming retrovirus which causes Adult T-cell Leukemia (ATL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). Cellular transformation can be caused by a single viral trans-activating protein, Tax. Tax may contribute to transformation through interaction with components of the DNA damage response pathway, promoting cellular genomic instability. We examined cellular Tax complexes in an effort to elucidate potential protein-protein interactions that can model the Tax-induced molecular events. We also investigated the role of post-translational modification in regulating Tax function. We employed a direct physical analysis of Tax complexes isolated from mammalian cells using affinity purification followed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis, in order to identify both Tax-interacting proteins as well as post-translational modifications of the Tax protein itself. We identified the DNA-dependent Protein Kinase catalytic subunit (DNA-PKcs) as a novel Tax-interacting protein. Using bioinformatics analysis, we created a database of Tax-interacting proteins and, using this tool, identified DNA-PKcs as a predicted member of the Tax complex. Physical mapping of purified Tax protein revealed novel phosphorylation sites at T48, T184, T215 and S336. Mutational analysis demonstrated phosphorylation at T215 is associated with loss of Tax trans-activation of CREB and NF-κB-responsive promoters, while T48 preferentially affects NF-κB-responsive promoters, and T184 and 5336 have no effect on these Tax functions. We confirm the presence of DNA-PKcs and the regulatory protein Ku70 in the Tax complex by co-immunoprecipitation. Tax increases phosphorylation of DNA-PKcs and co-localizes with phosphor-DNA-PKcs within nuclear Tax Speckled Structures (TSS). Cytoplasmically-localized Tax deletion mutants cause a redistribution of phosphor-DNA-PK to the cytoplasm. Tax-expressing cells harbor significantly increased DNA-PK kinase activity, as measured in an in vitro kinase assay. Inhibition of DNA-PK activity dramatically reduces Tax-induced autophosphorylation of Chk2, a known DNA-PK substrate. Suppression of DNA-PKcs expression by siRNA has no significant effect on Tax-induced G2/M arrest. Tax shows no significant effect on cellular end-joining repair as measured by a plasmid-based in vivo end-joining assay. Tax-expressing cells show delayed dissolution of damage-induced nuclear speckles containing DNA-PK, γ-H2AX and Tax, suggesting a mechanism for impaired repair response

Look Who\u27s Talking: Exploring the DEI STEM Librarianship Conversation

Librarian research on diversity, equity, and inclusion (DEI) in its various iterations dates back over a decade; however, in the context of science, technology, engineering, and math (STEM) librarianship this work still seems very limited. We collect and analyze papers published in STEM library and information science journals over the past 10 years to better understand the nature of this work. In our research we consider the content of existing conversations, what is missing from this discourse, and areas for further research

Sounds Good To Me: How Communication Mode and Framing Affect Audit Quality

In the audit process, audit associates seek explanations from clients, follow-up to corroborate or refute these explanations, and document their findings. Throughout these interactions with the client, auditors must focus on the task at hand and maintain their professional skepticism. This paper examines whether (i) communication mode (face-to-face or email) and (ii) the frame of the client’s response (as explanation or fact) affect the ability of auditors to appropriately assess the quality of client communications and follow-up when necessary. We use professional auditors and conduct a 2x2 between-participants experiment. We find that auditors receiving a face-to-face response from the client that is framed as an ‘explanation’ assess the response as higher quality than auditors in the other three conditions. As a result, these auditors are less likely to follow-up with additional questions, likely impairing audit quality and increasing audit risk. We attribute the decrease in auditor performance to distractions causing the auditors to accept the client’s story at face value rather than applying appropriate skepticism. Our recommendations are that auditor associates (a) use email communication where feasible and (b) prime themselves to listen for ‘facts’ and ask the client to respond with ‘facts’ when face-to-face meetings are unavoidable or otherwise desirable

Sounds Good to Me: How Communication Mode and Priming Affect Auditor Performance

Audit associates routinely interact with clients to request explanations and evidence regarding financial statement account balances. Client explanations may be vague or incomplete. We examine whether auditors\u27 assessments of the quality of client explanations and their decision to follow-up with the client are influenced by (1) communication modes that vary in media richness, and (2) a prime that is intended to stimulate skeptical behavior. Media richness refers to the amount of data inherent in the communication mode. We predict that richer communication modes, such as video, can be more distracting than less rich communication modes, such as email. More distracted auditors will assess the quality of the client\u27s response as higher and are less likely to follow-up with the client—potentially impairing audit quality and increasing audit risk. We predict and find that a prime that focuses auditors on the verifiability of the client\u27s response will mitigate this behavior

Physical and in silico approaches identify DNA-PK in a Tax DNA-damage response interactome

<p>Abstract</p> <p>Background</p> <p>We have initiated an effort to exhaustively map interactions between HTLV-1 Tax and host cellular proteins. The resulting Tax interactome will have significant utility toward defining new and understanding known activities of this important viral protein. In addition, the completion of a full Tax interactome will also help shed light upon the functional consequences of these myriad Tax activities. The physical mapping process involved the affinity isolation of Tax complexes followed by sequence identification using tandem mass spectrometry. To date we have mapped 250 cellular components within this interactome. Here we present our approach to prioritizing these interactions via an <it>in silico </it>culling process.</p> <p>Results</p> <p>We first constructed an <it>in silico </it>Tax interactome comprised of 46 literature-confirmed protein-protein interactions. This number was then reduced to four Tax-interactions suspected to play a role in DNA damage response (Rad51, TOP1, Chk2, 53BP1). The first-neighbor and second-neighbor interactions of these four proteins were assembled from available human protein interaction databases. Through an analysis of betweenness and closeness centrality measures, and numbers of interactions, we ranked proteins in the first neighborhood. When this rank list was compared to the list of physical Tax-binding proteins, DNA-PK was the highest ranked protein common to both lists. An overlapping clustering of the Tax-specific second-neighborhood protein network showed DNA-PK to be one of three bridge proteins that link multiple clusters in the DNA damage response network.</p> <p>Conclusion</p> <p>The interaction of Tax with DNA-PK represents an important biological paradigm as suggested via consensus findings <it>in vivo </it>and <it>in silico</it>. We present this methodology as an approach to discovery and as a means of validating components of a consensus Tax interactome.</p

Harm reduction: A missing piece to the holistic care of patients who inject drugs

BACKGROUND: The rise in injection drug use (IDU) has led to an increase in drug-related infections. Harm reduction is an important strategy for preventing infections among people who inject drugs (PWID). We attempted to evaluate the harm reduction counseling that infectious diseases physicians provide to PWID presenting with infections. METHODS: An electronic survey was distributed to physician members of the Emerging Infections Network to inquire about practices used when caring for patients with IDU-related infections. RESULTS: In total, 534 ID physicians responded to the survey. Of those, 375 (70%) reported routinely caring for PWID. Most respondents report screening for human immunodeficiency virus (HIV) and viral hepatitis (98%) and discussing the risk of these infections (87%); 63% prescribe immunization against viral hepatitis, and 45% discuss HIV preexposure prophylaxis (PrEP). However, 55% of respondents (n = 205) reported not counseling patients on safer injection strategies. Common reasons for not counseling included limited time and a desire to emphasize antibiotic therapy/medical issues (62%), lack of training (55%), and believing that it would be better addressed by other services (47%). Among respondents who reported counseling PWID, most recommended abstinence from IDU (72%), handwashing and skin cleansing before injection (62%), and safe disposal of needles/drug equipment used before admission (54%). CONCLUSIONS: Almost all ID physicians report screening PWID for HIV and viral hepatitis and discussing the risks of these infections. Despite frequently encountering PWID, fewer than half of ID physicians provide safer injection advice. Opportunities exist to standardize harm reduction education, emphasizing safer injection practices in conjunction with other strategies to prevent infections (eg, HIV PrEP or hepatitis A virus/hepatitis B virus vaccination)

What is the role of tobacco control advertising intensity and duration in reducing adolescent smoking prevalence? Findings from 16 years of tobacco control mass media advertising in Australia

ABSTRACTObjective: To examine how the intensity and duration of tobacco control advertising relate to adolescent smoking prevalence.Methods: Australian students (aged 12&ndash;17 years) participating in a national survey conducted triennially between 1993 and 2008 (sample size range 12 314&ndash;16 611). The outcome measure was students&rsquo; smoking in the previous 4 weeks collected through anonymous, self-completed surveys. For each student, monthly targeted rating points (TRPs, a measure of television advertising exposure) for tobacco control advertising was calculated for the 3 and 12 months prior to surveying. For each time period, cumulative TRPs exposure and exposure to three intensity levels (&ge;100 TRPs/month; &ge;400 TRPs/month; &ge;800 TRPs/month) over increasing durations (eg, 1 month, 2 months, etc) were calculated. Logistic regression examined associations between TRPs and adolescent smoking after controlling for demographic and policy variables.Results: Past 3-month cumulative TRPs were found to have an inverse relationship with smoking prevalence. Low TRPs exposure in the past 12 months was positively associated with adolescent smoking prevalence. However, smoking prevalence reduced with cumulative exposure levels above 5800 cumulative TRPs. Additionally, exposure to &ge;400 TRPs/month and &ge;800 TRPs/month were associated with reduced likelihood of smoking, although the duration needed for this effect differed for the two intensity levels. When intensity was &ge;400 TRPs/month, the odds of smoking only reduced with continuous exposure. When intensity was &ge;800 TRPs/month, exposure at levels less than monthly was associated with reductions in smoking prevalence.Conclusions: Both antismoking advertising intensity and duration are important for ensuring reductions in adolescent smoking prevalence.<br /

Short-term changes in quitting-related cognitions and behaviours after the implementation of plain packaging with larger health warnings : findings from a national cohort study with Australian adult smokers

ABSTRACTBackground: Plain packaging (PP) with larger graphic health warnings (GHWs) was implemented in Australia in late 2012. This study examined effects of these packaging changes on short-term changes in quitting-related cognitions and behaviours.Methods: We used a series of cohorts of Australian adult cigarette smokers originally sourced from a nationally representative cross-sectional tracking survey, followed up approximately 1 month after their baseline interview (n(weighted)=5441). Logistic regression analyses compared changes in seven quitting-related outcomes over this 1-month follow-up period for the cohorts surveyed before PP, over the period of transition to PP, and during the first year of PP, adjusting for baseline levels of the outcome and covariates.Results: Compared to the referent group of smokers who completed their follow-up survey pre-PP, those who were followed-up in the early transition period showed greater increases in rates of stopping themselves from smoking (OR=1.51, 95% CI (1.08 to 2.10)) and higher quit attempt rates (OR=1.43, 95% CI (1.00 to 2.03)), those followed-up in the late transition period showed greater increases in intentions to quit (OR=1.42, 95% CI (1.06 to 1.92)) and pack concealment (OR=1.55, 95% CI (1.05 to 2.31)), and those followed- up in the first year of PP showed higher levels of pack concealment (OR=1.65, 95% CI (1.01 to 2.72)), more premature stubbing out of cigarettes (OR=1.55, 95% CI (1.01 to 2.36)), and higher quit attempt rates (OR=1.52, 95% CI (1.01 to 2.30)).Conclusions: These findings provide some of the strongest evidence to date that implementation of PP with larger GHWs was associated with increased rates of quitting cognitions, microindicators of concern and quit attempts among adult cigarette smokers.<br /

Australian adult smokers\u27 responses to plain packaging with larger graphic health warnings 1 year after implementation: results from a national cross-sectional tracking survey

AbstractBackground We assessed whether the Australian plain packs with larger graphic health warnings (GHWs) achieved three specific objectives of reducing the appeal of tobacco, increasing health warning effectiveness and reducing the ability of packaging to mislead about smoking harms.Methods We compared responses from continuous cross-sectional telephone surveys of n=2176 cigarette smokers during pre-plain packaging (April&ndash;September 2012, pre-PP) with n=759 surveyed in the transition period (October&ndash;November 2012) and n=4240 during the first year of implementation (December 2012&ndash;November 2013, PP year 1), using multivariate logistic regression analyses.Results From pre-PP to PP year 1, more smokers disliked their pack (p&lt;0.001), perceived lower pack appeal (p&lt;0.001), lower cigarette quality (p&lt;0.001), lower satisfaction (p&lt;0.001) and lower value (p&lt;0.001) and disagreed brands differed in prestige (p=0.003). There was no change in perceived differences in taste of different brands. More smokers noticed GHWs (p&lt;0.001), attributed much motivation to quit to GHWs (p&lt;0.001), avoided specific GHWs when purchasing (p&lt;0.001), and covered packs (p&lt;0.001), with no change in perceived exaggeration of harms. PP year 1 saw an increased proportion believing that brands do not differ in harmfulness (p=0.004), but no change in the belief that variants do not differ in strength or the perceived harmfulness of cigarettes compared with a year ago. Interactions signified greater change for four outcomes assessing aspects of appeal among young adults and two appeal outcomes among mid-aged adults.Conclusions The specific objectives of plain packaging were achieved and generally sustained among adult smokers up to 12 months after implementation.<br /