GMMA Can Stabilize Proteins Across Different Functional Constraints.

Stabilizing proteins without otherwise hampering their function is a central task in protein engineering and design. PYR1 is a plant hormone receptor that has been engineered to bind diverse small molecule ligands. We sought a set of generalized mutations that would provide stability without affecting functionality for PYR1 variants with diverse ligand-binding capabilities. To do this we used a global multi-mutant analysis (GMMA) approach, which can identify substitutions that have stabilizing effects and do not lower function. GMMA has the added benefit of finding substitutions that are stabilizing in different sequence contexts and we hypothesized that applying GMMA to PYR1 with different functionalities would identify this set of generalized mutations. Indeed, conducting FACS and deep sequencing of libraries for PYR1 variants with two different functionalities and applying a GMMA analysis identified 5 substitutions that, when inserted into four PYR1 variants that each bind a unique ligand, provided an increase of 2-6 °C in thermal inactivation temperature and no decrease in functionality.

Reductions in community noise levels in vancouver, Canada, during pandemic lockdown and association with land cover type.

BACKGROUND: Urban transportation noise is a major public concern because of its adverse effects on health. The determinants of urban noise exposure have not been widely explored but the "natural experiment" presented by the COVID-19 lockdowns presented a unique opportunity. This study examined the relationship between environmental characteristics and urban noise pollution during the COVID-19 related lockdown in Metro Vancouver, Canada, from March 21st to May 18th, 2020. METHODS: We used noise exposure data from the Vancouver International Airport (YVR) noise management program, comparing the noise levels during "Phase One" of the COVID-19 lockdown in 2020 to the corresponding time period in 2019 from 21 Noise Monitoring Terminals (NMTs) located throughout Metro Vancouver. We modelled the relationship between the change in noise level and the physical NMT environments, including land cover, and total length of roads at four different time periods (24Hr, daytime, evening and nighttime) and within three different buffer zones (100 m, 250 m, and 500 m). RESULTS: Of 59,472 hourly measurements of community noise, the 24-h noise level was reduced by an average of 2.20 dBA between 2019 and 2020. Higher proportions of greenspace, barren areas, and soil-cover around NMTs resulted in stronger noise reductions and higher density of building, pavement, and water weakened the amount of noise reduction. Proximity of high-volume traffic roads (highways) were associated with weaker noise reduction. CONCLUSION: The COVID-19 related lockdown was associated with reduced noise in Metro Vancouver, and the relative reduction depended on the types of the environment surrounding the NMT. Future research on the effects of urban environmental characteristics on geographic inequality in noise levels and health consequences of the COVID-19 related lockdown is merited.

The Canadian Environmental Quality Index (Can-EQI): Development and calculation of an index to assess spatial variation of environmental quality in Canada's 30 largest cities.

BACKGROUND: Multiple characteristics of the urban environment have been shown to influence population health and health-related behaviours, though the distribution and combined effects of these characteristics on health is less understood. A composite measure of multiple environmental conditions would allow for comparisons among different urban areas; however, this measure is not available in Canada. OBJECTIVES: To develop an index of environmental quality for Canada's largest urban areas and to assess the influence of population size on index values. METHODS: We conducted a systematic search of potential datasets and consulted with experts to refine and select datasets for inclusion. We identified and selected nine datasets across five domains (outdoor air pollution, natural environments, built environments, radiation, and climate/weather). Datasets were chosen based on known impacts on human health across the life course, complete geographic coverage of the cities of interest, and temporal alignment with the 2016 Canadian census. Each dataset was then summarized into dissemination areas (DAs). The Canadian Environmental Quality Index (Can-EQI) was created by summing decile ranks of each variable based on hypothesized relationships to health outcomes. RESULTS: We selected 30 cities with a population of more than 100,000 people which included 28,026 DAs and captured approximately 55% of the total Canadian population. Can-EQI scores ranged from 21.1 to 88.9 out of 100, and in Canada's largest cities were 10.2 (95% CI: -10.7, -9.7) points lower than the smallest cities. Mapping the Can-EQI revealed high geographic variability within and between cities. DISCUSSION: Our work demonstrates a valuable methodology for exploring variations in environmental conditions in Canada's largest urban areas and provides a means for exploring the role of environmental factors in explaining urban health inequalities and disparities. Additionally, the Can-EQI may be of value to municipal planners and decision makers considering the allocation of investments to improve urban conditions.

The influence of early-life residential exposure to different vegetation types and paved surfaces on early childhood development: A population-based birth cohort study.

BACKGROUND: Growing evidence suggests that exposure to green space is associated with improved childhood health and development, but the influence of different green space types remains relatively unexplored. In the present study, we investigated the association between early-life residential exposure to vegetation and early childhood development and evaluated whether associations differed according to land cover types, including paved land. METHODS: Early childhood development was assessed via kindergarten teacher-ratings on the Early Development Instrument (EDI) in a large population-based birth cohort (n = 27,539) in Metro Vancouver, Canada. The residential surrounding environment was characterized using a high spatial resolution land cover map that was linked to children by six-digit residential postal codes. Early-life residential exposure (from birth to time of EDI assessment, mean age = 5.6 years) was calculated as the mean of annual percentage values of different land cover classes (i.e., total vegetation, tree cover, grass cover, paved surfaces) within a 250 m buffer zone of postal code centroids. Multilevel models were used to analyze associations between respective land cover classes and early childhood development. RESULTS: In adjusted models, one interquartile range increase in total vegetation percentage was associated with a 0.33 increase in total EDI score (95% CI: 0.21, 0.45). Similar positive associations were observed for tree cover (ß-coefficient: 0.26, 95% CI: 0.15, 0.37) and grass cover (ß-coefficient: 0.12, 95% CI: 0.02, 0.22), while negative associations were observed for paved surfaces (ß-coefficient: -0.35, 95% CI: -0.47, -0.23). CONCLUSIONS: Our findings indicate that increased early-life residential exposure to vegetation is positively associated with early childhood developmental outcomes, and that associations may be stronger for residential exposure to tree cover relative to grass cover. Our results further indicate that childhood development may be negatively associated with residential exposure to paved surfaces. These findings can inform urban planning to support early childhood developmental health.

Neighborhood environmental exposures and incidence of attention deficit/hyperactivity disorder: A population-based cohort study.

BACKGROUND: Emerging studies have associated low greenspace and high air pollution exposure with risk of child attention deficit/hyperactivity disorder (ADHD). Population-based studies are limited, however, and joint effects are rarely evaluated. We investigated associations of ADHD incidence with greenspace, air pollution, and noise in a population-based birth cohort. METHODS: We assembled a cohort from administrative data of births from 2000 to 2001 (N âˆ¼ 37,000) in Metro Vancouver, Canada. ADHD was identified by hospital records, physician visits, and prescriptions. Cox proportional hazards models were applied to assess associations between environmental exposures and ADHD incidence adjusting for available covariates. Greenspace was estimated using vegetation percentage derived from linear spectral unmixing of Landsat imagery. Fine particulate matter (PM2.5) and nitrogen dioxide (NO2) were estimated using land use regression models; noise was estimated using a deterministic model. Exposure period was from birth until the age of three. Joint effects of greenspace and PM2.5 were analysed in two-exposure models and by categorizing values into quintiles. RESULTS: During seven-year follow-up, 1217 ADHD cases were diagnosed. Greenspace was associated with lower incidence of ADHD (hazard ratio, HR: 0.90 [0.81-0.99] per interquartile range increment), while PM2.5 was associated with increased incidence (HR: 1.11 [1.06-1.17] per interquartile range increment). NO2 (HR: 1.01 [0.96, 1.07]) and noise (HR: 1.00 [0.95, 1.05]) were not associated with ADHD. There was a 50% decrease in the HR for ADHD in locations with the lowest PM2.5 and highest greenspace exposure, compared to a 62% increase in HR in locations with the highest PM2.5 and lowest greenspace exposure. Effects of PM2.5 were attenuated by greenspace in two-exposure models. CONCLUSIONS: We found evidence suggesting environmental inequalities where children living in greener neighborhoods with low air pollution had substantially lower risk of ADHD compared to those with higher air pollution and lower greenspace exposure.

Assessing the association between lifetime exposure to greenspace and early childhood development and the mediation effects of air pollution and noise in Canada: a population-based birth cohort study.

BACKGROUND: Exposure to greenspace is associated with improved childhood development, but the pathways behind this relationship are insufficiently understood. Therefore, we aimed to investigate the association between lifetime residential exposure to greenspace and early childhood development and evaluate the extent to which this association is mediated by reductions in traffic-related air pollution and noise. METHODS: This population-based birth cohort study comprised singleton births in Metro Vancouver, BC, Canada, between April 1, 2000, and Dec 31, 2005. Children and mothers had to be registered with the mandatory provincial health insurance programme, Medical Services Plan, and have lived within the study area from the child's birth to the time of outcome assessment. Early childhood development was assessed via teacher ratings on the Early Development Instrument (EDI), and we used the total EDI score as the primary outcome variable. We estimated greenspace using percentage vegetation derived from spectral unmixing of annual Landsat satellite image composites. Lifetime residential exposure to greenspace was estimated as the mean of annual percentage vegetation values within 250 m of participants' residential postal codes. Multilevel modelling, adjusted for eight covariates, was used to investigate associations between greenspace exposure and EDI scores. We estimated the mediation effects of nitrogen dioxide (NO2), fine particulate matter (PM2·5), and noise levels using causal mediation analyses. FINDINGS: Of the 37 745 children born in Metro Vancouver between April 1, 2000, and Dec 31, 2005, 27 372 were included in our final study sample. In the adjusted model, 1 IQR increase in percentage vegetation was associated with a 0·16 (95% CI 0·04-0·28; p=0·0073) increase in total EDI score, indicating small improvements in early childhood development. We estimated that 97·1% (95% CI 43·0-396·0), 29·5% (12·0-117·0), and 35·2% (17·9-139·0) of the association was mediated through reductions in NO2, PM2·5, and noise, respectively. INTERPRETATION: Increased exposure to residential greenspace might improve childhood development by reducing the adverse developmental effects of traffic-related exposures, especially NO2 air pollution. Our study supports the implementation of healthy urban planning and green infrastructure interventions. FUNDING: Canadian Institutes of Health Research.

The association between natural environments and childhood mental health and development: A systematic review and assessment of different exposure measurements.

BACKGROUND: Several studies have assessed the relationship between exposure to natural environments (NEs) and childhood mental health and development. In most cases, a positive association has been found, but results are inconsistent, and the strength of association is unclear. This inconsistency may reflect the heterogeneity in measurements used to assess NE. OBJECTIVES: This systematic review aims to identify the most common NE metrics used in childhood mental health and development research. Our second aim is to identify the metrics that are most consistently associated with health and assess the relative strength of association depending on type of NE exposure measurement, in terms of metric used (i.e., measurement technique, such as remote sensing), but also rate (i.e., spatial and temporal exposure). METHODS: We used the PRISMA protocol to identify eligible studies, following a set of pre-defined inclusion criteria based on the PECOS strategy. A number of keywords were used for retrieving relevant articles from Medline, Embase, PsychINFO, and Web of Science databases between January 2000-November 2020. From these, we extracted data on type of NE measurement and relative association to a number of indicators of childhood mental health and development. We conducted a systematic assessment of quality and risk of bias in the included articles to evaluate the level of evidence. Case studies and qualitative studies were excluded. RESULTS: After screening of title (283 studies included), abstract, and full article, 45 studies were included in our review. A majority of which were conducted in North America and Europe (n = 36; 80%). The majority of studies used land use or land covers (LULC, n = 24; 35%) to determine exposures to NEs. Other metrics included the normalized difference vegetation index (NDVI), expert measures (e.g., surveys of data collection done by experts), surveys (e.g., self-reported assessments), and use of NE (e.g., measures of a participant's use of NE such as through GPS tracts or parent reports). Rate was most commonly determined by buffer zones around residential addresses or postal codes. The most consistent association to health outcomes was found for buffers of 100 m, 250 m, 500 m, and within polygons boundaries (e.g., census tracts). Six health categories, academic achievement, prevalence of doctor diagnosed disorders, emotional and behavioral functioning, well-being, social functioning, and cognitive skills, were created post hoc. We found sufficient evidence between NDVI (Landsat) and emotional and behavioral well-being. Additionally, we found limited evidence between LULC datasets and academic achievement; use of NE, parent/guardian reported greenness, and expert measures of greenness and emotional and behavioral functioning; and use of NE and social functioning. DISCUSSION: This review demonstrates that several NE measurements must be evaluated further before sufficient evidence for a potential association between distinct NE exposure metrics and childhood mental health and development can be established. Further, we suggest increased coordination between research efforts, for example, by replication of studies and comparing different NE measurements systematically, so that effect sizes can be confirmed for various health outcomes. Finally, we recommend implementing research designs that assess underlying pathways of nature-health relations and utilize measurement techniques that adequately assess exposure, access, use, and perception of NEs in order to contribute to a better understanding of health impacts of surrounding natural environments.

Protein products of nonstop mRNA disrupt nucleolar homeostasis.

Stalled mRNA translation results in the production of incompletely synthesized proteins that are targeted for degradation by ribosome-associated quality control (RQC). Here we investigated the fate of defective proteins translated from stall-inducing, nonstop mRNA that escape ubiquitylation by the RQC protein LTN1. We found that nonstop protein products accumulated in nucleoli and this localization was driven by polylysine tracts produced by translation of the poly(A) tails of nonstop mRNA. Nucleolar sequestration increased the solubility of invading proteins but disrupted nucleoli, altering their dynamics, morphology, and resistance to stress in cell culture and intact flies. Our work elucidates how stalled translation may affect distal cellular processes and may inform studies on the pathology of diseases caused by failures in RQC and characterized by nucleolar stress.

The gammaherpesviral TATA-box-binding protein directly interacts with the CTD of host RNA Pol II to direct late gene transcription.

ß- and γ-herpesviruses include the oncogenic human viruses Kaposi's sarcoma-associated virus (KSHV) and Epstein-Barr virus (EBV), and human cytomegalovirus (HCMV), which is a significant cause of congenital disease. Near the end of their replication cycle, these viruses transcribe their late genes in a manner distinct from host transcription. Late gene transcription requires six virally encoded proteins, one of which is a functional mimic of host TATA-box-binding protein (TBP) that is also involved in recruitment of RNA polymerase II (Pol II) via unknown mechanisms. Here, we applied biochemical protein interaction studies together with electron microscopy-based imaging of a reconstituted human preinitiation complex to define the mechanism underlying Pol II recruitment. These data revealed that the herpesviral TBP, encoded by ORF24 in KSHV, makes a direct protein-protein contact with the C-terminal domain of host RNA polymerase II (Pol II), which is a unique feature that functionally distinguishes viral from cellular TBP. The interaction is mediated by the N-terminal domain (NTD) of ORF24 through a conserved motif that is shared in its ß- and γ-herpesvirus homologs. Thus, these herpesviruses employ an unprecedented strategy in eukaryotic transcription, wherein promoter recognition and polymerase recruitment are facilitated by a single transcriptional activator with functionally distinct domains.

MISTERMINATE Mechanistically Links Mitochondrial Dysfunction with Proteostasis Failure.

Mitochondrial dysfunction and proteostasis failure frequently coexist as hallmarks of neurodegenerative disease. How these pathologies are related is not well understood. Here, we describe a phenomenon termed MISTERMINATE (mitochondrial-stress-induced translational termination impairment and protein carboxyl terminal extension), which mechanistically links mitochondrial dysfunction with proteostasis failure. We show that mitochondrial dysfunction impairs translational termination of nuclear-encoded mitochondrial mRNAs, including complex-I 30kD subunit (C-I30) mRNA, occurring on the mitochondrial surface in Drosophila and mammalian cells. Ribosomes stalled at the normal stop codon continue to add to the C terminus of C-I30 certain amino acids non-coded by mRNA template. C-terminally extended C-I30 is toxic when assembled into C-I and forms aggregates in the cytosol. Enhancing co-translational quality control prevents C-I30 C-terminal extension and rescues mitochondrial and neuromuscular degeneration in a Parkinson's disease model. These findings emphasize the importance of efficient translation termination and reveal unexpected link between mitochondrial health and proteome homeostasis mediated by MISTERMINATE.

Born to be Wise: a population registry data linkage protocol to assess the impact of modifiable early-life environmental exposures on the health and development of children.

INTRODUCTION: Deficiencies in childhood development is a major global issue and inequalities are large. The influence of environmental exposures on childhood development is currently insufficiently explored. This project will analyse the impact of various modifiable early life environmental exposures on different dimensions of childhood development. METHODS: Born to be Wise will study a Canadian cohort of approximately 34 000 children who have completed an early development test at the age of 5. Land use regression models of air pollution and spatially defined noise models will be linked to geocoded data on early development to analyse any harmful effects of these exposures. The potentially beneficial effect on early development of early life exposure to natural environments, as measured by fine-grained remote sensing data and various land use indexes, will also be explored. The project will use data linkages and analyse overall and age-specific impact, including variability depending on cumulative exposure by assigning time-weighted exposure estimates and by studying subsamples who have changed residence and exposure. Potentially moderating effects of natural environments on air pollution or noise exposures will be studied by mediation analyses. A matched case-control design will be applied to study moderating effects of natural environments on the association between low socioeconomic status and early development. The main statistical approach will be mixed effects models, applying a specific software to deal with multilevel random effects of nested data. Extensive confounding control will be achieved by including data on a range of detailed health and sociodemographic variables. ETHICS AND DISSEMINATION: The study protocol has been ethically approved by the Behavioural Research Ethics Board at the University of British Columbia. The findings will be published in peer-reviewed journals and presented at scholarly conferences. Through stakeholder engagement, the results will also reach policy and a broader audience.

Lung ultrasonography as a direct measure of evolving respiratory dysfunction and disease severity in patients with acute pancreatitis.

BACKGROUND: The value of lung ultrasonography in the diagnosis of respiratory dysfunction and severity stratification in patients with acute pancreatitis (AP) was investigated. METHODS: Over a 3-month period, 41 patients (median age: 59.1 years; 21 males) presenting with a diagnosis of potential AP were prospectively recruited. Each participant underwent lung ultrasonography and the number of comet tails was linked with contemporaneous clinical data. Group comparisons, areas under the curve (AUC) and respective measures of diagnostic accuracy were investigated. RESULTS: A greater number of comet tails were evident in patients with respiratory dysfunction (P = 0.021), those with severe disease (P < 0.001) and when contemporaneous and maximum CRP exceeded 100 mg/L (P = 0.048 and P = 0.003 respectively). Receiver-operator characteristic plot area under the curve (AUC) was greater when examining upper lung quadrants, using respiratory dysfunction and AP severity as variables of interest (AUC = 0.783, 95% C.I.: 0.544-0.962, and AUC = 0.996, 95% C.I.: 0.982-1.000, respectively). Examining all lung quadrants except for the lower lateral resulted in greater AUCs for contemporaneous and maximum CRP (AUC = 0.708, 95% C.I.: 0.510-0.883, and AUC = 0.800, 95% C.I.: 0.640-0.929). DISCUSSION: Ultrasonography of non-dependent lung parenchyma can reliably detect evolving respiratory dysfunction in AP. This simple bedside technique shows promise as an adjunct to severity stratification.

Interaction between ORF24 and ORF34 in the Kaposi's Sarcoma-Associated Herpesvirus Late Gene Transcription Factor Complex Is Essential for Viral Late Gene Expression.

Transcription of herpesviral late genes is stimulated after the onset of viral DNA replication but otherwise restricted. Late gene expression in gammaherpesviruses requires the coordination of six early viral proteins, termed viral transactivation factors (vTFs). Here, we mapped the organization of this protein complex for Kaposi's sarcoma-associated herpesvirus. Disruption of this complex via point mutation of the interaction interface between the open reading frame 24 (ORF24) and ORF34 vTFs ablated both late gene expression and viral replication.

Lung ultrasonography as a direct measure of evolving respiratory dysfunction and disease severity in patients with acute pancreatitis.

BACKGROUND: The value of lung ultrasonography in the diagnosis of respiratory dysfunction and severity stratification in patients with acute pancreatitis (AP) was investigated. METHODS: Over a 3-month period, 41 patients (median age: 59.1 years; 21 males) presenting with a diagnosis of potential AP were prospectively recruited. Each participant underwent lung ultrasonography, and the number of comet tails present on scans was linked with contemporaneous clinical data. Group comparisons, areas under the curve (AUC) and respective measures of diagnostic accuracy were investigated. RESULTS: A greater number of comet tails were evident in patients with respiratory dysfunction (P = 0.013), those with severe disease (P = 0.001) and when contemporaneous and maximum in-patient C-reactive protein (CRP) exceeded 150 mg/l (P = 0.018 and P = 0.049, respectively). Receiver-operator characteristic plot area under the curve (AUC) was greater when examining upper lung quadrants, using respiratory dysfunction and AP severity as variables of interest (AUC = 0.803, 95% CI: 0.583-1.000, and AUC = 0.996, 95% CI: 0.983-1.000, respectively). Examining all lung quadrants resulted in greater AUCs for contemporaneous and maximum CRP (AUC = 0.764, 95% CI: 0.555-0.972, and AUC = 0.704, 95% CI: 0.510-0.898). DISCUSSION: Ultrasonography of non-dependent lung parenchyma can reliably detect evolving respiratory dysfunction in AP. This simple bedside technique shows promise as an adjunct to severity stratification.

Modulation of the cGAS-STING DNA sensing pathway by gammaherpesviruses.

Infection of cells with DNA viruses triggers innate immune responses mediated by DNA sensors. cGMP-AMP synthase (cGAS) is a key DNA sensor that produces the cyclic dinucleotide cGMP-AMP (cGAMP) upon activation, which binds to and activates stimulator of interferon genes (STING), leading to IFN production and an antiviral response. Kaposi's sarcoma-associated herpesvirus (KSHV) is a DNA virus that is linked to several human malignancies. We report that KSHV infection activates the cGAS-STING pathway, and that cGAS and STING also play an important role in regulating KSHV reactivation from latency. We screened KSHV proteins for their ability to inhibit this pathway and identified six viral proteins that block IFN-ß activation through this pathway. This study is the first report identifying multiple viral proteins encoded by a human DNA virus that inhibit the cGAS-STING DNA sensing pathway. One such protein, viral interferon regulatory factor 1 (vIRF1), targets STING by preventing it from interacting with TANK binding kinase 1 (TBK1), thereby inhibiting STING's phosphorylation and concomitant activation, resulting in an inhibition of the DNA sensing pathway. Our data provide a unique mechanism for the negative regulation of STING-mediated DNA sensing. Moreover, the depletion of vIRF1 in the context of KSHV infection prevented efficient viral reactivation and replication, and increased the host IFN response to KSHV. The vIRF1-expressing cells also inhibited IFN-ß production following infection with DNA pathogens. Collectively, our results demonstrate that gammaherpesviruses encode inhibitors that block cGAS-STING-mediated antiviral immunity, and that modulation of this pathway is important for viral transmission and the lifelong persistence of herpesviruses in the human population.

Global mapping of herpesvirus-host protein complexes reveals a transcription strategy for late genes.

Mapping host-pathogen interactions has proven instrumental for understanding how viruses manipulate host machinery and how numerous cellular processes are regulated. DNA viruses such as herpesviruses have relatively large coding capacity and thus can target an extensive network of cellular proteins. To identify the host proteins hijacked by this pathogen, we systematically affinity tagged and purified all 89 proteins of Kaposi's sarcoma-associated herpesvirus (KSHV) from human cells. Mass spectrometry of this material identified over 500 virus-host interactions. KSHV causes AIDS-associated cancers, and its interaction network is enriched for proteins linked to cancer and overlaps with proteins that are also targeted by HIV-1. We found that the conserved KSHV protein ORF24 binds to RNA polymerase II and brings it to viral late promoters by mimicking and replacing cellular TATA-box-binding protein (TBP). This is required for herpesviral late gene expression, a complex and poorly understood phase of the viral lifecycle.

Unconventional sequence requirement for viral late gene core promoters of murine gammaherpesvirus 68.

Infection with the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), is associated with several cancers. During lytic replication of herpesviruses, viral genes are expressed in an ordered cascade. However, the mechanism by which late gene expression is regulated has not been well characterized in gammaherpesviruses. In this study, we have investigated the cis element that mediates late gene expression during de novo lytic infection with murine gammaherpesvirus 68 (MHV-68). A reporter system was established and used to assess the activity of viral late gene promoters upon infection with MHV-68. It was found that the viral origin of lytic replication, orilyt, must be on the reporter plasmid to support activation of the late gene promoter. Furthermore, the DNA sequence required for the activation of late gene promoters was mapped to a core element containing a distinct TATT box and its neighboring sequences. The critical nucleotides of the TATT box region were determined by systematic mutagenesis in the reporter system, and the significance of these nucleotides was confirmed in the context of the viral genome. In addition, EBV and KSHV late gene core promoters could be activated by MHV-68 lytic replication, indicating that the mechanisms controlling late gene expression are conserved among gammaherpesviruses. Therefore, our results on MHV-68 establish a solid foundation for mechanistic studies of late gene regulation.

Genotoxicity of the cancer chemopreventive drug candidates CP-31398, SHetA2, and phospho-ibuprofen.

The genotoxic activities of three cancer chemopreventive drug candidates, CP-31398 (a cell permeable styrylquinazoline p53 modulator), SHetA2 (a flexible heteroarotinoid), and phospho-ibuprofen (PI, a derivative of ibuprofen) were tested. None of the compounds were mutagenic in the Salmonella/Escherichia coli/microsome plate incorporation test. CP-31398 and SHetA2 did not induce chromosomal aberrations (CA) in Chinese hamster ovary (CHO) cells, either in the presence or absence of rat hepatic S9 (S9). PI induced CA in CHO cells, but only in the presence of S9. PI, its parent compound ibuprofen, and its moiety diethoxyphosphoryloxybutyl alcohol (DEPBA) were tested for CA and micronuclei (MN) in CHO cells in the presence of S9. PI induced CA as well as MN, both kinetochore-positive (Kin+) and -negative (Kin-), in the presence of S9 at ≤100µg/ml. Ibuprofen was negative for CA, positive for MN with Kin+ at 250µg/ml, and positive for MN with Kin- at 125 and 250µg/ml. DEPBA induced neither CA nor MN at ≤5000µg/ml. The induction of chromosomal damage in PI-treated CHO cells in the presence of S9 may be due to its metabolites. None of the compounds were genotoxic, in the presence or absence of S9, in the GADD45α-GFP Human GreenScreen assay and none induced MN in mouse bone marrow erythrocytes.

Investigations of iridium-mediated reversible C-H bond cleavage: characterization of a 16-electron iridium(III) methyl hydride complex.

New iridium complexes of a tridentate pincer ligand, 2,6-bis(di-tert-butylphosphinito)pyridine (PONOP), have been prepared and used in the study of hydrocarbon C-H bond activation. Intermolecular oxidative addition of a benzene C-H bond was directly observed with [(PONOP)Ir(I)(cyclooctene)][PF(6)] at ambient temperature, resulting in a cationic five-coordinate iridium(III) phenyl hydride product. Protonation of the (PONOP)Ir(I) methyl complex yielded the corresponding iridium(III) methyl hydride cation, a rare five-coordinate, 16-valence electron transition metal alkyl hydride species which was characterized by X-ray diffraction. Kinetic studies of C-H bond coupling and reductive elimination reactions from the five-coordinate complexes have been carried out. Exchange NMR spectroscopy measurements established a barrier of 17.8(4) kcal/mol (22 degrees C) for H-C(aryl) bond coupling in the iridium(III) phenyl hydride cation and of 9.3(4) kcal/mol (-105 degrees C) for the analogous H-C(alkyl) coupling in the iridium(III) methyl hydride cation. The origin of the higher barrier of H-C(aryl) relative to H-C(alkyl) bond coupling is proposed to be influenced by a hindered rotation about the Ir-C(aryl) bond, a result of the sterically demanding PONOP ligand.