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One of several challenges in design of clinical chemoprevention trials is the selection of the dose, formulation, and dose schedule of the intervention agent. Therefore, a cross-over clinical trial was undertaken to compare the bioavailability and tolerability of sulforaphane from two of broccoli sprout–derived beverages: one glucoraphanin-rich (GRR) and the other sulforaphane-rich (SFR). Sulforaphane was generated from glucoraphanin contained in GRR by gut microflora or formed by treatment of GRR with myrosinase from daikon (Raphanus sativus) sprouts to provide SFR. Fifty healthy, eligible participants were requested to refrain from crucifer consumption and randomized into two treatment arms. The study design was as follows: 5-day run-in period, 7-day administration of beverages, 5-day washout period, and 7-day administration of the opposite intervention. Isotope dilution mass spectrometry was used to measure levels of glucoraphanin, sulforaphane, and sulforaphane thiol conjugates in urine samples collected daily throughout the study. Bioavailability, as measured by urinary excretion of sulforaphane and its metabolites (in approximately 12-hour collections after dosing), was substantially greater with the SFR (mean = 70%) than with GRR (mean = 5%) beverages. Interindividual variability in excretion was considerably lower with SFR than with GRR beverage. Elimination rates were considerably slower with GRR, allowing for achievement of steady-state dosing as opposed to bolus dosing with SFR. Optimal dosing formulations in future studies should consider blends of sulforaphane and glucoraphanin as SFR and GRR mixtures to achieve peak concentrations for activation of some targets and prolonged inhibition of others implicated in the protective actions of sulforaphane. Cancer Prev Res; 4(3); 384–95. ©2011 AACR.

Supplementary Tables from Bioavailability of Sulforaphane from Two Broccoli Sprout Beverages: Results of a Short-term, Cross-over Clinical Trial in Qidong, China

Supplementary Figure from Bioavailability of Sulforaphane from Two Broccoli Sprout Beverages: Results of a Short-term, Cross-over Clinical Trial in Qidong, China

Supplementary Figure 2 from Insulin-like Growth Factor-II Methylation Status in Lymphocyte DNA and Colon Cancer Risk in the Northern Sweden Health and Disease Cohort

Supplementary Figure 1 from Insulin-like Growth Factor-II Methylation Status in Lymphocyte DNA and Colon Cancer Risk in the Northern Sweden Health and Disease Cohort

Supplementary Materials from Insulin-like Growth Factor-II Methylation Status in Lymphocyte DNA and Colon Cancer Risk in the Northern Sweden Health and Disease Cohort

Supplementary Figure 3 from Insulin-like Growth Factor-II Methylation Status in Lymphocyte DNA and Colon Cancer Risk in the Northern Sweden Health and Disease Cohort

Inorganic pyrophosphatase catalyzes the conversion of pyrophosphate to phosphate and is often critical for driving reactions forward in cellular processes such as nucleic acid and protein synthesis. Commonly used methods for quantifying pyrophosphatase enzyme activity employ reacting liberated phosphate with a second molecule to produce absorbance changes or employing a second enzyme in coupled reactions to produce a product with a detectable absorbance. In this investigation, a novel [31P]-NMR spectroscopy-based assay was used to quantitatively measure the formation of phosphate and evaluate the activity of inorganic pyrophosphatase from the thermoacidophilic Crenarchaeota Sulfolobus islandicus. The enzymatic activity was directly measured via integration of the [31P] resonance associated with the phosphate product (δ = 2.1 ppm). Sulfolobus islandicus inorganic pyrophosphatase preferentially utilized Mg2+ as divalent cation and had pH and temperature optimums of 6.0 of 50 °C, respectively. The Vmax value was 850 μmol/min/mg and the Km for pyrophosphate was 1.02 mM. Sequence analysis indicates the enzyme is a Family I pyrophosphatase. Sulfolobus islandicus inorganic pyrophosphatase was shown to be inhibited by sodium fluoride with a IC50 of 2.26 mM, compared to a IC50 of 0.066 mM for yeast inorganic pyrophosphatase. These studies reveal that a [31P]-NMR spectroscopy-based assay is an effective method for analyzing catalysis by phosphate-producing enzymes.

Multiplication of the invertebrate DNA baculoviruses activates the host DNA damage response (DDR), which promotes virus DNA replication. DDR signaling is initiated by the host insect’s phosphatidylinositol-3 kinase-related kinases (PIKKs), including ataxia telangiectasia-mutated kinase (ATM). Like other PIKKs, ATM phosphorylates an array of host DDR proteins at serine/threonine glutamine (S/TQ) motifs, the result of which leads to cell cycle arrest, DNA repair, or apoptosis. To define the role of host PIKKs in baculovirus replication, we compared replication levels of the baculovirus prototype species Autographa californica multiple nucleopolyhedrovirus in permissive Spodoptera frugiperda (SF21) cells with and without ATM function. Caffeine, which inhibits multiple DDR kinases, and the ATM-specific inhibitors KU-55933 and KU-60019 each prevented phosphorylation of Spodoptera histone H2AX (SfH2AX), a recognized indicator of ATM activity. However, only caffeine reduced autographa californica multiple nucleopolyhedrovirus (AcMNPV)-induced bulk phosphorylation of S/TQ protein motifs. Furthermore, only caffeine, not KU-55933 or KU-60019, reduced AcMNPV yields, suggesting a limited role for ATM. To investigate further, we identified and edited the Spodoptera ATM gene (sfatm). Consistent with ATM’s known functions, CRISPR/Cas9-mediated knockout of sfatm eliminated DNA damage-induced phosphorylation of DDR marker SfH2AX in SF21 cells. However, loss of sfatm failed to affect the levels of AcMNPV multiplication. These findings suggested that in the absence of the kinase SfATM, another caffeine-sensitive host DDR kinase promotes S/TQ phosphorylation and baculovirus multiplication. Thus, baculoviruses activate and utilize the host insect DDR in an ATM-independent manner. IMPORTANCE The DDR, while necessary for the maintenance and fidelity of the host genome, represents an important cellular response to viral infection. The prolific DNA baculoviruses activate and manipulate the invertebrate DDR by using mechanisms that positively impact virus multiplication, including virus DNA replication. As the key DDR initiator kinase, ATM was suspected to play a critical role in this host response. However, we show here that baculovirus AcMNPV activates an ATM-independent DDR. By identifying the insect host ATM ortholog (Spodoptera frugiperda SfATM) and evaluating genetic knockouts, we show that SfATM is dispensable for AcMNPV activation of the DDR and for virus replication. Thus, another PIKK, possibly the closely related kinase ATR (ATM- and Rad3-related kinase), is responsible for efficient baculovirus multiplication. These findings better define the host pathways used by invertebrates to engage viral pathogens, including DNA viruses.

Fluent recall of basic facts is essential to the development of more complex math skills. Therefore, failure to develop fluency with basic facts may impede the development of these skills. The present study used a between groups experimental design to investigate whether a basic facts fluency program, implemented within a self-regulated learner (SRL) framework, could lead to increased fluency with multiplication facts for Year 5 and Year 6 New Zealand students (9–10 years old). This study also investigated the extent to which the SRL program altered students’ basic facts practice behavior outside of school hours. The study found that the SRL program resulted in rapid fluency development that was maintained over time. Nomothetic and idiographic analysis confirmed that the program was suitable for use within Tier 1 of the response to intervention framework. In addition, the study also found that students who received the program altered their practice behavior outside school hours. The results from this study show how elements of self-regulated learning and precision teaching can be successfully combined to enhance students’ mathematics achievement.

This paper includes an introduction to the concept of spreadsheet optimization and modeling as it specifically applies to combinatorial problems. One of the best known of the classic combinatorial problems is the “Traveling Salesman Problem” (TSP). The classic Traveling Salesman Problem has the objective of minimizing some value, usually distance, while defining a sequence of locations where each is visited once. An additional requirement is that the tour ends in the same location where the tour started. Variants of the classic Traveling Salesman Problem are developed including the Bottleneck TSP and the Variation Bottleneck TSP.

The increasing use of high-strength materials in sheet metal processing results in challenges for cutting presses. The required cutting force increases with the strength of the material. After material separation, a sudden release of the energy stored in the machine leads to intense system vibrations and noise emission. Noise protection and reduction solutions such as machine cabins or active vibration damping systems are expensive, require much space or frequent maintenance. Due to this, forming machines must be acoustically optimized. In this article, selected structure measures for the machine-side noise reduction are derived and evaluated in the context of simulation studies using an acoustic machine model.

Background A growing body of research has documented the positive effects of gratitude programs on participants' mental health and well-being. For children and adolescents, these programs typically rely on school-based group designs tied with a health curriculum, whereas innovative technology-based programs are relatively understudied. Methods This experiment investigated the feasibility and efficacy of a gratitude text-messaging program for promoting adolescent mental health relative to a positive reflective control condition. Results Young people showed positive changes over the course of the program in their general sense of gratitude, subjective well-being, and reduced depressive symptoms, with some evidence that those with higher levels of depressive symptoms benefited more from the gratitude program. However, there were no significant differences across the two groups in the magnitude of these mental health changes. Participants in both conditions valued and were highly engaged with the interactive text-messaging approach. Conclusion This interactive e-health promotion strategy seemed to promote strong engagement and showed promising effectiveness with young people, with some challenges to feasibility due to the labor intensive nature of sending and responding to a large number of text-messages. The importance of carefully considering risk management strategies when developing such programs was also highlighted.

The production and decomposition of litter in mangroves plays a significant role in the nutrient and organic carbon cycles. These can be highly variable both spatially and temporally as a result of numerous factors including tidal range, forest type, abundance and type of herbivorous fauna, temperature, and microbial activity. Mangroves also play an important role in blue carbon sequestration, with their status as carbon sinks crucial in mitigating against greenhouse gas-induced climate change. Blue carbon is a term used to describe the carbon captured by oceans and coastal ecosystems. We review and discuss the current available knowledge regarding sources of organic matter (OM) in mangroves as well as the roles of benthic macrofauna, water and microbial activity in the decomposition of OM in order to gain a better understanding of the decompositional processes that take place. Macrofauna break down and bury litter, thereby improving litter quality, in turn increasing decomposition rates via leaching and microbial activity. Microbial decomposition in mangroves is slow as a result of phenolic concentrations in the litter. A build-up of phenolic compounds inhibits microbial activity leading to the accumulation of OM in mangroves. Although knowledge has improved, there are still gaps in the information available and we still have an incomplete picture of the decompositional process in mangroves, and in particular the formation of blue carbon stores, necessitating further research.

Tertiary learning advisors (TLA) are key support staff for university students in Aotearoa New Zealand and serve a major role by promoting effective strategies for students’ studying and learning. For TLAs to effectively assist students, particularly first-year university students, institutions must have a greater understanding of the academic transition experience as reported by students. The current study used a mixed-method approach to consider students’ reflections on their learning and study after their first semester of university. Results show that 2 out of 3 students reported feeling underprepared for their university courses, and most students reported attempts to modify their learning and study approaches in their adaptations to university. Further, students’ perceptions of the learning environment as clear and organised, interesting and personally relevant, and supportive of building a peer-based learning network were linked to positive changes in their learning and study practices. Such findings highlight the importance of the psychosocial climate on learning and learners during the student transition to university. While the majority of participants reported that their modified learning strategies were effective, a small minority felt incapable of overcoming this challenge. The implications of these results are discussed about stress and coping theory. Recommendations for TLAs to support transitioning students are included.

Using life-course longitudinal data from the Christchurch Health and Development Study (CHDS), we examined ethnic differences in rates of psychiatric disorder among New Zealand adolescents, comparing New Zealand Māori to their European peers. The CHDS includes a large birth cohort of New Zealand children who have been regularly assessed throughout childhood, adolescence, and adulthood. Ethnicity (New Zealand Māori vs. non-Māori) was assessed at age 14 years. Internalizing and externalizing disorders were assessed at age 15, 16, and 18 years. A diverse range of risk factors were included to test if differences in mental health difficulties were explained by greater exposure to early life-course challenges. We found significant differences in rates of mental disorder during adolescence between New Zealand Māori and non-Māori, but these differences were largely explained by early developmental challenges and adolescent peer affiliations. Differences across the two ethnic groups in rates of internalizing disorders were explained by the increased exposure amongst Māori to socioeconomic disadvantage during childhood, while differences in externalizing disorders were explained by greater exposure amongst Māori to childhood family adversity and deviant peer affiliation during adolescence. The findings point to the significant influence of cumulative early life-course risk factors in accounting for the ethnic differences between Māori and non-Māori in the development of adolescent internalizing and externalizing disorders.

The present study examined how teachers functioned as first responders in a natural disaster and their perceptions of their emotional management both in the immediate aftermath of a large earthquake and their personal and professional functioning 18 months after the disaster. Employing a mixed-methods design, the results showed mixed effects for earthquake related factors and emotional management in associations with personal and professional functioning. The findings point to the importance of teacher training in emotional management and the role that school management can play in training teachers for their role as first responders and sensitively managing their post-disaster work environment.

Baculovirus-encoded inhibitor of apoptosis (IAP) proteins likely evolved from their host cell IAP homologs, which function as critical regulators of cell death. Despite their striking relatedness to cellular IAPs, including the conservation of two baculovirus IAP repeat (BIR) domains and a C-terminal RING, viral IAPs use an unresolved mechanism to suppress apoptosis in insects. To define this mechanism, we investigated Op-IAP3, the prototypical IAP from baculovirus OpMNPV. We found that Op-IAP3 forms a stable complex with SfIAP, the native, short-lived IAP of host insect Spodoptera frugiperda . Long-lived Op-IAP3 prevented virus-induced SfIAP degradation, which normally causes caspase activation and apoptosis. In uninfected cells, Op-IAP3 also increased SfIAP steady-state levels and extended SfIAP's half-life. Conversely, SfIAP stabilization was lost or reversed in the presence of mutated Op-IAP3 that was engineered for reduced stability. Thus, Op-IAP3 stabilizes SfIAP and preserves its antiapoptotic function. In contrast to SfIAP, Op-IAP3 failed to bind or inhibit native Spodoptera caspases. Furthermore, BIR mutations that abrogate binding of well-conserved IAP antagonists did not affect Op-IAP3's capacity to prevent virus-induced apoptosis. Remarkably, Op-IAP3 also failed to prevent apoptosis when endogenous SfIAP was ablated by RNA silencing. Thus, Op-IAP3 requires SfIAP as a cofactor. Our findings suggest a new model wherein Op-IAP3 interacts directly with SfIAP to maintain its intracellular level, thereby suppressing virus-induced apoptosis indirectly. Consistent with this model, Op-IAP3 has evolved an intrinsic stability that may serve to repress signal-induced turnover and autoubiquitination when bound to its targeted cellular IAP. IMPORTANCE The IAPs were first discovered in baculoviruses because of their potency for preventing apoptosis. However, the antiapoptotic mechanism of viral IAPs in host insects has been elusive. We show here that the prototypical viral IAP, Op-IAP3, blocks apoptosis indirectly by associating with unstable, autoubiquitinating host IAP in such a way that cellular IAP levels and antiapoptotic activities are maintained. This mechanism explains Op-IAP3's requirement for native cellular IAP as a cofactor and the dispensability of caspase inhibition. Viral IAP-mediated preservation of the host IAP homolog capitalizes on normal IAP-IAP interactions and is likely the result of viral IAP evolution in which degron-mediated destabilization and ubiquitination potential have been reduced. This mechanism illustrates another novel means by which DNA viruses incorporate host death regulators that are modified for resistance to host regulatory controls for the purpose of suppressing host cell apoptosis and acquiring replication advantages.

Inhibitor-of-apoptosis (IAP) proteins are key regulators of the innate antiviral response by virtue of their capacity to respond to signals affecting cell survival. In insects, wherein the host IAP provides a primary restriction to apoptosis, diverse viruses trigger rapid IAP depletion that initiates caspase-mediated apoptosis, thereby limiting virus multiplication. We report here that the N-terminal leader of two insect IAPs, Spodoptera frugiperda SfIAP and Drosophila melanogaster DIAP1, contain distinct instability motifs that regulate IAP turnover and apoptotic consequences. Functioning as a protein degron, the cellular IAP leader dramatically shortened the life span of a long-lived viral IAP (Op-IAP3) when fused to its N terminus. The SfIAP degron contains mitogen-activated kinase (MAPK)-like regulatory sites, responsible for MAPK inhibitor-sensitive phosphorylation of SfIAP. Hyperphosphorylation correlated with increased SfIAP turnover independent of the E3 ubiquitin-ligase activity of the SfIAP RING, which also regulated IAP stability. Together, our findings suggest that the SfIAP phospho-degron responds rapidly to a signal-activated kinase cascade, which regulates SfIAP levels and thus apoptosis. The N-terminal leader of dipteran DIAP1 also conferred virus-induced IAP depletion by a caspase-independent mechanism. DIAP1 instability mapped to previously unrecognized motifs that are not found in lepidopteran IAPs. Thus, the leaders of cellular IAPs from diverse insects carry unique signal-responsive degrons that control IAP turnover. Rapid response pathways that trigger IAP degradation and initiate apoptosis independent of canonical prodeath gene (Reaper-Grim-Hid) expression may provide important innate immune advantages. Furthermore, the elimination of these response motifs within viral IAPs, including those of baculoviruses, explains their unusual stability and their potent antiapoptotic activity. IMPORTANCE Apoptosis is an effective means by which a host controls virus infection. In insects, inhibitor-of-apoptosis (IAP) proteins act as regulatory sentinels by responding to cellular signals that determine the fate of infected cells. We discovered that lepidopteran (moth and butterfly) IAPs, which are degraded upon baculovirus infection, are controlled by a conserved phosphorylation-sensitive degron within the IAP N-terminal leader. The degron likely responds to virus-induced kinase-specific signals for degradation through SKP1/Cullin/F-box complex-mediated ubiquitination. Such signal-induced destruction of cellular IAPs is distinct from degradation caused by well-known IAP antagonists, which act to expel IAP-bound caspases. The major implication of this study is that insects have multiple signal-responsive mechanisms by which the sentinel IAPs are actively degraded to initiate host apoptosis. Such diversity of pathways likely provides insects with rapid and efficient strategies for pathogen control. Furthermore, the absence of analogous degrons in virus-encoded IAPs explains their relative stability and antiapoptotic potency.

Choline kinase catalyzes the conversion of choline to phosphocholine (PC) by transferring a phosphate group from adenosine triphosphate (ATP) as the first step in the biosynthetic pathway for the membrane phospholipid phosphatidylcholine, an essential pathway in the Leishmania parasitic protozoan. Commonly used methods for kinetically quantifying the enzyme include a radioisotope assay utilizing labeled choline and a coupled spectrophotometric assay with multiple enzymes and substrates that indirectly measures choline kinase activity. When testing potential inhibitors with the coupled assay, results can cast doubt on whether choline kinase is being inhibited or one of the coupled enzymes. Therefore, 31P NMR spectroscopy was used to quantitatively measure the formation of the key product, phosphocholine, and to evaluate choline kinase activity. Interrogation of 31P NMR spectroscopy offers a number of benefits. Since this isotope is 100% abundant and has a relatively large gyromagnetic ratio, it is considered one of the more sensitive nuclides. As such, the need for costly isotopic enriched phosphorous is not required and detection of the 31P signal is possible even at relatively low concentrations. The enzymatic activity of Leishmania infantum choline kinase was able to be directly measured via integration of the 31P resonance associated with the phosphocholine product (δ = 3.94 ppm). These initial studies reveal that a 31P NMR spectroscopic-based assay could be used for testing substrate or transition state analogs as competitive inhibitors of Leishmania choline kinase that may prevent phosphatidylcholine synthesis in the parasite.

The present study examined the development of teacher identity in a cohort of first year student teachers from a developmental and social psychological perspective, relying on Erikson's (1964) theory of identity development and Turner, Oakes, Haslam, and McGarty's (1994) self-categorization theory. As hypothesized, aspects of both personal identity development and social identity were significantly associated with higher teacher identity after controlling for several covariates. The findings have several implications for teacher education programs and point to the potential utility of applying well-researched psychological theory when examining the processes by which student teachers develop a professional teaching identity.

Population level shifts in the timing of parenting onset and family composition have been well documented. However, the impacts of these changes on the life course experiences of high risk, teen and younger mothers remain poorly understood. To address this issue, this paper examined intergenerational changes in the family formation and parenting experiences of teen and younger mothers who gave birth in the 1970s and the 2000s (G2, N = 108). The study sample consisted of two generations of early parenting women studied as part of the Christchurch Health and Development Study in New Zealand. Early motherhood (

Over 100 single nucleotide polymorphisms (SNP) are validated in the TP53 tumor suppressor gene. They define haplotypes, which may differ in their activities. Therefore, mutation in cancer may occur at different rates depending upon haplotypes. However, these associations may be masked by differences in mutations types and causes of mutagenesis. We have analyzed the associations between 19 SNPs spanning the TP53 locus and a single specific aflatoxin-induced TP53 mutation (R249S) in 85 in hepatocellular carcinoma cases and 132 controls from Thailand. An association with R249S mutation (P = 0.007) was observed for a combination of two SNPs (rs17882227 and rs8064946) in a linkage disequilibrium block extending from upstream of exon 1 to the first half of intron 1. This domain contains two coding sequences overlapping with TP53 (WRAP53 and Hp53int1) suggesting that sequences in TP53 intron 1 encode transcripts that may modulate R249S mutation rate in HCC.

Objectives This study aimed to evaluate the trends and regional variation of stroke hospital care in 30-day in-hospital mortality, hospital length of stay (LOS), and 1-year total hospitalization cost after implementation of the Alberta Provincial Stroke Strategy. Methods New ischemic stroke patients (N = 7632) admitted to Alberta acute care hospitals between 2006 and 2011 were followed for 1 year. We analyzed in-hospital mortality with logistic regression, LOS with negative binomial regression, and the hospital costs with generalized gamma model (log link). The risk-adjusted results were compared over years and between zones using observed/expected results. Results The risk-adjusted mortality rates decreased from 12.6% in 2006/2007 to 9.9% in 2010/2011. The regional variations in mortality decreased from 8.3% units in 2008/2009 to 5.6 in 2010/2011. The LOS of the first episode dropped significantly in 2010/2011 after a 4-year slight increase. The regional variation in LOS was 15.5 days in 2006/2007 and decreased to 10.9 days in 2010/2011. The 1-year hospitalization cost increased initially, and then kept on declining during the last 3 years. The South and Calgary zones had the lowest costs over the study period. However, this gap was diminishing. Conclusions After implementation of the Alberta Provincial Stroke Strategy, both mortality and hospital costs demonstrated a decreasing trend during the later years of study. The LOS increased slightly during the first 4 years but had a significant drop at the last year. In general, the regional variations in all 3 indicators had a diminishing trend.

Background Previous research has documented that exposure to parental separation/divorce during childhood can be associated with long-term consequences into adulthood. This study sought to extend this literature by examining associations between childhood exposure to parental separation/divorce and later parenting behavior as an adult in a New Zealand birth cohort. Methods Data were drawn from the Christchurch Health and Development Study (CHDS), a longitudinal study of a birth cohort of 1,265 children born in 1977 in Christchurch, New Zealand. Information about exposure to parental separation and divorce was gathered annually from birth to 15 years. At the 30-year follow-up, all cohort members who had become parents (biological or nonbiological) were assessed on several parenting dimensions (sensitivity, warmth, overreactivity, inconsistency, quality of child management, and physical punishment). Results The analyses showed that exposure to more frequent parental separation in childhood and adolescence was associated with lower levels of parental sensitivity and warmth, greater overreactivity, and an increased use of physical punishment as a parent, after controlling for a wide range of family socioeconomic and psychosocial factors, and individual child characteristics. Conclusions The findings suggest that as exposure to parental separation increases, so does the likelihood of experiencing multiple developmental challenges in childhood and adolescence. As an adult, these life-course experiences can have small but significant associations with the quality of parenting behavior.

We report a measurement of the nuclear polarization of laser-cooled, optically-pumped $^{37}\mathrm{K}$ atoms which will allow us to precisely measure angular correlation parameters in the beta-decay of the same atoms. These results will be used to test the $V-A$ framework of the weak interaction at high precision. At the TRIUMF Neutral Atom Trap (TRINAT), a magneto-optical trap (MOT) confines and cools neutral $^{37}\mathrm{K}$ atoms and optical pumping spin-polarizes them. We monitor the nuclear polarization of the same atoms that are decaying in situ by photoionizing a small fraction of the partially polarized atoms and then use the standard optical Bloch equations to model their population distribution. We obtain an average nuclear polarization of $P = 0.9913\pm0.0008$, which is significantly more precise than previous measurements with this technique. Since our current measurement of the beta-asymmetry has $0.2\%$ statistical uncertainty, the polarization measurement reported here will not limit its overall uncertainty. This result also demonstrates the capability to measure the polarization to $, 32 pages, 11 figures, submitted to New Journal of Physics

The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) initiates apoptosis in diverse insects through events triggered by virus DNA (vDNA) replication. To define the proapoptotic pathway and its role in antivirus defense, we investigated the link between the host's DNA damage response (DDR) and apoptosis. We report here that AcMNPV elicits a DDR in the model insect Drosophila melanogaster . Replication of vDNA activated DDR kinases, as evidenced by ATM-driven phosphorylation of the Drosophila histone H2AX homolog (H2Av), a critical regulator of the DDR. Ablation or inhibition of ATM repressed H2Av phosphorylation and blocked virus-induced apoptosis. The DDR kinase inhibitors caffeine and KU55933 also prevented virus-induced apoptosis in cells derived from the permissive AcMNPV host, Spodoptera frugiperda . This block occurred at a step upstream of virus-mediated depletion of the cellular inhibitor-of-apoptosis protein, an event that initiates apoptosis in Spodoptera and Drosophila . Thus, the DDR is a conserved, proapoptotic response to baculovirus infection. DDR inhibition also repressed vDNA replication and reduced virus yields 100,000-fold, demonstrating that the DDR contributes to virus production, despite its recognized antivirus role. In contrast to virus-induced phosphorylation of Drosophila H2Av, AcMNPV blocked phosphorylation of the Spodoptera H2AX homolog (SfH2AX). Remarkably, AcMNPV also suppressed SfH2AX phosphorylation following pharmacologically induced DNA damage. These findings indicate that AcMNPV alters canonical DDR signaling in permissive cells. We conclude that AcMNPV triggers a proapoptotic DDR that is subsequently modified, presumably to stimulate vDNA replication. Thus, manipulation of the DDR to facilitate multiplication is an evolutionarily conserved strategy among DNA viruses of insects and mammals.

Linear solvation theories are well established to describe electrostatic hydration of small solutes when the hydration free energy is dominated by the electrostatic free energy of the solute multipole. In contrast, hydration of nanometer solutes is driven by surface hydration. We address the question of whether the linear-response thermodynamics established for small multipolar solutes applies to surface hydration. To this end, molecular dynamics simulations are carried out on a model C180 solute that carries no global multipole, but the surface of which is decorated with radially pointing dipoles. Linear response is dramatically violated in this case. Further, two crossovers in the solvation thermodynamics are discovered as the surface polarity is increased. Both transformations produce strongly nonlinear solvation response. The second, more collective, crossover leads to a dramatic slowing down of the interfacial dynamics, reaching the time-scales of nanoseconds. Our picture offers the possibility of flipping water domains at interfaces of nanoparticles and biomolecules.

IE1 is the principal transcriptional regulator of the baculoviruses. Like multifunctional transcription factors of other large DNA viruses, IE1 is an essential, site-specific DNA-binding phosphoprotein that activates virus gene expression and promotes genome replication. To define the poorly understood mechanisms by which IE1 achieves its diverse functions, we identified IE1 domains that contribute to productive infection of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), the baculovirus prototype. Site-directed mutagenesis revealed that the N-terminal 23 residues of IE1 are required for origin-specific DNA replication and AcMNPV propagation, but not for DNA-binding-dependent transcriptional activation. Within this defined replication domain, we identified an invariant TPXR/H motif that resembles a consensus cyclin-dependent kinase phosphorylation site. Amino acid substitutions of potential phosphorylation sites within or near this motif caused loss of IE1-mediated DNA replication activity. Remarkably, substitution of the single threonine (residue 15) within the TPXR/H motif caused complete loss of AcMNPV multiplication. The replication domain was required for IE1 phosphorylation. It was also sufficient for conferring phosphorylation of a heterologous protein. Importantly, IE1 hyperphosphorylation coincided exclusively with AcMNPV DNA replication. The temporal regulation of IE1 phosphorylation and the essential nature of the TPXR/H motif suggest that phosphorylation critically alters and possibly activates DNA replication activity of IE1 during infection. The striking conservation of the TPXR/H motif among IE1 proteins further suggests that this molecular switch may be a common mechanism by which the alphabaculoviruses coordinate DNA replication and gene expression by using a single regulator.

Data from the Christchurch Health and Development Study, a 30-year prospective longitudinal study, were used to examine the associations between the quality of parent–child relations in adolescence and adult parenting behaviour 15 years later. At ages 14 and 15 years, cohort members were interviewed about the quality of their relationship with their parents. At age 30, those who had become parents underwent a parenting assessment using self-report and observational ratings of positive (warmth, sensitivity) and negative parenting (overreactive, inconsistency, and physical punishment/abuse). Results showed that adolescents who reported higher quality parent–child relationships were later characterized by higher levels of parental warmth, sensitivity, and effective child management, and lower levels of overreactive parenting. These associations remained after extensive covariate adjustment. Study findings highlight the importance of close parent–child relations during adolescence in preparing an individual for the challenges of caring for and parenting their own children when they themselves become parents.

In regions with high prevalence of chronic hepatitis B virus (HBV) infection and dietary aflatoxin B(1) (AFB(1)) exposure, hepatocellular carcinomas (HCCs) often contain TP53 mutation at codon 249 (R249S). Furthermore, a C-terminal truncated HBx protein expressed from hepatocyte integrated HBV is associated with HCC development. This study evaluates the association between R249S and HBX status in relation to HCC in West African population. HBX (complete or 3'-truncated) and HBS genes were assessed by PCR in cell-free DNA (CFDNA) from plasma of subjects recruited in a hospital-based case-control study (325 controls, 78 cirrhotic patients and 198 HCC cases) conducted in The Gambia. These samples had been previously analyzed for R249S and HBV serological status. Complete HBX sequence was frequently detected in CFDNA of HCC-R249S positive (77%, 43/56) compared with HCC-R249S-negative cases (44%, 22/50). Conversely, the proportion of 3'-truncated HBX gene was significantly higher in HCC-R249S negative than positive cases (34%, 17/50, compared with 12%, 7/56) (χ(2) = 12.12; P = 0.002; distribution of R249S negative and positive according to HBX status). Occult HBV infection (detected by PCR) was present in 24% of HCC previously considered as negative by HBV serology. Moreover, HBV mutation analysis revealed that double mutation at nucleotides 1762(T)/1764(A) was associated with diagnosis of cirrhosis or HCC {cirrhosis: odds ratio (OR): 9.50 [95% confidence interval (CI) 1.50-60.11]; HCC: OR: 11.29 [95% CI 2.07-61.47]}. These findings suggest that in HCC from The Gambia, complete HBX sequences are often associated with the presence of TP53 R249S mutation.

Background: Chronic hepatitis B virus (HBV) infection and dietary aflatoxin B1 (AFB1) exposure are etiological factors for hepatocellular carcinoma (HCC) in countries with hot, humid climates. HCC often harbors a TP53 (tumor protein p53) mutation at codon 249 (R249S). In chronic carriers, 1762T/1764A mutations in the HBV X gene are associated with increased HCC risk. Both mutations have been detected in circulating cell-free DNA (CFDNA) from asymptomatic HBV carriers. Objective: We evaluated seasonal variation in R249S and HBV in relation to AFB1 exposure. Methods: R249S was quantitated by mass spectrometry in CFDNA in a cross-sectional survey of 473 asymptomatic subjects (237 HBV carriers and 236 noncarriers) recruited in three villages in the Gambia over a 10-month period. 1762T/1764A HBV mutations were detected by quantitative polymerase chain reaction. In addition, the HBV S gene was sequenced in 99 subjects positive for HBV surface antigen (HBsAg). Results: We observed a seasonal variation of serum R249S levels. Positivity for R249S and average concentration were significantly higher in HBsAg-positive subjects surveyed during April–July (61%; 5,690 ± 11,300 R249S copies/mL serum) than in those surveyed October–March [32% and 480 ± 1,030 copies/mL serum (odds ratio = 3.59; 95% confidence interval: 2.05, 6.30; p < 0.001)]. Positivity for HBV e antigen (HBeAg) (a marker of HBV replication) and viral DNA load also varied seasonally, with 15–30% of subjects surveyed between April and June HBeAg positive, compared with < 10% surveyed during other months. We detected 1762T/1764A mutations in 8% of carriers, half of whom were positive for R249S. We found HBV genotype E in 95 of 99 HBsAg-positive subjects. Conclusion: R249S is detectable in CFDNA of asymptomatic subjects. Evidence of temporal and quantitative variations suggests an interaction among AFB1 exposure, HBV positivity, and replication on TP53 mutation formation or persistence.

An isotope dilution mass spectrometry method has been developed for the simultaneous measurement of picolinoyl derivatives of testosterone (T), dihydrotestosterone (DHT), 17β-estradiol (E(2)), and 5α-androstan-3α,17β-diol (3α-diol) in rat intratesticular fluid. The method uses reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Following derivatization of 10-μL samples of testicular fluid with picolinoyl chloride hydrochloride, the samples were purified by solid phase extraction before analysis. The accuracy of the method was satisfactory for the 4 analytes at 3 concentrations, and both inter- and intraday reproducibility were satisfactory for T, DHT, and E(2). Measurements of intratesticular T concentrations in a group of 8 untreated adult rats by this method correlated well with measurements of the same samples by radioimmunoassay. As in men, there was considerable rat-to-rat variability in T concentration, despite the fact that the rats were inbred. Although its levels were more than an order of magnitude lower than those of T, DHT was measured reliably in all 8 intratesticular fluid samples. DHT concentration also varied from rat to rat and was highly correlated with T levels. The levels of E(2) and 3α-diol also were measurable. The availability of a sensitive method by which to measure steroids accurately and rapidly in the small volumes of intratesticular fluid obtainable from individual rats will make it possible to examine the effects, over time, of such perturbations as hormone and drug administration and environmental toxicant exposures on the intratesticular hormonal environment of exposed individual males and thereby to begin to understand differences in response between individuals.

Apoptosis is an important antivirus defense by virtue of its impact on virus multiplication and pathogenesis. To define molecular mechanisms by which viruses are detected and the apoptotic response is initiated, we examined the antiviral role of host inhibitor-of-apoptosis (IAP) proteins in insect cells. We report here that the principal IAPs, DIAP1 and SfIAP, of the model insects Drosophila melanogaster and Spodoptera frugiperda , respectively, are rapidly depleted and thereby inactivated upon infection with the apoptosis-inducing baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Virus-induced loss of these host IAPs triggered caspase activation and apoptotic death. Elevation of IAP levels by ectopic expression repressed caspase activation. Loss of host IAP in both species was triggered by AcMNPV DNA replication. By using selected inhibitors, we found that virus-induced IAP depletion was mediated in part by the proteasome but not by caspase cleavage. Consistent with this conclusion, mutagenic disruption of the SfIAP RING motif, which acts as an E3 ubiquitin ligase, stabilized SfIAP during infection. Importantly, SfIAP was also stabilized upon the removal of its 99-residue N-terminal leader, which serves as a critical determinant of IAP turnover. These data indicated that a host pathway initiated by virus DNA replication and acting through instability motifs embedded within IAP triggers IAP depletion and thereby causes apoptosis. Taken together, the results of our study suggest that host modulation of cellular IAP levels is a conserved mechanism by which insects mount an apoptotic antiviral response. Thus, host IAPs may function as critical sentinels of virus invasion in insects.

We present the results of numerical simulations of the electrostatics and dynamics of water surrounding Kihara solutes described by a Lennard-Jones layer at the surface of a hard-sphere core. The dipolar response of the hydration layer peaks at the solute surface, significantly exceeding bulk water in polarity. This effect can be observed by atomic force microscopy. The dynamics of water shells are slow close to the surface, but become faster with the growing layer thickness and approach the bulk limit for layers 1 nm thick. Slowing of the inner hydration shells strongly depends on the strength of solute–solvent attraction, offering a possibility of highly heterogeneous interfacial dynamics.

One of several challenges in design of clinical chemoprevention trials is the selection of the dose, formulation, and dose schedule of the intervention agent. Therefore, a cross-over clinical trial was undertaken to compare the bioavailability and tolerability of sulforaphane from two of broccoli sprout–derived beverages: one glucoraphanin-rich (GRR) and the other sulforaphane-rich (SFR). Sulforaphane was generated from glucoraphanin contained in GRR by gut microflora or formed by treatment of GRR with myrosinase from daikon (Raphanus sativus) sprouts to provide SFR. Fifty healthy, eligible participants were requested to refrain from crucifer consumption and randomized into two treatment arms. The study design was as follows: 5-day run-in period, 7-day administration of beverages, 5-day washout period, and 7-day administration of the opposite intervention. Isotope dilution mass spectrometry was used to measure levels of glucoraphanin, sulforaphane, and sulforaphane thiol conjugates in urine samples collected daily throughout the study. Bioavailability, as measured by urinary excretion of sulforaphane and its metabolites (in approximately 12-hour collections after dosing), was substantially greater with the SFR (mean = 70%) than with GRR (mean = 5%) beverages. Interindividual variability in excretion was considerably lower with SFR than with GRR beverage. Elimination rates were considerably slower with GRR, allowing for achievement of steady-state dosing as opposed to bolus dosing with SFR. Optimal dosing formulations in future studies should consider blends of sulforaphane and glucoraphanin as SFR and GRR mixtures to achieve peak concentrations for activation of some targets and prolonged inhibition of others implicated in the protective actions of sulforaphane. Cancer Prev Res; 4(3); 384–95. ©2011 AACR.