Species‐specific responses to wetland mitigation among amphibians in the Greater Yellowstone Ecosystem

Habitat loss and degradation are leading causes of biodiversity declines, therefore assessing the capacity of created mitigation wetlands to replace habitat for wildlife has become a management priority. We used single season occupancy models to compare the occurrence of larvae of four species of pond‐breeding amphibians in wetlands created for mitigation, wetlands impacted by road construction, and unimpacted reference wetlands along a highway corridor in the Greater Yellowstone Ecosystem, United States. Created wetlands were shallow and had less aquatic vegetation and surface area than impacted and reference wetlands. Occupancy of barred tiger salamander (Ambystoma mavortium) and boreal chorus frog (Pseudacris maculata) larvae was similar across wetland types, whereas boreal toads (Anaxyrus boreas) occurred more often in created wetlands than reference and impacted wetlands. However, the majority of created wetlands (>80%) dried partially or completely before amphibian metamorphosis occurred in both years of our study, resulting in heavy mortality of larvae and, we suspect, little to no recruitment. Columbia spotted frogs (Rana luteiventris), which require emergent vegetation that is not common in newly created wetlands, occurred commonly in impacted and reference wetlands but were found in only one created wetland. Our results show that shallow created wetlands with little aquatic vegetation may be attractive breeding areas for some amphibians, but may result in high mortality and little recruitment if they fail to hold water for the entire larval period.     

Effect of human papillomavirus (HPV) vaccination on clinical indicators of sexual behaviour among adolescent girls: the Ontario Grade 8 HPV Vaccine Cohort Study

Background:

Suboptimal human papillomavirus (HPV) vaccine coverage in some jurisdictions is partly attributed to fears that vaccination may increase risky sexual behaviour. We assessed the effect of HPV vaccination on clinical indicators of sexual behaviour among adolescent girls in Ontario.

Methods:

Using Ontario’s administrative health databases, we identified a population-based cohort of girls in grade 8 in the 2 years before (2005/06 and 2006/07) and after (2007/08 and 2008/09) implementation of Ontario’s grade 8 HPV vaccination program. For each girl, we then obtained data on vaccine receipt in grades 8 and 9 and data on indicators of sexual behaviour (pregnancy and non–HPV-related sexually transmitted infections) in grades 10–12. Using a quasi-experimental method known as regression discontinuity, we estimated, for each outcome, the risk difference (RD) and relative risk (RR) attributable to vaccination and to program eligibility.

Results:

The cohort comprised 260 493 girls, of whom 131 781 were ineligible for the program and 128 712 were eligible. We identified 15 441 (5.9%) cases of pregnancy and sexually transmitted infection and found no evidence that vaccination increased the risk of this composite outcome: RD per 1000 girls −0.61 (95% confidence interval [CI] −10.71 to 9.49) and RR 0.96 (95% CI 0.81 to 1.14). Similarly, we found no discernible effect of program eligibility: RD per 1000 girls −0.25 (95% CI −4.35 to 3.85) and RR 0.99 (95% CI 0.93 to 1.06). The findings were similar when outcomes were assessed separately.

Interpretation:

We present strong evidence that HPV vaccination does not have any significant effect on clinical indicators of sexual behaviour among adolescent girls. These results suggest that concerns over increased promiscuity following HPV vaccination are unwarranted and should not deter from vaccinating at a young age.Infection with the human papillomavirus (HPV) is the most commonly diagnosed sexually transmitted infection in Canada and around the world.¹ Although most of these infections are transient and self-resolving, others persist and can cause important health outcomes, including cervical cancer and anogenital warts.In 2006, Canada was among 49 countries to license Gardasil (Merck, Whitehouse Station, New Jersey), a quadrivalent HPV vaccine designed to protect against 4 types of HPV (6, 11, 16, 18) that cause 70% of cases of cervical cancer and most cases of anogenital warts.^2–4 As one of the first cancer-preventing vaccines, this vaccine received expedited approval in several countries and was the subject of intensive marketing, lobbying and public health campaigns around the world.⁵ By 2012, it had been approved in almost 100 countries, many of which also implemented nationwide HPV vaccination programs aimed primarily at immunizing young girls before the onset of sexual activity.⁶Despite the popularity of large-scale immunization programs, HPV vaccination has faced a great deal of controversy regarding unanswered questions about the real-world effects of this vaccine.^7,8 A major topic of public debate has been the possibility that HPV vaccination might lead to sexual disinhibition,⁹ that is, that receipt of the vaccine might give women and girls a false sense of protection against all sexually transmitted infections and that this false sense of protection might lead them to engage in more risky sexual behaviours than they would otherwise (e.g., be more promiscuous or neglect to use condoms). Increases in these risky behaviours could have important clinical consequences, including increased risk of pregnancy and sexually transmitted infections. Although there is little empirical support for the notion that sexual health interventions promote risky sexual behaviours,^10,11 this possible unintended effect of the HPV vaccine would undermine its value for reducing the burden of sexual health–related diseases. Moreover, parental fears of increased promiscuity following HPV vaccination have been reported as a major determinant of vaccine refusal,¹² which may help to explain suboptimal HPV vaccine coverage in some jurisdictions.^6,13 Evidently, both actual and perceived sexual disinhibition can have a negative effect on the potential health benefits of HPV vaccination. Therefore, we conducted a population-based, retrospective cohort study to assess the effect of HPV vaccination on clinical indicators of sexual behaviour among adolescent girls in Ontario.     

The pro-apoptotic protein Prostate Apoptosis Response Protein-4 (Par-4) can be activated in colon cancer cells by treatment with Src inhibitor and 5-FU

The overexpression of the pro-apoptotic protein Prostate Apoptosis Response Protein-4 in colon cancer has been shown to increase response to the chemotherapeutic agent 5-fluorouracil (5-FU). Although colon cancer cells endogenously express Par-4, the presence or overexpression of Par-4 alone does not cause apoptosis. We hypothesize that Par-4 is inactivated in colon cancer. In colon cancer, the levels and the kinase activity of the nonreceptor tyrosine kinase c-Src increase with tumor progression. One of the downstream effectors of c-Src is Akt1. Akt1 has been shown to inhibit the pro-apoptotic activity of Par-4 in prostate cancer cells. We therefore investigated the potential of activating Par-4 by inhibiting c-Src. Colon carcinoma cell lines were treated with the Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2) in combination with the chemotherapeutic agent 5-FU. Treating cells with PP2 and 5-FU resulted in reduced interaction of Par-4 with Akt1 and with the scaffolding protein 14-3-3σ, and mobilization of Par-4 to the nucleus. Par-4 was shown to interact not only with Akt1 and 14-3-3σ, but also with c-Src. Overexpression of c-Src induced the phosphorylation of Par-4 at tyrosine site/s. Thus, in this study, we have shown that Par-4 can be activated by inhibiting Src with a pharmacological inhibitor and adding a chemotherapeutic agent. The activation of the pro-apoptotic protein Par-4 as reported in this study is a novel mechanism by which apoptosis occurs with a Src kinase inhibitor and 5-FU. In addition, we have demonstrated that the pro-apoptotic activity of endogenously expressed Par-4 can be increased in colon cancer cells.     

Development of methods for the field evaluation of Oobius agrili (Hymenoptera: Encyrtidae) in North America,a newly introduced egg parasitoid of the emerald ash borer (Coleoptera: Buprestidae)

A field study was conducted in forested plots near Lansing, Michigan in 2008 and 2009 to evaluate the newly introduced egg parasitoid Oobius agrili Zhang and Huang (Hymenoptera: Encyrtidae) for control of the invasive emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coleoptera: Buprestidae). To measure parasitism by O. agrili, laboratory-reared “sentinel EAB eggs” were deployed under bark flaps on trunks of selected ash trees in both parasitoid-release and non-release control plots. In addition, naturally occurring EAB eggs were collected in both parasitoid-release and control plots to measure parasitism. While no parasitism was detected with either sentinel or naturally occurring EAB eggs in control plots in either 2008 or 2009, a low level of parasitism by O. agrili was detected in the parasitoid-release plots in both artificially deployed sentinel eggs (?1%) and field-collected, naturally occurring eggs (1.1–4.2%) in both years. In addition to losses due to parasitism by O. agrili, a large proportion (37–52%) of the field-deployed sentinel eggs disappeared, possibly due to predators such as ants, in both parasitoid-release and control plots. While no statistical differences in parasitism by O. agrili were detected between parasitoid release and control plots, other sources of egg mortality such as disappearance due to predation on eggs, varied significantly across study sites in both 2008 and 2009. The relevance of these findings to future release and evaluation strategies for O. agrili for biological control of the invasive emerald ash borer in the US is discussed.     

Gfer inhibits Jab1-mediated degradation of p27kip1 to restrict proliferation of hematopoietic stem cells

Growth factor erv1-like (Gfer) is an evolutionarily conserved sulfhydryl oxidase that is enriched in embryonic and adult stem cells and plays an essential prosurvival role in pluripotent embryonic stem cells. Here we show that knockdown (KD) of Gfer in hematopoietic stem cells (HSCs) compromises their in vivo engraftment potential and triggers a hyper-proliferative response that leads to their exhaustion. KD of Gfer in HSCs does not elicit a significant alteration of mitochondrial morphology or loss of cell viability. However, these cells possess significantly reduced levels of the cyclin-dependent kinase inhibitor p27(kip1). In contrast, overexpression of Gfer in HSCs results in significantly elevated total and nuclear p27(kip1). KD of Gfer results in enhanced binding of p27(kip1) to its inhibitor, the COP9 signalosome subunit jun activation-domain binding protein 1 (Jab1), leading to its down-regulation. Conversely, overexpression of Gfer results in its enhanced binding to Jab1 and inhibition of the Jab1-p27(kip1) interaction. Furthermore, normalization of p27(kip1) in Gfer-KD HSCs rescues their in vitro proliferation deficits. Taken together, our data demonstrate the presence of a novel Gfer-Jab1-p27(kip1) pathway in HSCs that functions to restrict abnormal proliferation.     

The hydroxyl functionality and a rigid proximal N are required for forming a novel non-covalent quinine-heme complex

Quinoline antimalarial drugs bind both monomeric and dimeric forms of free heme, with distinct preferences depending on the chemical environment. Under biological conditions, chloroquine (CQ) appears to prefer to bind to μ-oxo dimeric heme, while quinine (QN) preferentially binds monomer. To further explore this important distinction, we study three newly synthesized and several commercially available QN analogues lacking various functional groups. We find that removal of the QN hydroxyl lowers heme affinity, hemozoin (Hz) inhibition efficiency, and antiplasmodial activity. Elimination of the rigid quinuclidyl ring has similar effects, but elimination of either the vinyl or methoxy group does not. Replacing the quinuclidyl N with a less rigid tertiary aliphatic N only partially restores activity. To further study these trends, we probe drug-heme interactions via NMR studies with both Fe and Zn protoporphyrin IX (FPIX, ZnPIX) for QN, dehydroxyQN (DHQN), dequinuclidylQN (DQQN), and deamino-dequinuclidylQN (DADQQN). Magnetic susceptibility measurements in the presence of FPIX demonstrate that these compounds differentially perturb FPIX monomer-dimer equilibrium. We also isolate the QN-FPIX complex formed under mild aqueous conditions and analyze it by mass spectrometry, as well as fluorescence, vibrational, and solid-state NMR spectroscopies. The data elucidate key features of QN pharmacology and allow us to propose a refined model for the preferred binding of QN to monomeric FPIX under biologically relevant conditions. With this model in hand, we also propose how QN, CQ, and amodiaquine (AQ) differ in their ability to inhibit Hz formation.     

Calorie restriction attenuates LPS-induced sickness behavior and shifts hypothalamic signaling pathways to an anti-inflammatory bias

Calorie restriction (CR) has been demonstrated to alter cytokine levels; however, its potential to modify sickness behavior (fever, anorexia, cachexia) has not. The effect of CR on sickness behavior was examined in male C57BL/6J mice fed ad libitum or restricted 25% (CR25%) or restricted 50% (CR50%) in food intake for 28 days and injected with 50 μg/kg of LPS on day 29. Changes in body temperature, locomotor activity, body weight, and food intake were determined. A separate cohort of mice were fed ad libitum or CR50% for 28 days, and hypothalamic mRNA expression of inhibitory factor κB-α (IκB-α), cyclooxygenase-2 (COX-2), prostaglandin E(2) (PGE(2)), suppressor of cytokine signaling 3 (SOCS3), IL-10, neuropeptide Y (NPY), leptin, proopiomelanocortin (POMC), and corticotrophin-releasing hormone (CRH) were determined at 0, 2, and 4 h post-LPS. CR50% mice did not develop fevers, whereas the CR25% mice displayed a fever shorter in duration but with the same peak as the controls. Both CR25% and CR50% mice showed no sign of anorexia and reduced cachexia after LPS administration. Hypothalamic mRNA expression of NPY and CRH were both increased by severalfold in CR50% animals preinjection compared with controls. The CR50% mice did not demonstrate the expected rise in hypothalamic mRNA expression of COX-2, microsomal prostaglandin E synthase-1, POMC, or CRH 2 h post-LPS, and leptin expression was decreased at this time point. Increases in SOCS3, IL-10, and IκB-α expression in CR50% animals were enhanced compared with ad libitum-fed controls at 4 h post-LPS. CR results in a suppression of sickness behavior in a dose-dependent manner, which may be due to CR attenuating proinflammatory pathways and enhancing anti-inflammatory pathways.     

Negative emotionality, depressive symptoms and cortisol diurnal rhythms: analysis of a community sample of middle-aged males

Prior research suggests that individuals with particular personality traits, like negative emotionality, are at greater risk for adverse health outcomes. Despite bivariate associations between negative emotionality, depressive symptoms and the hypothalamic pituitary adrenal axis (HPA axis), few studies have sought to understand the biological pathways through which negative emotionality, depressive symptomatology and cortisol—one of the primary hormonal products of the HPA axis—are associated. The present study explored whether negative emotionality influenced cortisol dysregulation through current depressive symptomatology and whether negative emotionality served as a moderator of the relationship between depressive symptoms and cortisol. In the community-based Vietnam Era Twin Study of Aging, 783 male twins completed two days of cortisol saliva sampling in their natural environments. Three measures of cortisol were analyzed: waking levels, the cortisol awakening response, and the peak to bed slope. Depressive symptoms significantly mediated the associations between negative emotionality and the peak to bed slope. A 2-way interaction between depressive symptoms and negative emotionality was significant for the peak to bed slope and for waking levels of cortisol. Exploration of the interactions illustrated that depressive symptoms only affected cortisol slopes at average or high levels of negative emotionality and only affected waking levels at low levels of negative emotionality. Negative emotionality and depressive symptoms were not related to the cortisol awakening response. This is the first study to find indirect associations between negative emotionality and peak to bed cortisol slopes through depressive symptoms. These findings illustrate the complex interplay between personality characteristics, depressive symptoms and different indices of the cortisol diurnal rhythm.     

Synergistic Ca2+ responses by G{alpha}i- and G{alpha}q-coupled G-protein-coupled receptors require a single PLC{beta} isoform that is sensitive to both G{beta}{gamma} and G{alpha}q

Cross-talk between Gα(i)- and Gα(q)-linked G-protein-coupled receptors yields synergistic Ca(2+) responses in a variety of cell types. Prior studies have shown that synergistic Ca(2+) responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cβ3 (PLCβ3), with a possible contribution of PLCβ2, whereas signaling through PLCβ4 interferes with synergy. We here show that synergy can be induced by the combination of Gβγ and Gα(q) activation of a single PLCβ isoform. Synergy was absent in macrophages lacking both PLCβ2 and PLCβ3, but it was fully reconstituted following transduction with PLCβ3 alone. Mechanisms of PLCβ-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCβ2. RNAi-mediated knockdown of endogenous PLCβs demonstrated that synergy in these cells was dependent on PLCβ3, but PLCβ1 and PLCβ4 did not contribute, and overexpression of either isoform inhibited Ca(2+) synergy. When synergy was blocked by RNAi of endogenous PLCβ3, it could be reconstituted by expression of either human PLCβ3 or mouse PLCβ2. In contrast, it could not be reconstituted by human PLCβ3 with a mutation of the Y box, which disrupted activation by Gβγ, and it was only partially restored by human PLCβ3 with a mutation of the C terminus, which partly disrupted activation by Gα(q). Thus, both Gβγ and Gα(q) contribute to activation of PLCβ3 in cells for Ca(2+) synergy. We conclude that Ca(2+) synergy between Gα(i)-coupled and Gα(q)-coupled receptors requires the direct action of both Gβγ and Gα(q) on PLCβ and is mediated primarily by PLCβ3, although PLCβ2 is also competent.     

Targeting TLR4 signaling by TLR4 Toll/IL-1 receptor domain-derived decoy peptides: identification of the TLR4 Toll/IL-1 receptor domain dimerization interface

Agonist-induced dimerization of TLR4 Toll/IL-1R (TIR) domains initiates intracellular signaling. Therefore, identification of the TLR4-TIR dimerization interface is one key to the rational design of therapeutics that block TLR4 signaling. A library of cell-permeating decoy peptides, each of which represents a nonfragmented patch of the TLR4 TIR surface, was designed such that the peptides entirely encompass the TLR4 TIR surface. Each peptide was synthesized in tandem with a cell-permeating Antennapedia homeodomain sequence and tested for the ability to inhibit early cytokine mRNA expression and MAPK activation in LPS-stimulated primary murine macrophages. Five peptides--4R1, 4R3, 4BB, 4R9, and 4αE--potently inhibited all manifestations of TLR4, but not TLR2 signaling. When tested for their ability to bind directly to TLR4 TIR by F?rster resonance energy transfer using time-resolved fluorescence spectroscopy, Bodipy-TMR-X-labeled 4R1, 4BB, and 4αE quenched fluorescence of TLR4-Cerulean expressed in HeLa or HEK293T cells, whereas 4R3 was partially active, and 4R9 was least active. These findings suggest that the area between the BB loop of TLR4 and its fifth helical region mediates TLR4 TIR dimerization. Moreover, our data provide direct evidence for the utility of the decoy peptide approach, in which peptides representing various surface-exposed segments of a protein are initially probed for the ability to inhibit protein function, and then their specific targets are identified by F?rster resonance energy transfer to define recognition sites in signaling proteins that may be targeted therapeutically to disrupt functional transient protein interactions.