Rigorous theory-based intervention research is the bold action needed to address oral health disparities and inequities.

The Consensus Statement about behavioral and social sciences in oral health encourages future research to draw on testable theories that specify causal pathways that reflect the complex nature of oral health. In this commentary, we amplify the importance of explicit and well-specified theory in oral health intervention research, acknowledging that problematic use of theory has limited its utility in developing effective public health interventions. Also, we affirm the need to focus on determinants of oral health-and health inequities-most likely to drive meaningful change, and to understand the causal pathways that connect drivers of change from the individual to the global level. We view theory-based, causal mechanisms research as a powerful approach to building successful public health interventions, and suggest resources to inspire such research, including exemplary studies, methodologies, and collaborative initiatives that facilitate strong theory-based public health research.

The molecular architecture of Drosophila melanogaster defense against Beauveria bassiana explored through evolve and resequence and quantitative trait locus mapping.

Little is known about the genetic architecture of antifungal immunity in natural populations. Using two population genetic approaches, quantitative trait locus (QTL) mapping and evolve and resequence (E&R), we explored D. melanogaster immune defense against infection with the fungus Beauveria bassiana. The immune defense was highly variable both in the recombinant inbred lines from the Drosophila Synthetic Population Resource used for our QTL mapping and in the synthetic outbred populations used in our E&R study. Survivorship of infection improved dramatically over just 10 generations in the E&R study, and continued to increase for an additional nine generations, revealing a trade-off with uninfected longevity. Populations selected for increased defense against B. bassiana evolved cross resistance to a second, distinct B. bassiana strain but not to bacterial pathogens. The QTL mapping study revealed that sexual dimorphism in defense depends on host genotype, and the E&R study indicated that sexual dimorphism also depends on the specific pathogen to which the host is exposed. Both the QTL mapping and E&R experiments generated lists of potentially causal candidate genes, although these lists were nonoverlapping.

HIV screening in dental settings: Challenges, opportunities, and a call to action.

HIV is responsible for tremendous suffering and loss around the world, but many advances in HIV screening, diagnosis, treatment, and prevention provide hope for an end to the HIV epidemic. Global and national campaigns facilitate access to these HIV advances, but some individuals and communities still lack access, particularly in developing countries. To reach those who remain under-served, campaigns encourage greater integration of HIV services with non-HIV services. As members of the healthcare team with a clinical stake in HIV, dental care providers have a unique contribution to make. Much research on the role of dental care providers in HIV has focused on HIV screening in the dental setting, and researchers have identified possible ways forward but also daunting challenges. Approaches for screening, brief intervention, and referral to treatment used in primary care and dental care settings for other health risks may help overcome challenges related to provider scope of practice and need for training. Approaches to managing distress and uncertainty in other clinical contexts may help overcome challenges related to patient acceptability, equipping providers to manage sensitive topics and emotional aspects of HIV screening. While not panaceas, these approaches may be useful to dental care providers interested in answering the global "call to action" for contributing to ending the HIV epidemic.

Computer-Assisted Tracking of Chlamydomonas Species.

The green algae Chlamydomonas reinhardtii is a model system for motility in unicellular organisms. Photo-, gravi-, and chemotaxis have previously been associated with C. reinhardtii, and observing the extent of these responses within a population of cells is crucial for refining our understanding of how this organism responds to changing environmental conditions. However, manually tracking and modeling a statistically viable number of samples of these microorganisms is an unreasonable task. We hypothesized that automated particle tracking systems are now sufficiently advanced to effectively characterize such populations. Here, we present an automated method to observe C. reinhardtii motility that allows us to identify individual cells as well as global information on direction, speed, and size. Nutrient availability effects on wild-type C. reinhardtii swimming speeds, as well as changes in speed and directionality in response to light, were characterized using this method. We also provide for the first time the swimming speeds of several motility-deficient mutant lines. While our present effort is focused around the unicellular green algae, C. reinhardtii, we confirm the general utility of this approach using Chlamydomonas moewusii, another member of this genus which contains over 300 species. Our work provides new tools for evaluating and modeling motility in this model organism and establishes the methodology for conducting similar experiments on other unicellular microorganisms.

NIH research opportunities for the prevention and treatment for chronic conditions.

Chronic conditions constitute the leading cause of death and disability in the USA and constitute 86 per cent of the nation's annual healthcare expenses. Approximately half of all American adults have at least one chronic condition; 25 per cent of these Americans have two or more chronic conditions. The National Institutes of Health have funded many projects that explain epidemiology, risk factors, and prevention and treatment of chronic conditions, though research questions remain. This commentary discusses some past projects, current areas of interest, and funding opportunities from many NIH Institutes, Centers, and Offices.

NIH Policies on Experimental Studies with Humans.

The U.S. National Institutes of Health clinical trials policies will apply broadly to studies involving experimental manipulations of humans. These studies will require registration and reporting in ClinicalTrials.gov, grant application submission under a clinical trials funding opportunity announcement, and Good Clinical Practice training for investigators.

The NIH Science of Behavior Change Program: Transforming the science through a focus on mechanisms of change.

The goal of the NIH Science of Behavior Change (SOBC) Common Fund Program is to provide the basis for an experimental medicine approach to behavior change that focuses on identifying and measuring the mechanisms that underlie behavioral patterns we are trying to change. This paper frames the development of the program within a discussion of the substantial disease burden in the U.S. attributable to behavioral factors, and details our strategies for breaking down the disease- and condition-focused silos in the behavior change field to accelerate discovery and translation. These principles serve as the foundation for our vision for a unified science of behavior change at the NIH and in the broader research community.

Reenvisioning Clinical Science: Unifying the Discipline to Improve the Public Health.

We present a vision of clinical science, based on a conceptual framework of intervention development endorsed by the Delaware Project. This framework is grounded in an updated stage model that incorporates basic science questions of mechanisms into every stage of clinical science research. The vision presented is intended to unify various aspects of clinical science toward the common goal of developing maximally potent and implementable interventions, while unveiling new avenues of science in which basic and applied goals are of equally high importance. Training in this integrated, translational model may help students learn how to conduct research in every domain of clinical science and at each stage of intervention development. This vision aims to propel the field to fulfill the public health goal of producing implementable and effective treatment and prevention interventions.

Hydrogen sulfide dilates rat mesenteric arteries by activating endothelial large-conductance Ca²âº-activated K⁺ channels and smooth muscle Ca²âº sparks.

We have previously shown that hydrogen sulfide (H2S) reduces myogenic tone and causes relaxation of phenylephrine (PE)-constricted mesenteric arteries. This effect of H2S to cause vasodilation and vascular smooth muscle cell (VSMC) hyperpolarization was mediated by large-conductance Ca(2+)-activated potassium channels (BKCa). Ca(2+) sparks are ryanodine receptor (RyR)-mediated Ca(2+)-release events that activate BKCa channels in VSMCs to cause membrane hyperpolarization and vasodilation. We hypothesized that H2S activates Ca(2+) sparks in small mesenteric arteries. Ca(2+) sparks were measured using confocal microscopy in rat mesenteric arteries loaded with the Ca(2+) indicator fluo-4. VSMC membrane potential (Em) was measured in isolated arteries using sharp microelectrodes. In PE-constricted arteries, the H2S donor NaHS caused vasodilation that was inhibited by ryanodine (RyR blocker), abluminal or luminal iberiotoxin (IbTx, BKCa blocker), endothelial cell (EC) disruption, and sulfaphenazole [cytochrome P-450 2C (Cyp2C) inhibitor]. The H2S donor NaHS (10 µmol/l) increased Ca(2+) sparks but only in the presence of intact EC and this was blocked by sulfaphenazole or luminal IbTx. Inhibiting cystathionine γ-lyase (CSE)-derived H2S with ß-cyano-l-alanine (BCA) also reduced VSMC Ca(2+) spark frequency in mesenteric arteries, as did EC disruption. However, excess CSE substrate homocysteine did not affect spark activity. NaHS hyperpolarized VSMC Em in PE-depolarized mesenteric arteries with intact EC and also hyperpolarized EC Em in arteries cut open to expose the lumen. This hyperpolarization was prevented by ryanodine, sulfaphenazole, and abluminal or luminal IbTx. BCA reduced IbTx-sensitive K(+) currents in freshly dispersed mesenteric ECs. These results suggest that H2S increases Ca(2+) spark activity in mesenteric artery VSMC through activation of endothelial BKCa channels and Cyp2C, a novel vasodilatory pathway for this emerging signaling molecule.

Regulation of endothelial BK channels by heme oxygenase-derived carbon monoxide and caveolin-1.

A novel vasodilatory influence of endothelial cell (EC) large-conductance Ca(2+)-activated K(+) (BK) channels is present after in vivo exposure to chronic hypoxia (CH) and may exist in other pathological states. However, the mechanism of channel activation that results in altered vasoreactivity is unknown. Previously, we demonstrated that inhibition of either BK channels or heme oxygenase (HO) restores vasoconstrictor reactivity after CH. Additionally, administration of the scaffolding domain of caveolin (Cav)-1 inhibits EC BK activity and restores vasoconstrictor reactivity in this setting. These results led us to hypothesize that CH exposure results in a loss in Cav-1 inhibition of EC BK channels, resulting in their activation by HO-derived carbon monoxide (CO). Experiments were conducted on freshly dispersed aortic ECs from control and CH-exposed (barometric pressure: 380 mmHg for 48 h) rats. In electrophysiology experiments, outward currents were greater in cells from CH rats as well as from cells from control rats treated with the cholesterol-depleting agent methyl-ß-cyclodextrin. These enhanced currents were returned to control by HO inhibition. Channel activity could be restored by the CO donor CO-releasing molecule (CORM)-2 during HO inhibition. Administration of the Cav-1 scaffolding domain eliminated BK currents in cells from CH rats, and current was not restored by the addition of CORM-2. Colocalization experiments in ECs from control and CH rats demonstrated an association between HO-2, Cav-1, and BK. We conclude that EC BK channel activity is HO dependent in the absence of the inhibitory effect of the Cav-1 scaffolding domain.

Role of caveolin-1 in endothelial BKCa channel regulation of vasoreactivity.

A novel vasodilatory influence of endothelial cell (EC) large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels is present following in vivo exposure to chronic hypoxia (CH) and may exist in other pathological states. However, the mechanism of channel activation that results in altered vasoreactivity is unknown. We tested the hypothesis that CH removes an inhibitory effect of the scaffolding domain of caveolin-1 (Cav-1) on EC BK(Ca) channels to permit activation, thereby affecting vasoreactivity. Experiments were performed on gracilis resistance arteries and ECs from control and CH-exposed (380 mmHg barometric pressure for 48 h) rats. EC membrane potential was hyperpolarized in arteries from CH-exposed rats and arteries treated with the cholesterol-depleting agent methyl-ß-cyclodextrin (MBCD) compared with controls. Hyperpolarization was reversed by the BK(Ca) channel antagonist iberiotoxin (IBTX) or by a scaffolding domain peptide of Cav-1 (AP-CAV). Patch-clamp experiments documented an IBTX-sensitive current in ECs from CH-exposed rats and in MBCD-treated cells that was not present in controls. This current was enhanced by the BK(Ca) channel activator NS-1619 and blocked by AP-CAV or cholesterol supplementation. EC BK(Ca) channels displayed similar unitary conductance but greater Ca(2+) sensitivity than BK(Ca) channels from vascular smooth muscle. Immunofluorescence imaging demonstrated greater association of BK(Ca) α-subunits with Cav-1 in control arteries than in arteries from CH-exposed rats, although fluorescence intensity for each protein did not differ between groups. Finally, AP-CAV restored myogenic and phenylephrine-induced constriction in arteries from CH-exposed rats without affecting controls. AP-CAV similarly restored diminished reactivity to phenylephrine in control arteries pretreated with MBCD. We conclude that CH unmasks EC BK(Ca) channel activity by removing an inhibitory action of the Cav-1 scaffolding domain that may depend on cellular cholesterol levels.

Altered membrane lipid domains limit pulmonary endothelial calcium entry following chronic hypoxia.

Agonist-induced Ca(2+) entry into the pulmonary endothelium depends on activation of both store-operated Ca(2+) (SOC) entry and receptor-operated Ca(2+) (ROC) entry. We previously reported that pulmonary endothelial cell SOC entry and ROC entry are reduced in chronic hypoxia (CH)-induced pulmonary hypertension. We hypothesized that diminished endothelial Ca(2+) entry following CH is due to derangement of caveolin-1 (cav-1) containing cholesterol-enriched membrane domains important in agonist-induced Ca(2+) entry. To test this hypothesis, we measured Ca(2+) influx by fura-2 fluorescence following application of ATP (20 µM) in freshly isolated endothelial cells pretreated with the caveolar-disrupting agent methyl-ß-cyclodextrin (mßCD; 10 mM). Cholesterol depletion with mßCD attenuated agonist-induced Ca(2+) entry in control endothelial cells to the level of that from CH rats. Interestingly, endothelial membrane cholesterol was lower in cells isolated from CH rats compared with controls although the density of caveolae did not differ between groups. Cholesterol repletion with a cholesterol:mßCD mixture or the introduction of the cav-1 scaffolding peptide (AP-cav; 10 µM) rescued ATP-induced Ca(2+) entry in endothelia from CH arteries. Agonist-induced Ca(2+) entry assessed by Mn(2+) quenching of fura-2 fluorescence was also significantly elevated by luminal AP-cav in pressurized intrapulmonary arteries from CH rats to levels of controls. Similarly, patch-clamp experiments revealed diminished inward current in response to ATP in cells from CH rats compared with controls that was restored by AP-cav. These data suggest that CH-induced pulmonary hypertension leads to reduced membrane cholesterol that limits the activity of ion channels necessary for agonist-activated Ca(2+) entry.

Novel role of endothelial BKCa channels in altered vasoreactivity following hypoxia.

The systemic vasculature exhibits attenuated vasoconstriction following hypobaric chronic hypoxia (CH) that is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization. We hypothesized that increased activity of endothelial cell (EC) large-conductance, calcium-activated potassium (BK(Ca)) channels contributes to this response. Gracilis resistance arteries from hypobaric CH (barometric pressure = 380 mmHg for 48 h) rats demonstrated reduced myogenic reactivity and hyperpolarized VSM membrane potential (E(m)) compared with controls under normoxic ex vivo conditions. These differences were eliminated by endothelial disruption. In the presence of cyclooxygenase and nitric oxide synthase inhibition, combined intraluminal administration of the intermediate and small-conductance, calcium-activated K(+) channel blockers TRAM-34 and apamin was without effect on myogenic responsiveness and VSM E(m) in both groups; however, these variables were normalized in CH arteries by intraluminal administration of the BK(Ca) inhibitor iberiotoxin (IBTX). Basal EC E(m) was hyperpolarized in arteries from CH rats compared with controls and was restored by IBTX, but not by TRAM-34/apamin. K(+) channel blockers were without effect on EC basal E(m) in controls. Similarly, IBTX blocked acetylcholine-induced dilation in arteries from CH rats, but was without effect in controls, whereas TRAM-34/apamin eliminated dilation in controls. Acetylcholine-induced EC hyperpolarization and calcium responses were inhibited by IBTX in CH arteries and by TRAM-34/apamin in controls. Patch-clamp experiments on freshly isolated ECs demonstrated greater K(+) current in cells from CH rats that was normalized by IBTX. IBTX was without effect on K(+) current in controls. We conclude that hypobaric CH induces increased endothelial BK(Ca) channel activity that contributes to reduced myogenic responsiveness and EC and VSM cell hyperpolarization.

Altered protein kinase C regulation of pulmonary endothelial store- and receptor-operated Ca2+ entry after chronic hypoxia.

Chronic hypoxia (CH)-induced pulmonary hypertension is associated with decreased basal pulmonary artery endothelial cell (EC) Ca(2+), which correlates with reduced store-operated Ca(2+) (SOC) entry. Protein kinase C (PKC) attenuates SOC entry in ECs. Therefore, we hypothesized that PKC has a greater inhibitory effect on EC SOC and receptor-operated Ca(2+) entry after CH. To test this hypothesis, we assessed SOC in the presence or absence of the nonselective PKC inhibitor GF109203X [2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide] in freshly isolated, Fura-2-loaded ECs obtained from intrapulmonary arteries of control and CH rats (4 weeks at 0.5 atm). We found that SOC entry and 1-oleoyl-2-acetyl-sn-glycerol (OAG)- and ATP-induced Ca(2+) influx were attenuated in ECs from CH rats versus controls, and GF109203X restored SOC and OAG responses to the level of controls. In contrast, nonselective PKC inhibition with GF109203X or the selective PKC(epsilon) inhibitor myristoylated V1-2 attenuated ATP-induced Ca(2+) entry in ECs from control but not CH pulmonary arteries. ATP-induced Ca(2+) entry was also attenuated by the T-type voltage-gated Ca(2+) channel (VGCC) inhibitor mibefradil in control cells. Consistent with the presence of endothelial T-type VGCC, we observed depolarization-induced Ca(2+) influx in control cells that was inhibited by mibefradil. This response was largely absent in ECs from CH arteries. We conclude that CH enhances PKC-dependent inhibition of SOC- and OAG-induced Ca(2+) entry. Furthermore, these data suggest that CH may reduce the ATP-dependent Ca(2+) entry that is mediated, in part, by PKCepsilon and mibefradil-sensitive Ca(2+) channels in control cells.