Project Kealahou: Improving Hawai‘i's System of Care for At-Risk Girls and Young Women through Gender-Responsive,Trauma-Informed Care

Project Kealahou (PK) is a six-year, federally-funded program aimed at improving services and outcomes for Hawai‘i''s female youth who are at risk for running away, truancy, abuse, suicide, arrest and incarceration. PK builds upon two decades of sustained cross-agency efforts among the state''s mental health, juvenile justice, education, and child welfare systems to promote system-of-care (SOC) principles of community-based, individualized, culturally and linguistically competent, family driven, youth-guided, and evidence-based services. In addition, PK emphasizes trauma-informed and gender-responsive care in serving its target population of females ages 11–18 years who have experienced psychological trauma.Results from the first four years of the implementation of PK in the Department of Health''s (DOH) Child and Adolescent Mental Health Division (CAMHD) highlight the serious familial, socioeconomic, functional, and interpersonal challenges faced by the young women who receive services in Hawai‘i''s SOC. Despite the challenges faced by PK youth and their families, preliminary results of the evaluation of PK show significant improvements across multiple clinical and functional domains of service recipients. A financial analysis indicates that these outcomes were obtained with a minimal overall increase in costs when compared to standard care alone. Overall, these results suggest that PK may offer a cost effective way to improve access, care, and outcomes for at-risk youth and their families in Hawai‘i.     

Modulation of α1β3γ2 GABAA receptors expressed in X. laevis oocytes using a propofol photoswitch tethered to the transmembrane helix

Tethered photoswitches are molecules with two photo-dependent isomeric forms, each with different actions on their biological targets. They include reactive chemical groups capable of covalently binding to their target. Our aim was to develop a β-subunit-tethered propofol photoswitch (MAP20), as a tool to better study the mechanism of anesthesia through the GABA_A α1β3γ2 receptor. We used short spacers between the tether (methanethiosulfonate), the photosensitive moiety (azobenzene), and the ligand (propofol), to allow a precise tethering adjacent to the putative propofol binding site at the β⁺α⁻ interface of the receptor transmembrane helices (TMs). First, we used molecular modeling to identify possible tethering sites in β3TM3 and α1TM1, and then introduced cysteines in the candidate positions. Two mutant subunits [β3(M283C) and α1(V227C)] showed photomodulation of GABA responses after incubation with MAP20 and illumination with lights at specific wavelengths. The α1β3(M283C)γ2 receptor showed the greatest photomodulation, which decreased as GABA concentration increased. The location of the mutations that produced photomodulation confirmed that the propofol binding site is located in the β⁺α⁻ interface close to the extracellular side of the transmembrane helices. Tethering the photoswitch to cysteines introduced in the positions homologous to β3M283 in two other subunits (α1W288 and γ2L298) also produced photomodulation, which was not entirely reversible, probably reflecting the different nature of each interface. The results are in agreement with a binding site in the β⁺α⁻ interface for the anesthetic propofol.

While photoswitches have been a popular topic in numerous reviews (1), their application in research is still very rare. Photoswitches are freely diffusible molecules containing a photosensitive moiety which can alternate between two isomeric forms depending on light irradiation at specific wavelengths. These isomeric forms of the photoswitch possess different affinities or efficacies toward their biological target, such that their pharmacological activity can be turned on or off depending on the light wavelength used, thus providing temporal and spatial control. When photoswitches covalently tether to a native or engineered residue at the specific biological target, the resulting conformation would determine the corresponding pharmacological activity. One way the tethered photoswitch can be activated is by positioning the tether in such a way that the ligand moiety can reach its binding site in only one of the photoswitch conformations. Additional major advantages of the tethered photoswitches are high local concentration (the residue cannot diffuse away) and spatial restriction within the biological target. The ligand groups of photoswitches can possess diverse pharmacological activities, acting as agonists, inverse agonists, or antagonists at specific binding sites.The GABA_A receptor is formed by five subunits (usually two α, two β, and one γ or δ) arranged in pseudosymmetry around a central channel, in the following order (counterclockwise, viewed from the extracellular side): γ-β-α-β-α (2). Each interface is named after the subunits that form it, with “+” and “−” designated following the counterclockwise order (a model of the α1β3γ2 GABA_A receptor can be found in SI Appendix, Fig. S1). Each subunit consists of an extracellular domain, attached to a sequence of four transmembrane helices (TMs), with a large intracellular loop inserted between TM3 and TM4. Multiple anesthetic drugs that act through this receptor possess relatively low binding affinities; therefore, precise identification and characterization of their binding sites require the use of techniques like mutagenesis, substituted cysteine modification protection (SCAMP), and photolabeling with photoreactive anesthetic analogs (3). Structures of the GABA_A receptor with bound anesthetic drugs have recently been made available (4), but one unexplored way of obtaining valuable, functional information would be by using appropriately designed tethered photoswitches. In previous studies of GABA_A receptors, the photoswitches used have included diffusible propofol photoswitches (5, 6) and a tethered propofol photoswitch with a very long spacer between the tethering cysteine (located in the extracellular domain) and the propofol moiety (6). Though all three positively modulated GABA_A receptors, none could be used to define the propofol binding site.Multiple studies point to propofol binding cavities being located in the TM interfaces between GABA_A receptor subunits. The observation that specific mutations in TM2 or TM3 of GABA_A β subunits could dramatically decrease propofol potentiation of GABA responses, and even direct activation of the receptor (7–9) led to the development of the β3(N265M) knockin mouse, which showed either a greatly decreased or absent hypnotic effect of propofol, depending on the test used (10). More recent studies have focused on a more complete characterization of binding sites for propofol as well as other intravenous anesthetics. A photoreactive propofol analog labeled three amino acids in the β⁺α⁻ interface (β3M286, α1M236, and α1I239) and one in the α⁺β⁻ interface (β3M227) in α1β3 receptors. Using etomidate and R-mTFD-MPAB [R-5-allyl-1-methyl-5-(m-trifluoromethyldiazirinylphenyl)barbituric acid] to inhibit photolabeling established that propofol also binds to the β⁺β⁻ interface, suggesting that propofol shows little selectivity for either interface (11). Another study mutated photolabeled residues (α1M236, β3M227, and their homologs, all located in TM1) to tryptophan (causing steric occupancy of the pocket) and cysteine (to test propofol protection against cysteine-specific labeling) (12). SCAMP studies confirmed that propofol binds to the β⁺α⁻ and α⁺β⁻ interfaces, and also to the γ⁺β⁻ interface; there was no evidence of binding to the α⁺γ⁻ interface. A more recent study has expanded the analysis of residues at the β⁺α⁻ interface that line a putative propofol binding site (13) (SI Appendix, Fig. S1). And cryoelectron microscopy (cryo-EM) structures of α1β2γ2 GABA_A receptors bound to intravenous anesthetics and benzodiazepines have recently been published (4), consolidating the evidence toward a propofol binding site at the β⁺α⁻ interfaces.Our aim was to develop a β-subunit-tethered propofol photoswitch, as a tool to better study the mechanism of anesthesia through the GABA_A α1β3ɣ2 receptor, merging both structural and functional approaches. This tethered photoswitch (Fig. 1A) consists of propofol as the ligand group, linked to an azobenzene group (which is photosensitive and can change between cis and trans isomeric forms, Fig. 1B), and finally a tethering group (methanethiosulfonate, that spontaneously forms a covalent bond with the thiolate group of cysteines located in water-filled cavities). This photoswitch was abbreviated as MAP20 (methanethiosulfonate azobenzene propofol 2020). These three basic components of this tethered photoswitch are connected by short spacers, decreasing the range between tethering and target residues. We used β3 subunits in our study because the immobilizing and hypnotic effects of propofol are mostly mediated by β3-containing receptors (10).Open in a separate windowFig. 1.Tethered photoswitch. (A) Methanethiosulfonate azobenzene propofol 2020 (MAP20) consists of propofol (ligand group), an azobenzene moiety (photosensitive), and a tether (methanethiosulfonate). (B) The azobenzene moiety can change between cis and trans isomeric forms depending on the wavelength of the light irradiated.     

Detection of the hemoglobin E mutation using the color complementation assay: application to complex genotyping

The color complementation assay (CCA) is a method of allele-specific DNA amplification by which competitive priming and extension of fluorescently labeled oligonucleotide primers determine the color of DNA amplification product. This diagnostic method precludes the need for radioisotopes, electrophoresis, and multiple high-stringency reaction conditions. The multiplicity of mutant globin genes present in Southeast Asians complicates clinical diagnosis and underscores the importance of DNA-based diagnostic methods. We have applied CCA to distinguish beta A and beta E alleles. Competing 15mer primers were a fluorescein-labeled complement to beta A and a rhodamine-labeled complement to beta E, identical except for their central nucleotides. A common unlabeled primer was used to amplify DNA product, the color of which was determined by the perfectly complementary primer. Color photography and spectrofluorometry, as well as a method of black-white photography that we developed to distinguish fluorescein- and rhodamine- labeled DNA, were used to record results. We applied CCA to define the complex genotype of a Thai woman with thalassemia intermedia, 96% HbE, and 4% HbF whose possible genotypes included several permutations of alpha-thalassemia, beta-thalassemia, and beta E genes. zeta-Globin gene mapping of DNA doubly digested with Bg/II and Asp 718 showed the -alpha 3.7/--SEA genotype, and CCA confirmed homozygous beta E/beta E. The CCA is useful for diagnosing the compound hemoglobin genotypes of Southeast Asians and could be applied also to prenatal diagnosis in this population.     

Ordered restriction endonuclease maps of yeast artificial chromosomes created by optical mapping on surfaces.

We have developed a surface mounting technology for the rapid construction of ordered restriction maps from individual DNA molecules. Optical restriction maps constructed from yeast artificial chromosome DNA molecules mounted on specially derivatized glass surfaces are accurate and reproducible, and the technology is amenable to automation. The mounting procedures described here should also be useful for fluorescence in situ hybridization studies. We believe these improvements to optical mapping will further stimulate the development of nonelectrophoretic approaches to genome analysis.     

Transient protein kinase C activation primes long-term depression and suppresses long-term potentiation of synaptic transmission in hippocampus.

Activity-dependent long-lasting plasticity in hippocampus and neocortex includes long-term potentiation (LTP) and long-term depression (LTD) of synaptic strength. Recent studies have confirmed theoretical predictions that the sensitivity of LTP- and LTD-inducing mechanisms is dynamically regulated by previous synaptic history. In particular, prior induction of either repeated short-term potentiations or LTP lowers the threshold for induction of LTD and raises the threshold for LTP. In the current study, transient activation of protein kinase C with phorbol 12,13-diacetate was able to substitute for synaptic activity in priming synapses to exhibit enhanced homosynaptic LTD and to suppress the induction of LTP at Schaffer collateral synapses in area CA1 of hippocampal slices. This priming lasted 30 min, but not 3 hr, following phorbol 12,13-diacetate bath application. These data suggest that a protein kinase C-sensitive phosphorylation site may be an activity-sensitive target mediating the rapid expression of LTP and LTD.     

Soil Dust Aerosols and Wind as Predictors of Seasonal Meningitis Incidence in Niger

Background: Epidemics of meningococcal meningitis are concentrated in sub-Saharan Africa during the dry season, a period when the region is affected by the Harmattan, a dry and dusty northeasterly trade wind blowing from the Sahara into the Gulf of Guinea.Objectives: We examined the potential of climate-based statistical forecasting models to predict seasonal incidence of meningitis in Niger at both the national and district levels.Data and methods: We used time series of meningitis incidence from 1986 through 2006 for 38 districts in Niger. We tested models based on data that would be readily available in an operational framework, such as climate and dust, population, and the incidence of early cases before the onset of the meningitis season in January–May. Incidence was used as a proxy for immunological state, susceptibility, and carriage in the population. We compared a range of negative binomial generalized linear models fitted to the meningitis data.Results: At the national level, a model using early incidence in December and averaged November–December zonal wind provided the best fit (pseudo-R² = 0.57), with zonal wind having the greatest impact. A model with surface dust concentration as a predictive variable performed indistinguishably well. At the district level, the best spatiotemporal model included zonal wind, dust concentration, early incidence in December, and population density (pseudo-R² = 0.41).Conclusions: We showed that wind and dust information and incidence in the early dry season predict part of the year-to-year variability of the seasonal incidence of meningitis at both national and district levels in Niger. Models of this form could provide an early-season alert that wind, dust, and other conditions are potentially conducive to an epidemic.Citation: Pérez García-Pando C, Stanton MC, Diggle PJ, Trzaska S, Miller RL, Perlwitz JP, Baldasano JM, Cuevas E, Ceccato P, Yaka P, Thomson MC. 2014. Soil dust aerosols and wind as predictors of seasonal meningitis incidence in Niger. Environ Health Perspect 122:679–686; http://dx.doi.org/10.1289/ehp.1306640     

Social Branding to Decrease Smoking Among Young Adults in Bars

Objectives. We evaluated a Social Branding antitobacco intervention for “hipster” young adults that was implemented between 2008 and 2011 in San Diego, California.Methods. We conducted repeated cross-sectional surveys of random samples of young adults going to bars at baseline and over a 3-year follow-up. We used multinomial logistic regression to evaluate changes in daily smoking, nondaily smoking, and binge drinking, controlling for demographic characteristics, alcohol use, advertising receptivity, trend sensitivity, and tobacco-related attitudes.Results. During the intervention, current (past 30 day) smoking decreased from 57% (baseline) to 48% (at follow-up 3; P = .002), and daily smoking decreased from 22% to 15% (P < .001). There were significant interactions between hipster affiliation and alcohol use on smoking. Among hipster binge drinkers, the odds of daily smoking (odds ratio [OR] = 0.44; 95% confidence interval [CI] = 0.30, 0.63) and nondaily smoking (OR = 0.57; 95% CI = 0.42, 0.77) decreased significantly at follow-up 3. Binge drinking also decreased significantly at follow-up 3 (OR = 0.64; 95% CI = 0.53, 0.78).Conclusions. Social Branding campaigns are a promising strategy to decrease smoking in young adult bar patrons.Tobacco companies1 and public health authorities2–5 recognize young adulthood as a critical time when experimenters either quit or transition to regular tobacco use. Young adults are also aspirational role models for youths.1,6,7 Tobacco companies devote considerable resources to reaching young adults to encourage tobacco use,1,8–11 and young adults have a high prevalence of smoking.12 In California in 2011, young adults had the highest smoking prevalence of any age group, and the Department of Health estimated that 32% of California smokers started smoking between the ages of 18 and 26 years.13 Although they are more likely to intend to quit and successfully quit than older adults,14–17 young adults are less likely to receive assistance with smoking cessation.18,19 Although there are few proven interventions to discourage young adult smoking,20 cessation before age 30 years avoids virtually all of the long-term adverse health effects of smoking.21Tobacco companies have a long history of using bars and nightclubs to reach young adults and to encourage smoking.1,6,9–11,22–24 Bar attendance and exposure to tobacco bar marketing is strongly associated with smoking.25 The 1998 Master Settlement Agreement and Food and Drug Administration regulations that limit tobacco advertising to youths, explicitly permit tobacco marketing in “adult only” venues, including bars and nightclubs.26,27Aggressive tobacco marketing may actually be more intensive in smoke-free bars: a 2010 study of college students attending bars found that students in the community with a smoke-free bar law were more likely to be approached by tobacco marketers, offered free gifts, and to take free gifts for themselves than in communities without a smoke-free bar law.28 Bars and nightclubs also attract young adults who are more likely to exhibit personality traits such as sensation seeking,29 increasing their risk30 independently of receptivity to tobacco advertising; tobacco promotional messages resonate with these personality traits.8,31 Tobacco marketing campaigns are tailored to specific segments of the population defined by psychographics (e.g., values, attitudes, shared interests, such as tastes in music and fashion, and friend groups) and demographic criteria, and they aim to create positive smoker images, identities, and social norms for smoking.1,8 Tobacco marketing campaigns also focus on young adult trendsetters to leverage peer influence to promote smoking.6,10In contrast to the tobacco companies’ efforts, most young adult health interventions take place in colleges or health centers rather than social environments.32–39 Bars and nightclub venues represent an opportunity to reach those at highest risk for long-term smoking morbidity and mortality.40 We evaluated the effectiveness of an intervention to decrease cigarette smoking by countering tobacco industry marketing strategies targeting young adults attending bars and nightclubs in the San Diego, California, “hipster” scene. Because tobacco and alcohol use are strongly linked,41,42 we also examined the effects of the intervention on alcohol use and among binge drinkers. We found a significant decrease in smoking in the community where the intervention took place, including significant decreases among nondaily smokers and binge drinkers, as well as a significant decrease in binge drinking.     

When Health Policy and Empirical Evidence Collide: The Case of Cigarette Package Warning Labels and Economic Consumer Surplus

In its graphic warning label regulations on cigarette packages, the Food and Drug Administration severely discounts the benefits of reduced smoking because of the lost “pleasure” smokers experience when they stop smoking; this is quantified as lost “consumer surplus.” Consumer surplus is grounded in rational choice theory. However, empirical evidence from psychological cognitive science and behavioral economics demonstrates that the assumptions of rational choice are inconsistent with complex multidimensional decisions, particularly smoking. Rational choice does not account for the roles of emotions, misperceptions, optimistic bias, regret, and cognitive inefficiency that are germane to smoking, particularly because most smokers begin smoking in their youth. Continued application of a consumer surplus discount will undermine sensible policies to reduce tobacco use and other policies to promote public health.The 2009 Family Smoking Prevention and Tobacco Control Act (HR 1256, 2009) required the United States Food and Drug Administration (FDA) to issue a regulation requiring cigarette companies to place large graphic warnings on all cigarette packages. As part of the process of issuing this regulation, the FDA conducted a cost–benefit analysis of the graphic warning label regulation.1 In its analysis, the FDA estimated the benefits of graphic warning labels, including reduced tobacco-induced illness and premature death, then cut the estimated benefits of these warning labels in half to account for the cost of lost “pleasure” smokers incurred as a result of quitting (and lost pleasure would-be smokers would never experience) because of the new warning labels. The FDA quantified the cost of this lost pleasure using the economic concept of “consumer surplus,” which is the difference between what a utility maximizing individual would be willing to pay and the actual price.2–6Because of the extent that smokers are willing to pay more for cigarettes than their monetary cost, this willingness to pay more is an indication that smokers obtain a surplus benefit of smoking beyond the cost of the cigarettes. The FDA justified applying a large discount to the estimated health benefits of the warning labels, stating,

The concept of consumer surplus is a basic tool of welfare economics… . In an analysis of benefits based on willingness-to-pay, we cannot reject this tool and still fulfill our obligation to conduct a full and an objective economic analysis.1^(p36714)

Consumer surplus based on willingness to pay is a well-established concept in classical economics and is grounded in rational choice theory, a normative model of human decision-making.7 Rational choice theory represents human decision-making at its most logical, when decisions are the result of careful cost–benefit analysis, with people choosing the option that maximizes the utility of the choice after subtracting perceived costs.8–10When applied to smoking, this theory posits that smokers (and potential smokers) smoke because they computed that the current and future benefits of the pleasures of smoking outweigh the present value of future financial, social, and medical costs of smoking.11–13 These benefits may include both the physiologic responses and emotional or social advantages (either real or imagined) that smoking provides.By contrast, a large body of empirical evidence from cognitive behavioral sciences demonstrates that smokers (and would-be smokers) smoke because they are addicted and overestimate their ability to quit in the future.14 Rational choice theory (and the adjustments that have been proposed to deal with addictive behaviors) assumes stable preferences, foresight, knowledge, and adequate cognitive abilities to make the decision to start or continue smoking. Conversely, empirical evidence demonstrates that these assumptions are seriously violated by smoking behavior that almost always begins during adolescence15^(p179) and continues in adulthood through addictive consumption. In addition, there is no empirical literature that suggests adults who start smoking engage in deliberate decision-making processes in which they evaluate risks against benefits. The empirical literature suggests the opposite; even adults, who presumably are better equipped to consider the risks and benefits of smoking, do not anticipate regret or understand addiction.16–18Applying a significant loss in (real or potential) consumer surplus when measuring the value of antismoking initiatives has important implications for policy, including reducing the benefits of proposed health regulations. This reduction in the estimated benefits of the policy results in weakened regulations that are harder to defend when challenged in court.19,20 In using consumer surplus, a measure grounded in rational choice theory, to estimate a theoretical “cost” of not smoking,1^(p36772),4 the FDA is ignoring the strong empirical evidence against the validity of applying rational choice to smoking decisions, leading the FDA to seriously overestimate the costs of reducing smoking, and in turn, underestimate the net benefits.     

Infant rats can acquire,but not retain contextual associations in object-in-context and contextual fear conditioning paradigms

Context learning in postnatal day (PD) 16–18 rats has been taken by Revillo, Cotella, Paglini, and Arias (2015, Physiology & Behavior, 148 , 6–21) to challenge the view that the ontogeny of contextual learning is related to the development of the hippocampal system (Rudy, 1993, Behavioral Neuroscience, 107 (5), 887–891; Schiffino, Murawski, Rosen, & Stanton, 2011 Neurobiology of Learning and Memory, 95 (2), 190–198). Whether context learning is “incidental” or “reinforcement-driven” may determine the ontogeny and neural systems involved (Rudy, 2009, Learning & Memory (Cold Spring Harbor, N.Y.), 16 , 573–585). However, we have shown differential ontogeny of two different forms of incidental context learning, the context pre-exposure facilitation effect (CPFE; Jablonski, Schiffino, & Stanton, 2012, Developmental Psychobiology, 54 (7), 714–722), which emerges between PD 17 and 21; and object-in-context recognition (OiC, Ramsaran, Westbrook, & Stanton, 2016, Developmental Psychobiology, 58 (7), 883–895; Ramsaran, Sanders, & Stanton, 2016, Behavioural Brain Research, 298 , 37–47), which is present on PD17. We investigated whether this task-dissociation reflects an encoding or a retention deficit, by varying the sample-to-testing intervals for both tasks. Experiment 1A found that PD17 rats were able to perform the OiC task after short (5 min) but not long (24 hr) sample-to-test intervals. Experiments 1B and 1C found that PD17 rats trained on the CPFE are able to acquire and express context-shock associations after short but not long retention intervals. These findings suggest that pre-weanling rats encode contexts but show poor consolidation or retrieval after longer retention intervals.