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1.  Designing Preclinical Perceptibility Measures to Evaluate Topical Vaginal Gel Formulations: Relating User Sensory Perceptions and Experiences to Formulation Properties 
The effectiveness of any biomedical prevention technology relies on both biological efficacy and behavioral adherence. Microbicide trials have been hampered by low adherence, limiting the ability to draw meaningful conclusions about product effectiveness. Central to this problem may be an inadequate conceptualization of how product properties themselves impact user experience and adherence. Our goal is to expand the current microbicide development framework to include product “perceptibility,” the objective measurement of user sensory perceptions (i.e., sensations) and experiences of formulation performance during use. For vaginal gels, a set of biophysical properties, including rheological properties and measures of spreading and retention, may critically impact user experiences. Project LINK sought to characterize the user experience in this regard, and to validate measures of user sensory perceptions and experiences (USPEs) using four prototype topical vaginal gel formulations designed for pericoital use. Perceptibility scales captured a range of USPEs during the product application process (five scales), ambulation after product insertion (six scales), and during sexual activity (eight scales). Comparative statistical analyses provided empirical support for hypothesized relationships between gel properties, spreading performance, and the user experience. Project LINK provides preliminary evidence for the utility of evaluating USPEs, introducing a paradigm shift in the field of microbicide formulation design. We propose that these user sensory perceptions and experiences initiate cognitive processes in users resulting in product choice and willingness-to-use. By understanding the impact of USPEs on that process, formulation development can optimize both drug delivery and adherence.
PMCID: PMC3931431  PMID: 24180360
2.  ru2hot?: A public health education campaign for men who have sex with men to increase awareness of symptoms of acute HIV infection 
Sexually transmitted infections  2013;89(5):409-414.
Teach HIV-negative men who have sex with men (MSM) symptoms of acute HIV infection (AHI) and direct them to nucleic acid amplification testing (NAAT) though Public Health—Seattle & King County (PHSKC).
Cross-sectional surveys, retrospective database analysis and chart review.
Beginning in June 2009, the ru2hot? campaign described AHI symptoms and NAAT. Two preintervention and two postintervention surveys assessed campaign visibility, symptom knowledge, and healthcare-seeking behaviour. Regression analyses evaluated secular trends in case-finding.
366 MSM completed surveys. In survey 4, 23% of 100 men reported seeing the campaign, and 25% knew ‘ru2hot?’ referred to AHI. From survey 1 to survey 4, the proportion of subjects who knew ≥2 symptoms or that AHI was a ‘flu-like’ illness was unchanged (61% vs 57%, p=0.6). However, in survey 4, 13 (72%) of 18 subjects who saw the campaign named fever as a symptom of AHI compared with 19 (35%) of 55 subjects who had not seen the campaign (p=0.005). From 9/2003 to 12/2010, 622 (2.2%) of 27 661 MSM tested HIV-positive, and 111 (18%) were identified by the Public Health—Seattle & King County NAAT programme. In terms of the impact of the campaign on case-finding, diagnosis of EIA-negative/NAAT-positive and OraQuick-negative/EIA-positive cases increased from six in 2004 to 20 in 2010 (p=0.01), but postcampaign numbers were unchanged. 23 (51%) of 45 cases identified before and 8 (44%) of 18 cases identified after the campaign reported symptoms at initial testing (p=0.6).
Although a quarter of MSM surveyed saw the campaign and knowledge of fever (the symptom of emphasis) was high, case-finding was unchanged. Increasing campaign visibility could have had greater impact.
PMCID: PMC4257701  PMID: 23349338
3.  Seroadaptation among Men Who Have Sex with Men: Emerging Research Themes 
Current HIV/AIDS reports  2013;10(4):305-313.
Seroadaptation describes a diverse set of potentially harm-reducing behaviors that use HIV status to inform sexual decision making. Men who have sex with men (MSM) in many settings adopt these practices, but their effectiveness at preventing HIV transmission is debated. Past modeling studies have demonstrated that serosorting is only effective at preventing HIV transmission when most men accurately know their HIV status, but additional modeling is needed to address the effectiveness of broader seroadaptive behaviors. The types of information with which MSM make seroadaptive decisions is expanding to include viral load, treatment status, and HIV status based on home-use tests, and recent research has begun to examine the entire seroadaptive process, from an individual’s intentions to seroadapt to their behaviors to their risk of acquiring or transmitting HIV and other STIs. More research is needed to craft clear public health messages about the risks and benefits of seroadaptive practices.
PMCID: PMC3946930  PMID: 24234489
Serosorting; Seropositioning; HIV/AIDS; Prevention; Risk reduction; Behavioral aspects; Men who have sex with men (MSM); STIs; Seroadaptive practices
4.  Implementing Smoking Cessation Guidelines for Hospitalized Veterans: Effects on Nurse Attitudes and Performance 
Journal of General Internal Medicine  2013;28(11):1420-1429.
A minority of hospitalized smokers actually receives assistance in quitting during hospitalization or cessation counseling following discharge. This study aims to determine the impact of a guideline-based intervention on 1) nurses’ delivery of the 5A’s (Ask-Advise-Assess-Assist-Arrange follow-up) in hospitalized smokers, and 2) nurses’ attitudes toward the intervention.
We conducted a pre-post guideline implementation trial involving 205 hospitalized smokers on the inpatient medicine units at one US Department of Veterans Affairs (VA) medical center. The intervention included: 1) academic detailing of nurses on delivery of brief cessation counseling, 2) modification of the admission form to facilitate 5A’s documentation, and 3) referral of motivated inpatients to receive proactive telephone counseling. Based on subject interviews, we calculated a nursing 5A’s composite score for each patient (ranging from 0 to 9). We used linear regression with generalized estimating equations to compare the 5A’s composite score (and logistic regression to compare individual A’s) across periods. We compared 29 nurses’ ratings of their self-efficacy and decisional balance (“pros” and “cons”) with regard to cessation counseling before and after guideline implementation. Following implementation, we also interviewed a purposeful sample of nurses to assess their attitudes toward the intervention.
Of 193 smokers who completed the pre-discharge interview, the mean nursing 5A’s composite score was higher after guideline implementation (3.9 vs. 3.1, adjusted difference 1.0, 95 % CI 0.5–1.6). More patients were advised to quit (62 vs. 48 %, adjusted OR = 2.1, 95 % CI = 1.2–3.5) and were assisted in quitting (70 vs. 45 %, adjusted OR = 2.9, 95 % CI = 1.6–5.3) by a nurse during the post-implementation period. Nurses’ attitudes toward cessation counseling improved following guideline implementation (35.3 vs. 32.7 on “pros” subscale, p = 0.01), without significant change on the “cons” subscale.
A multifaceted intervention including academic detailing and adaptation of the nursing admission template is an effective strategy for improving nurses’ delivery of brief cessation counseling in medical inpatients.
PMCID: PMC3797327  PMID: 23649783
smoking cessation; Veterans; counseling; guideline-based intervention
7.  Why do men who have sex with men test for HIV infection? Results from a community-based testing program in Seattle 
Sexually transmitted diseases  2013;40(9):724-728.
The Centers for Disease Control and Prevention recommends at least annual HIV testing for men who have sex with men (MSM), but motivations for testing are not well understood.
We evaluated data from MSM testing for HIV at a community-based program in King County, Washington. Correlates of regular testing were examined using GEE regression models.
Between February 2004 and June 2011, 7176 MSM attended 12,109 HIV testing visits. When asked reasons for testing, 49% reported it was time for their regular test, 27% reported unprotected sex, 24% were starting relationships, 21% reported sex with someone new, 21% sought STI/hepatitis screening, 12% reported sex with an HIV-infected partner, 2% suspected primary HIV infection, and 16% reported other reasons. In multivariable analysis, factors associated with regular testing included having a regular healthcare provider and the following in the previous year: having only male partners, having ≥10 male partners, inhaled nitrite use, not injecting drugs, and not having unprotected anal intercourse with a partner of unknown/discordant status (p≤0.001 for all). Men reporting regular testing reported shorter intertest intervals than men who did not (median of 233 vs. 322 days, respectively; p<0.001).
Regular testing, sexual risk, and new partnerships were important drivers of HIV testing among MSM, and regular testing was associated with increased testing frequency. Promoting regular testing may reduce the time that HIV-infected MSM are unaware of their status, particularly among those who have sex with men and women or inject drugs.
PMCID: PMC4070507  PMID: 23949588
HIV screening; men who have sex with men; testing frequency; reasons for testing
8.  A BDNF loop-domain mimetic acutely reverses spontaneous apneas and respiratory abnormalities during behavioral arousal in a mouse model of Rett syndrome 
Disease Models & Mechanisms  2014;7(9):1047-1055.
Reduced levels of brain-derived neurotrophic factor (BDNF) are thought to contribute to the pathophysiology of Rett syndrome (RTT), a severe neurodevelopmental disorder caused by loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). In Mecp2 mutant mice, BDNF deficits have been associated with breathing abnormalities, a core feature of RTT, as well as with synaptic hyperexcitability within the brainstem respiratory network. Application of BDNF can reverse hyperexcitability in acute brainstem slices from Mecp2-null mice, suggesting that therapies targeting BDNF or its receptor, TrkB, could be effective at acute reversal of respiratory abnormalities in RTT. Therefore, we examined the ability of LM22A-4, a small-molecule BDNF loop-domain mimetic and TrkB partial agonist, to modulate synaptic excitability within respiratory cell groups in the brainstem nucleus tractus solitarius (nTS) and to acutely reverse abnormalities in breathing at rest and during behavioral arousal in Mecp2 mutants. Patch-clamp recordings in Mecp2-null brainstem slices demonstrated that LM22A-4 decreases excitability at primary afferent synapses in the nTS by reducing the amplitude of evoked excitatory postsynaptic currents and the frequency of spontaneous and miniature excitatory postsynaptic currents. In vivo, acute treatment of Mecp2-null and -heterozygous mutants with LM22A-4 completely eliminated spontaneous apneas in resting animals, without sedation. Moreover, we demonstrate that respiratory dysregulation during behavioral arousal, a feature of human RTT, is also reversed in Mecp2 mutants by acute treatment with LM22A-4. Together, these data support the hypothesis that reduced BDNF signaling and respiratory dysfunction in RTT are linked, and establish the proof-of-concept that treatment with a small-molecule structural mimetic of a BDNF loop domain and a TrkB partial agonist can acutely reverse abnormal breathing at rest and in response to behavioral arousal in symptomatic RTT mice.
PMCID: PMC4142725  PMID: 25147297
Mecp2; Brain-derived neurotrophic factor (BDNF); Respiration; Brainstem; Arousal
9.  The wisdom of Weismann 
Cell cycle (Georgetown, Tex.)  2009;8(14):2131-2132.
PMCID: PMC4086154  PMID: 19556871
10.  Trends in Reporting Methadone-Associated Cardiac Arrhythmia, 1997–2011 
Annals of internal medicine  2013;158(10):735-740.
Long-acting opioids are a leading cause of accidental death in the United States, and methadone is associated with greater mortality rates. Whether this increase is related to the proarrhythmic properties of methadone is unclear.
To describe methadone-associated arrhythmia events reported in the U.S. Food and Drug Administration Adverse Event Reporting System (FAERS).
Description of national adverse event registry data before and after publication of a 2002 report describing an association between methadone and arrhythmia.
FAERS, November 1997 and June 2011.
Adults with QTc prolongation or torsade de pointes and ventricular arrhythmia or cardiac arrest.
FAERS reports before and after the 2002 report.
1646 cases of ventricular arrhythmia or cardiac arrest and 379 cases of QTc prolongation or torsade de pointes were associated with methadone. Monthly reports of QTc prolongation or torsade de pointes increased from a mean of 0.3 (95% CI, 0.1 to 0.5) before the 2002 publication to a mean of 3.5 (CI, 2.5 to 4.8) after it. After 2000, methadone was the second-most common primary suspect in cases of QTc prolongation or torsade de pointes after dofetilide (a known proarrhythmic drug) and was associated with disproportionate reporting similar to that of antiarrhythmic agents known to promote torsade de pointes. Antiretroviral drugs for HIV were the most common coadministered drugs.
Reports to FAERs are voluntary and selective, and incidence rates cannot be determined from spontaneously reported data.
Since 2002, reports to FAERS of methadone-associated arrhythmia have increased substantially and are disproportionately represented relative to other events with the drug. Coadministration of methadone with antiretrovirals in patients with HIV may pose particular risk.
Primary Funding Source:
Colorado Clinical and Translational Sciences Institute, National Institutes of Health, and Agency for Healthcare Research and Quality.
PMCID: PMC3793842  PMID: 23689766
11.  Analysis of Vaginal Microbicide Film Hydration Kinetics by Quantitative Imaging Refractometry 
PLoS ONE  2014;9(4):e95005.
We have developed a quantitative imaging refractometry technique, based on holographic phase microscopy, as a tool for investigating microscopic structural changes in water-soluble polymeric materials. Here we apply the approach to analyze the structural degradation of vaginal topical microbicide films due to water uptake. We implemented transmission imaging of 1-mm diameter film samples loaded into a flow chamber with a 1.5×2 mm field of view. After water was flooded into the chamber, interference images were captured and analyzed to obtain high resolution maps of the local refractive index and subsequently the volume fraction and mass density of film material at each spatial location. Here, we compare the hydration dynamics of a panel of films with varying thicknesses and polymer compositions, demonstrating that quantitative imaging refractometry can be an effective tool for evaluating and characterizing the performance of candidate microbicide film designs for anti-HIV drug delivery.
PMCID: PMC3988224  PMID: 24736376
12.  Replacing Clinic-Based Tests With Home-Use Tests May Increase HIV Prevalence Among Seattle Men Who Have Sex With Men: Evidence From a Mathematical Model 
Home-use tests have potential to increase HIV testing but may increase the rate of false-negative tests and decrease linkage to HIV care. We sought to estimate the impact of replacing clinic-based testing with home-use tests on HIV prevalence among men who have sex with men (MSM) in Seattle, Washington.
We adapted a deterministic, continuous-time model of HIV transmission dynamics parameterized using a 2003 random digit dial study of Seattle MSM. Test performance was based on the OraQuick In-Home HIV Test (OraSure Technologies, Inc, Bethlehem, PA) for home-use tests and, on an average, of antigen-antibody combination assays and nucleic acid amplification tests for clinic-based testing.
Based on observed levels of clinic-based testing, our baseline model predicted an equilibrium HIV prevalence of 18.6%. If all men replaced clinic-based testing with home-use tests, prevalence increased to 27.5% if home-use testing did not impact testing frequency and to 22.4% if home-use testing increased testing frequency 3-fold. Regardless of how much home-use testing increased testing frequency, any replacement of clinic-based testing with home-use testing increased prevalence. These increases in HIV prevalence were mostly caused by the relatively long window period of the currently approved test. If the window period of a home-use test were 2 months instead of 3 months, prevalence would decrease if all MSM replaced clinic-based testing with home-use tests and tested more than 2.6 times more frequently.
Our model suggests that if home-use HIV tests replace supplement clinic-based testing, HIV prevalence may increase among Seattle MSM, even if home-use tests result in increased testing.
PMCID: PMC3955208  PMID: 24335742
13.  A suppressor screen in mouse Mecp2 implicates cholesterol metabolism in Rett Syndrome 
Nature genetics  2013;45(9):1013-1020.
Mutations in methyl CpG binding protein 2 (MECP2) cause Rett Syndrome, the most severe autism spectrum disorder. Re-expressing Mecp2 in symptomatic Mecp2 null mice dramatically improves function and longevity, providing hope that therapeutic intervention is possible in humans. To identify pathways in disease pathology for therapeutic intervention, a dominant ENU mutagenesis suppressor screen was carried out in Mecp2 null mice. Five suppressors that ameliorate symptoms of Mecp2 loss were isolated. Here we show that a stop codon mutation in squalene epoxidase (Sqle), a rate-limiting enzyme in cholesterol biosynthesis underlies suppression in one line. Subsequently, we show that lipid metabolism is perturbed in the brain and liver of Mecp2 null males. Consistently, statin drugs improve systemic perturbations of lipid metabolism, alleviate motor symptoms and confer increased longevity in Mecp2 mutant mice. The genetic screen therefore points to cholesterol homeostasis as a potential target for the treatment of Rett patients.
PMCID: PMC3837522  PMID: 23892605
14.  Restoring totipotency through epigenetic reprogramming 
Briefings in Functional Genomics  2012;12(2):118-128.
Epigenetic modifications are implicated in the maintenance and regulation of transcriptional memory by marking genes that were previously transcribed to facilitate transmission of these expression patterns through cell division. During germline specification and maintenance, extensive epigenetic modifications are acquired. Yet somehow at fertilization, the fusion of the highly differentiated sperm and egg results in formation of the totipotent zygote. This massive change in cell fate implies that the selective erasure and maintenance of epigenetic modifications at fertilization may be critical for the re-establishment of totipotency. In this review, we discuss recent studies that provide insight into the extensive epigenetic reprogramming that occurs around fertilization and the mechanisms that may be involved in the re-establishment of totipotency in the embryo.
PMCID: PMC4023274  PMID: 23117862
15.  Mutant mice reveal the molecular and cellular basis for specific sensory connections to inner ear epithelia and primary nuclei of the brain 
Hearing research  2005;206(0):52-63.
We review the in vivo evidence for afferent fiber guidance to the inner ear sensory epithelia and the central nuclei of termination. Specifically, we highlight our current molecular understanding for the role of hair cells and sensory epithelia in guiding afferents, how disruption of certain signals can alter fiber pathways, even in the presence of normal hair cells, and what role neurotrophins play in fiber guidance of sensory neurons to hair cells. The data suggest that the neurotrophin BDNF is the most important molecule known for inner ear afferent fiber guidance to hair cells in vivo. This suggestion is based on experiments on Ntf3 transgenic mice expressing BDNF under Ntf3 promoter that show deviations of fiber growth in the ear to areas that express BDNF but have no hair cells. However, fiber growth can occur in the absence of BDNF as demonstrated by double mutants for BDNF and Bax. We directly tested the significance of hair cells or sensory epithelia for fiber guidance in mutants that lose hair cells (Pou4f3) or do not form a posterior crista (Fgf10). While these data emphasize the role played by BDNF, normally released from hair cells, there is some limited capacity for directed growth even in the absence of hair cells, BDNF, or sensory epithelia. This directed growth may rely on semaphorins or other matrix proteins because targeted ablation of the sema3 docking site on the sema receptor Npn1 results in targeting errors of fibers even in the presence of hair cells and BDNF. Overall, our data support the notion that targeting of the afferent processes in the ear is molecularly distinct from targeting processes in the central nuclei. This conclusion is derived from data that show no recognizable central projection deviation, even if fibers are massively rerouted in the periphery, as in Ntf3tgBDNF mice in which vestibular fibers project to the cochlea.
PMCID: PMC3904737  PMID: 16080998
16.  Influence of ovarian and non-ovarian estrogens on weight gain in response to disruption of sweet taste – calorie relations in female rats 
Hormones and behavior  2012;63(1):40-48.
Regulation of energy balance in female rats is known to differ along a number of dimensions compared to male rats. Previous work from our lab has demonstrated that in female rats fed dietary supplements containing high-intensity sweeteners that may disrupt a predictive relation between sweet tastes and calories, excess weight gain is demonstrated only when females are also fed a diet high in fat and sugar, and is evidenced primarily in animals already prone to gain excess weight. In contrast, male rats show excess weight gain when fed saccharin-sweetened yogurt supplements when fed both standard chow diets and diets high in fat and sugar, and regardless of their proneness to excess weight gain. The goal of the present experiments was to determine whether ovarian, or other sources of estrogens, contributes to the resistance to excess weight gain in female rats fed standard chow diets along with dietary supplements sweetened with yogurt. Results of the first experiment indicated that when the ovaries were removed surgically in adult female rats, patterns of weight gain were similar in animals fed saccharin-sweetened compared to glucose-sweetened yogurt supplements. In the second experiment, when the ovaries were surgically removed in adult female rats, and local production of estrogens was suppressed with the aromatase inhibitor anastrozole, females fed the saccharin-sweetened yogurt consumed more energy and gained more weight than females fed the glucose-sweetened yogurt. However, when the ovaries were surgically removed prior to the onset of puberty (at 24 – 25 days of age), females given saccharin-sweetened yogurt along with vehicle gained excess weight. In contrast, weight gain was similar in those given saccharin-sweetened and glucose-sweetened yogurt along with anastrozole. The results suggest that behavioral differences between males and females in response to disruption of sweet→calorie relations may result from differences in patterns of local estrogen production. These differences may be established developmentally during the pubertal period in females.
PMCID: PMC3540164  PMID: 23146838
Energy balance; learning; estrogens; sweeteners; puberty
17.  Implementation of smoking cessation guidelines in the emergency department: a qualitative study of staff perceptions 
The US Public Health Service smoking cessation practice guideline specifically recommends that physicians and nurses strongly advise their patients who use tobacco to quit, but the best approach for attaining this goal in the emergency department (ED) remains unknown. The aim of this study was to characterize emergency physicians’ (EPs) and nurses’ (ENs) perceptions of cessation counseling and to identify barriers and facilitators to implementation of the 5 A’s framework (Ask-Advise-Assess-Assist-Arrange) in the ED.
We conducted semi-structured, face-to-face interviews of 11 EPs and 19 ENs following a pre-post implementation trial of smoking cessation guidelines in two study EDs. We used purposeful sampling to target EPs and ENs with different attitudes toward cessation counseling, based on their responses to a written survey (Decisional Balance Questionnaire). Conventional content analysis was used to inductively characterize the issues raised by study participants and to construct a coding structure, which was then applied to study transcripts.
The main findings of this study converged upon three overarching domains: 1) reactions to the intervention; 2) perceptions of patients’ receptivity to cessation counseling; and 3) perspectives on ED cessation counseling and preventive care. ED staff expressed ambivalence toward the implementation of smoking cessation guidelines. Both ENs and EPs agreed that the delivery of smoking cessation counseling is important, but that it is not always practical in the ED on account of time constraints, the competing demands of acute care, and resistance from patients. Participants also called attention to the need for improved role clarity and teamwork when implementing the 5 A’s in the ED.
There are numerous challenges to the implementation of smoking cessation guidelines in the ED. ENs are generally willing to take the lead in offering brief cessation counseling, but their efforts need to be reinforced by EPs. ED systems need to address workflow, teamwork, and practice policies that facilitate prescription of smoking cessation medication, referral for cessation counseling, and follow-up in primary care. The results of this qualitative evaluation can be used to guide the design of future ED intervention studies.
Trial registration registration number NCT00756704
PMCID: PMC3902188  PMID: 24460974
Emergency medical services; Smoking cessation; Attitude of health personnel; Qualitative research; Content analysis
18.  Testing the Effectiveness of an Abbreviated Version of the Nutrition Detectives Program 
Obese or overweight children have an increased risk for chronic diseases. Targeting diet and exercise in schools could help prevent childhood obesity. We have previously shown the effectiveness of a 90-minute nutrition program in improving elementary school students’ food-label literacy. The objective of this study was to investigate the effectiveness of a 45-minute version of the program.
We conducted a pre–post study in a public school district, with no control group. We provided teacher training and program materials. Participants were 5th-grade students in 5 schools who had parental consent and were willing to take part. We condensed the program to a 45-minute lesson with a presentation and hands-on activity. The lesson showed students why and how to make healthful food choices based on Nutrition Facts panels and ingredient lists. The district’s physical education teachers taught the lesson. The primary outcome measure was food-label literacy (ie, the ability to distinguish between more and less healthful foods using a validated test instrument with Nutrition Facts panels and ingredient lists).
A total of 212 students completed pre–post measures. Following program delivery, we observed a significant gain of 16.2 percentage points in scores overall, ranging from 4.3 percentage points to 23.6 percentage points among schools. Results were similar to those achieved with the 90-minute program.
The condensed nutrition program improved students’ food-label literacy while requiring a minimal allocation of time. Further studies in other school districts would be useful.
PMCID: PMC3984941  PMID: 24721217
19.  Quantitative Analysis of Microbicide Concentrations in Fluids, Gels and Tissues Using Confocal Raman Spectroscopy 
PLoS ONE  2013;8(12):e85124.
Topical vaginal anti-HIV microbicides are an important focus in female-based strategies to prevent the sexual transmission of HIV. Understanding microbicide pharmacokinetics is essential to development, characterization and implementation of efficacious microbicide drug delivery formulations. Current methods to measure drug concentrations in tissue (e.g., LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry) are highly sensitive, but destructive and complex. This project explored the use of confocal Raman spectroscopy to detect microbicide drugs and to measure their local concentrations in fluids, drug delivery gels, and tissues. We evaluated three candidate microbicide drugs: tenofovir, Dapivirine and IQP-0528. Measurements were performed in freshly excised porcine buccal tissue specimens, gel vehicles and fluids using two Horiba Raman microscopes, one of which is confocal. Characteristic spectral peak calibrations for each drug were obtained using serial dilutions in the three matrices. These specific Raman bands demonstrated strong linear concentration dependences in the matrices and were characterized with respect to their unique vibrational signatures. At least one specific Raman feature was identified for each drug as a marker band for detection in tissue. Sensitivity of detection was evaluated in the three matrices. A specific peak was also identified for tenofovir diphosphate, the anti-HIV bioactive product of tenofovir after phosphorylation in host cells. Z-scans of drug concentrations vs. depth in excised tissue specimens, incubated under layers of tenofovir solution in a Transwell assay, showed decreasing concentration with depth from the surface into the tissue. Time-dependent concentration profiles were obtained from tissue samples incubated in the Transwell assay, for times ranging 30 minutes - 6 hours. Calibrations and measurements from tissue permeation studies for tenofovir showed good correlation with gold standard LC-MS/MS data. These results demonstrate that confocal Raman spectroscopy holds promise as a tool for practical, minimally invasive, label-free measurement of microbicide drug concentrations in fluids, gels and tissues.
PMCID: PMC3875564  PMID: 24386455
20.  Preclinical discovery of candidate genes to guide pharmacogenetics during phase I development: the example of the novel anticancer agent ABT-751 
Pharmacogenetics and genomics  2013;23(7):10.1097/FPC.0b013e3283623e81.
ABT-751, a novel orally available antitubulin agent, is mainly eliminated as inactive glucuronide (ABT-751G) and sulfate (ABT-751S) conjugates. We performed a pharmacogenetic investigation of ABT-751 pharmacokinetics using in-vitro data to guide the selection of genes for genotyping in a phase I trial of ABT-751.
UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) enzymes were screened for ABT-751 metabolite formation in vitro. Forty-seven cancer patients treated with ABT-751 were genotyped for 21 variants in these genes.
UGT1A1, UGT1A4, UGT1A8, UGT2B7, and SULT1A1 were found to be involved in the formation of inactive ABT-751 glucuronide (ABT-751G) and sulfate (ABT-751S). SULT1A1 copy number (> 2) was associated with an average 34% increase in ABT-751 clearance (P= 0.044), an 18% reduction in ABT-751 AUC (P = 0.045), and a 50% increase in sulfation metabolic ratios (P=0.025). UGT1A8 rs6431558 was associated with a 28% increase in glucuronidation metabolic ratios (P =0.022), and UGT1A4*2 was associated with a 65% decrease in ABT-751 Ctrough (P = 0.009).
These results might represent the first example of a clinical pharmacokinetic effect of the SULT1A1 copy number variant on the clearance of a SULT1A1 substrate. A-priori selection of candidate genes guided by in-vitro metabolic screening enhanced our ability to identify genetic determinants of interpatient pharmacokinetic variability.
PMCID: PMC3858967  PMID: 23670235
ABT-751; drug development; drug metabolism; pharmacogenetics; phase I; sulfotransferase; UDP-glucuronosyltransferase
21.  Measuring Dilution of Microbicide Gels with Optical Imaging 
PLoS ONE  2013;8(12):e82213.
We present a novel approach for measuring topical microbicide gel dilution using optical imaging. The approach compares gel thickness measurements from fluorimetry and multiplexed low coherence interferometry in order to calculate dilution of a gel. As a microbicide gel becomes diluted at fixed thickness, its mLCI thickness measurement remains constant, while the fluorimetry signal decreases in intensity. The difference between the two measurements is related to the extent of gel dilution. These two optical modalities are implemented in a single endoscopic instrument that enables simultaneous data collection. A preliminary validation study was performed with in vitro placebo gel measurements taken in a controlled test socket. It was found that change in slope of the regression line between fluorimetry and mLCI based measurements indicates dilution. A dilution calibration curve was then generated by repeating the test socket measurements with serial dilutions of placebo gel with vaginal fluid simulant. This methodology can provide valuable dilution information on candidate microbicide products, which could substantially enhance our understanding of their in vivo functioning.
PMCID: PMC3858273  PMID: 24340006
22.  Does Improved Continuity of Primary Care Affect Clinician–Patient Communication in VA? 
Journal of General Internal Medicine  2013;29(Suppl 2):682-688.
Recent changes in health care delivery may reduce continuity with the patient’s primary care provider (PCP). Little is known about the association between continuity and quality of communication during ongoing efforts to redesign primary care in the Veterans Administration (VA).
To evaluate the association between longitudinal continuity of care (COC) with the same PCP and ratings of patient–provider communication during the Patient Aligned Care Team (PACT) initiative.
Cross-sectional survey.
Four thousand three hundred ninety-three VA outpatients who were assigned to a PCP, had at least three primary care visits to physicians or physician extenders during Fiscal Years 2009 and 2010 (combined), and who completed the Survey of Healthcare Experiences of Patients (SHEP) following a primary care visit in Fiscal Year (FY)2011.
Usual Provider of Continuity (UPC), Modified Modified Continuity Index (MMCI), and duration of PCP care were calculated for each primary care patient. UPC and MMCI values were categorized as follows: 1.0 (perfect), 0.75–0.99 (high), 0.50–0.74 (intermediate), and < 0.50 (low). Quality of communication was measured using the four-item Consumer Assessment of Healthcare Providers and Systems-Health Plan program (CAHPS-HP) communication subscale and a two-item measure of shared decision-making (SDM). Excellent care was defined using an “all-or-none” scoring strategy (i.e., when all items within a scale were rated “always”).
UPC and MMCI continuity remained high (0.81) during the early phase of PACT implementation. In multivariable models, low MMCI continuity was associated with decreased odds of excellent communication (OR = 0.74, 95 % CI = 0.58–0.95) and SDM (OR = 0.70, 95 % CI = 0.49, 0.99). Abbreviated duration of PCP care (< 1 year) was also associated with decreased odds of excellent communication (OR = 0.35, 95 % CI = 0.18, 0.71).
Reduced PCP continuity may significantly decrease the quality of patient–provider communication in VA primary care. By improving longitudinal continuity with the assigned PCP, while redesigning team-based roles, the PACT initiative has the potential to improve patient–provider communication.
PMCID: PMC4070228  PMID: 24072718
continuity of care; interpersonal communication; shared decision making; primary care
23.  Multicompartmental Pharmacokinetic Model of Tenofovir Delivery by a Vaginal Gel 
PLoS ONE  2013;8(9):e74404.
Trials of a vaginal Tenofovir gel for pre-exposure prophylaxis (PrEP) for HIV have given conflicting results. Knowledge of concentrations of Tenofovir and its active form Tenofovir diphosphate, at putative sites of anti-HIV functioning, is central to understanding trial outcomes and design of products and dosage regimens. Topical Tenofovir delivery to the vaginal environment is complex, multivariate and non-linear; determinants relate to drug, vehicle, dosage regimen, and environment. Experimental PK methods cannot yield mechanistic understanding of this process, and have uncontrolled variability in drug sampling. Mechanistic modeling of the process could help delineate its determinants, and be a tool in design and interpretation of products and trials.
Methods and Findings
We created a four-compartment mass transport model for Tenofovir delivery by a gel: gel, epithelium, stroma, blood. Transport was diffusion-driven in vaginal compartments; blood concentration was time-varying but homogeneous. Parameters for the model derived from in vitro and in vivo PK data, to which model predictions gave good agreement. Steep concentration gradients occurred in stroma ≤8 hours after gel release. Increasing epithelial thickness delayed initial TFV delivery to stroma and its decline: tmax increased but AUC at 24 hours was not significantly altered. At 24 and 48 hours, stromal concentrations were 6.3% and 0.2% of Cmax. Concentrations in simulated biopsies overestimated stromal concentrations, as much as ∼5X, depending upon time of sampling, biopsy thickness and epithelial thickness.
There was reasonably good agreement of model predictions with clinical PK data. Conversion of TFV to TFV-DP was not included, but PK data suggest a linear relationship between them. Thus contrasts predicted by this model can inform design of gels and dosage regimens in clinical trials, and interpretation of PK data. This mass transport based approach can be extended to TFV conversion to TFV-DP, and to other drugs and dosage forms.
PMCID: PMC3770582  PMID: 24040241
24.  Exploring the Factors That Affect Blood Cholesterol and Heart Disease Risk: Is Dietary Cholesterol as Bad for You as History Leads Us to Believe?12 
Advances in Nutrition  2012;3(5):711-717.
This paper summarizes presentations given at the 2011 Experimental Biology meetings about the latest research and a paleoanthropological perspective pertaining to the relationship between dietary cholesterol intake and cardiovascular disease risk. For much of the past 50 years, a great deal of the scientific literature regarding dietary fat and cholesterol intake has indicated a strong positive correlation with heart disease. In recent years, however, there have been a number of epidemiological studies that did not support a relationship between cholesterol intake and cardiovascular disease. Further, a number of recent clinical trials that looked at the effects of long-term egg consumption (as a vehicle for dietary cholesterol) reported no negative impact on various indices of cardiovascular health and disease. Coupled with data indicating that the impact of lowering dietary cholesterol intake on serum LDL levels is small compared with other dietary and lifestyle factors, there is a need to consider how otherwise healthy foods can be incorporated in the diet to meet current dietary cholesterol recommendations. Because eggs are a healthful food, it is particularly important that sensible strategies be recommended for inclusions of eggs in a healthy diet.
PMCID: PMC3648753  PMID: 22983850
25.  A TrkB Small Molecule Partial Agonist Rescues TrkB Phosphorylation Deficits and Improves Respiratory Function in a Mouse Model of Rett Syndrome 
Rett syndrome (RTT) results from loss-of-function mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2) and is characterized by abnormal motor, respiratory and autonomic control, cognitive impairment, autistic-like behaviors and increased risk of seizures. RTT patients and Mecp2 null mice exhibit reduced expression of Brain-Derived Neurotrophic Factor (BDNF), which has been linked in mice to increased respiratory frequency, a hallmark of RTT. The present study was undertaken to test the hypotheses that BDNF deficits in Mecp2 mutants are associated with reduced activation of the BDNF receptor, TrkB, and that pharmacologic activation of TrkB would improve respiratory function. We characterized BDNF protein expression, TrkB activation and respiration in heterozygous female Mecp2 mutant mice (Het), a model that recapitulates the somatic mosaicism for mutant Mecp2 found in typical RTT patients, and evaluated the ability of a small molecule TrkB agonist, LM22A-4, to ameliorate biochemical and functional abnormalities in these animals. We found that Het mice exhibit 1) reduced BDNF expression and TrkB activation in the medulla and pons and 2) breathing dysfunction, characterized by increased frequency due to periods of tachypnea, and increased apneas, as in RTT patients. Treatment of Het mice with LM22A-4 for 4 weeks rescued wildtype levels of TrkB phosphorylation in the medulla and pons and restored wildtype breathing frequency. These data provide new insight into the role of BDNF signaling deficits in the pathophysiology of RTT and highlight TrkB as a possible therapeutic target in this disease.
PMCID: PMC3710112  PMID: 22302819

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