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1.  Multiplexed ion beam imaging (MIBI) of human breast tumors 
Nature medicine  2014;20(4):436-442.
Immunohistochemistry (IHC) is a tool for visualizing protein expression employed as part of the diagnostic work-up for the majority of solid tissue malignancies. Existing IHC methods use antibodies tagged with fluorophores or enzyme reporters that generate colored pigments. Because these reporters exhibit spectral and spatial overlap when used simultaneously, multiplexed IHC is not routinely used in clinical settings. We have developed a method that uses secondary ion mass spectrometry to image antibodies tagged with isotopically pure elemental metal reporters. Multiplexed ion beam imaging (MIBI) is capable of analyzing up to 100 targets simultaneously over a five-log dynamic range. Here, we used MIBI to analyze formalin-fixed, paraffin-embedded (FFPE) human breast tumor tissue sections stained with ten labels simultaneously. The resulting data suggest that MIBI will provide new insights by integrating tissue microarchitecture with highly multiplexed protein expression patterns, and will be valuable for basic research, drug discovery and clinical diagnostics.
doi:10.1038/nm.3488
PMCID: PMC4110905  PMID: 24584119
2.  NMR cryoporometry characterisation studies of the relation between drug release profile and pore structural evolution of polymeric nanoparticles 
Graphical abstract
Nanoparticles for convective delivery.
PLGA/PLA polymeric nanoparticles could potentially enhance the effectiveness of convective delivery of drugs, such as carboplatin, to the brain, by enabling a more sustained dosage over a longer time than otherwise possible. However, the link between the controlled release nanoparticle synthesis route, and the subsequent drug release profile obtained, is not well-understood, which hinders design of synthesis routes and availability of suitable nanoparticles. In particular, despite pore structure evolution often forming a key aspect of past theories of the physical mechanism by which a particular drug release profile is obtained, these theories have not been independently tested and validated against pore structural information. Such validation is required for intelligent synthesis design, and NMR cryoporometry can supply the requisite information. Unlike conventional pore characterisation techniques, NMR cryoporometry permits the investigation of porous particles in the wet state. NMR cryoporometry has thus enabled the detailed study of the evolving, nanoscale structure of nanoparticles during drug release, and thus related pore structure to drug release profile in a way not done previously for nanoparticles. Nanoparticles with different types of carboplatin drug release profiles were compared, including burst release, and various forms of delayed release. ESEM and TEM images of these nanoparticles also provided supporting data showing the rapid initial evolution of some nanoparticles. Different stages, within a complex, varying drug release profile, were found to be associated with particular types of changes in the nanostructure which could be distinguished by NMR. For a core-coat nanoparticle formulation, the development of smaller nanopores, following an extended induction period with no structural change, was associated with the onset of substantial drug release. This information could be used to independently validate the rationale for a particular synthesis method. Hence, the specific reasons for the effectiveness of the synthesis route, for obtaining core-coat nanoparticles with delayed release, have been elucidated.
doi:10.1016/j.ijpharm.2014.04.018
PMCID: PMC4048935  PMID: 24726633
Drug; Controlled release; PLGA; Diffusion; Cryoporometry; NMR
3.  Generation and characterization of a unique reagent that recognizes a panel of recombinant human monoclonal antibody therapeutics in the presence of endogenous human IgG 
mAbs  2013;5(4):540-554.
Pharmacokinetic (PK) and immunohistochemistry (IHC) assays are essential to the evaluation of the safety and efficacy of therapeutic monoclonal antibodies (mAb) during drug development. These methods require reagents with a high degree of specificity because low concentrations of therapeutic antibody need to be detected in samples containing high concentrations of endogenous human immunoglobulins. Current assay reagent generation practices are labor-intensive and time-consuming. Moreover, these practices are molecule-specific and so only support one assay for one program at a time. Here, we describe a strategy to generate a unique assay reagent, 10C4, that preferentially recognizes a panel of recombinant human mAbs over endogenous human immunoglobulins. This “panel-specific” feature enables the reagent to be used in PK and IHC assays for multiple structurally-related therapeutic mAbs. Characterization revealed that the 10C4 epitope is conformational, extensive and mainly composed of non-CDR residues. Most key contact residues were conserved among structurally-related therapeutic mAbs, but the combination of these residues exists at low prevalence in endogenous human immunoglobulins. Interestingly, an indirect contact residue on the heavy chain of the therapeutic appears to play a critical role in determining whether or not it can bind to 10C4, but has no affect on target binding. This may allow us to improve the binding of therapeutic mAbs to 10C4 for assay development in the future. Here, for the first time, we present a strategy to develop a panel-specific reagent that can expedite the development of multiple clinical assays for structurally-related therapeutic mAbs.
doi:10.4161/mabs.24822
PMCID: PMC3906308  PMID: 23774668
monoclonal antibodies; PK assay; IHC assay; assay reagent; panel-specific
4.  Lower Exposure and Faster Clearance of Bevacizumab in Gastric Cancer and the Impact of Patient Variables: Analysis of Individual Data from AVAGAST Phase III Trial 
The AAPS Journal  2014;16(5):1056-1063.
Altered pharmacokinetics of antibody drugs has been reported in advanced gastric cancer (AGC). We aim to evaluate bevacizumab pharmacokinetics in AGC from the Phase III trial (AVAGAST), and explore the influence of patient variables. Bevacizumab concentrations (Cp) were measured in plasma samples taken following disease progression from 162 patients (7.5 mg/kg every 3 weeks). Predicted Cp [median and 90% prediction interval] was simulated using the population pharmacokinetic model established for other cancers (PPK model) and compared to observed Cp. Bevacizumab clearance was estimated using NONMEM and compared between subgroups. Patient characteristics of AGC are similar to other cancers except for lower body weight despite higher percentage of males. Eighty-five percent of observed Cp was below the median predicted Cp and 38% below the lower boundary of the 90% prediction interval. Median bevacizumab clearance in AGC was 4.5 versus 3 mL/day/kg in other cancers. Bevacizumab clearance was significantly faster (p < 0.05) in patients without gastrectomy (n = 42) or lower albumin. Clearance appeared to be faster in patients with lower total protein, higher ECOG scores, more metastatic sites, and poorer response. No significant difference in bevacizumab concentrations and clearance was observed between Asian and Non-Asian patients. AGC patients exhibited significantly lower bevacizumab exposure due to an approximate 50% increase in clearance versus other cancers. Bevacizumab is cleared faster in patients without prior gastrectomy. No significant difference in bevacizumab pharmacokinetics was observed between Asian and Non-Asian patients. The underlying mechanism for faster bevacizumab clearance in AGC is unknown and warrants further research.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-014-9631-6) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-014-9631-6
PMCID: PMC4147052  PMID: 24942210
advanced gastric cancer; bevacizumab; ethnicity; NONMEM; pharmacokinetics
5.  Copper Diamidocarbene Complexes: Characterization of Monomeric to Tetrameric Species 
Inorganic Chemistry  2014;53(5):2699-2707.
Treatment of CuCl with 1 equiv of the in situ prepared N-mesityl-substituted diamidocarbene 6-MesDAC produced a mixture of the dimeric and trimeric copper complexes [(6-MesDAC)CuCl]2 (1) and [(6-MesDAC)2(CuCl)3] (2). Combining CuCl with isolated, free 6-MesDAC in 1:1 and 3:2 ratios gave just 1 and 2, respectively, while increasing the ratio to >5:1 allowed the isolation of small amounts of the tetrameric copper complex [(6-MesDAC)2(CuCl)4] (3). Efforts to bring about metathesis reactions of 1 with MOtBu (M = Li, Na, K) proved successful only for M = Li to afford the spectroscopically characterized ate product [(6-MesDAC)CuCl·LiOtBu·2THF] (5). Attempts to crystallize this species instead gave a 1:1 mixture of 1 and the monomer [(6-MesDAC)CuCl] (6). The X-ray structures of 1–3 and 1 + 6, along with the cation [Cu(6-MesDAC)2]+ (4), have been determined.
Treatment of CuCl with the diamidocarbene 6-MesDAC generated [(6-MesDAC)CuCl]2, [(6-MesDAC)2(CuCl)3], and [(6-MesDAC)2(CuCl)4]. Efforts to convert the dimer to a Cu−OtBu complex led instead to a partially metathesized ate complex; attempts at crystallization afforded instead a 1:1 mixture of [(6-MesDAC)CuCl]2 and [(6-MesDAC)CuCl].
doi:10.1021/ic4031014
PMCID: PMC3989936  PMID: 24512071
6.  Ankyrin-G Participates in INa Remodeling in Myocytes from the Border Zones of Infarcted Canine Heart 
PLoS ONE  2013;8(10):e78087.
Cardiac Na channel remodeling provides a critical substrate for generation of reentrant arrhythmias in border zones of the infarcted canine heart. Recent studies show that Nav1.5 assembly and function are linked to ankyrin-G, gap, and mechanical junction proteins. In this study our objective is to expound the status of the cardiac Na channel, its interacting protein ankyrinG and the mechanical and gap junction proteins at two different times post infarction when arrhythmias are known to occur; that is, 48 hr and 5 day post coronary occlusion. Previous studies have shown the origins of arrhythmic events come from the subendocardial Purkinje and epicardial border zone. Our Purkinje cell (Pcell) voltage clamp study shows that INa and its kinetic parameters do not differ between Pcells from the subendocardium of the 48hr infarcted heart (IZPCs) and control non-infarcted Pcells (NZPCs). Immunostaining studies revealed that disturbances of Nav1.5 protein location with ankyrin-G are modest in 48 hr IZPCs. Therefore, Na current remodeling does not contribute to the abnormal conduction in the subendocardial border zone 48 hr post myocardial infarction as previously defined. In addition, immunohistochemical data show that Cx40/Cx43 co-localize at the intercalated disc (IDs) of control NZPCs but separate in IZPCs. At the same time, Purkinje cell desmoplakin and desmoglein2 immunostaining become diffuse while plakophilin2 and plakoglobin increase in abundance at IDs. In the epicardial border zone 5 days post myocardial infarction, immunoblot and immunocytochemical analyses showed that ankyrin-G protein expression is increased and re-localized to submembrane cell regions at a time when Nav1.5 function is decreased. Thus, Nav1.5 and ankyrin-G remodeling occur later after myocardial infarction compared to that of gap and mechanical junctional proteins. Gap and mechanical junctional proteins remodel in IZPCs early, perhaps to help maintain Nav1.5 subcellular location position and preserve its function soon after myocardial infarction.
doi:10.1371/journal.pone.0078087
PMCID: PMC3796465  PMID: 24155982
7.  Increased late sodium current contributes to long QT-related arrhythmia susceptibility in female mice 
Cardiovascular Research  2012;95(3):300-307.
Aims
Female gender is a risk factor for long QT-related arrhythmias, but the underlying mechanisms remain uncertain. Here, we tested the hypothesis that gender-dependent function of the post-depolarization ‘late’ sodium current (INa-L) contributes.
Methods and results
Studies were conducted in mice in which the canonical cardiac sodium channel Scn5a locus was disrupted, and expression of human wild-type SCN5A cDNA substituted. Baseline QT intervals were similar in male and female mice, but exposure to the sodium channel opener anemone toxin ATX-II elicited polymorphic ventricular tachycardia in 0/9 males vs. 6/9 females. Ventricular INa-L and action potential durations were increased in myocytes isolated from female mice compared with those from males before and especially after treatment with ATX-II. Further, ATX-II elicited potentially arrhythmogenic early afterdepolarizations in myocytes from 0/5 male mice and 3/5 female mice.
Conclusion
These data identify variable late INa as a modulator of gender-dependent arrhythmia susceptibility.
doi:10.1093/cvr/cvs160
PMCID: PMC3633400  PMID: 22562703
Mouse; Late sodium current; Gender; Arrhythmias
8.  Impact of Chlorine Dioxide Gas Sterilization on Nosocomial Organism Viability in a Hospital Room 
To evaluate the ability of ClO2 to decontaminate pathogens known to cause healthcare-associated infections in a hospital room strains of Acinetobacter baumannii, Escherichia coli, Enterococcus faecalis, Mycobacterium smegmatis, and Staphylococcus aureus were spot placed in duplicate pairs at 10 sites throughout a hospital room and then exposed to ClO2 gas. Organisms were collected and evaluated for reduction in colony forming units following gas exposure. Six sterilization cycles with varied gas concentrations, exposure limits, and relative humidity levels were conducted. Reductions in viable organisms achieved ranged from 7 to 10-log reductions. Two sterilization cycles failed to produce complete inactivation of organisms placed in a bathroom with the door closed. Reductions of organisms in the bathroom ranged from 6-log to 10-log reductions. Gas leakage between hospital floors did not occur; however, some minor gas leakage from the door of hospital room was measured which was subsequently sealed to prevent further leakage. Novel technologies for disinfection of hospital rooms require validation and safety testing in clinical environments. Gaseous ClO2 is effective for sterilizing environmental contamination in a hospital room. Concentrations of ClO2 up to 385 ppm were safely maintained in a hospital room with enhanced environmental controls.
doi:10.3390/ijerph10062596
PMCID: PMC3717754  PMID: 23792697
chlorine dioxide; sterilization; gas; nosocomial; hospital
10.  Managing Uncertainty in the Context of Clubfoot Care: Exploring the Value of Uncertainty Management Theory and the Sense of Virtual Community 
The Iowa Orthopaedic Journal  2013;33:142-148.
Serious health conditions, such as clubfoot, could be a major source of uncertainty and stress for parents of children affected. How parents deal with uncertainty and stress as related to their child’s health condition is of interest for medical professionals and health communicators alike. While physicians remain a preferred source of health information, during medical encounters or via phone and email communication, many individuals seek out health information on the Internet, including in online support communities. This study explored the connections between Uncertainty Management Theory (UMT) constructs and the potential contribution of the sense of virtual community (SOVC) to the UMT framework. The results of this research suggest that the UMT needs to be adapted for use in online contexts. One way is to include theoretical constructs, such as the sense of virtual community, specifically developed to measure online interactions. A modified and updated Uncertainty Management Theory could be useful in exploring, analyzing and understanding online behaviors related to health conditions such as clubfoot and thus contribute substantially to what we know about caregivers in their role as uncertainty managers.
PMCID: PMC3748870  PMID: 24027474
11.  The Development and Validation of the Client Expectations of Massage Scale 
Background:
Although there is evidence that client expectations influence client outcomes, a valid and reliable scale for measuring the range of client expectations for both massage therapy and the behaviors of their massage therapists does not exist. Understanding how client expectations influence client outcomes would provide insight into how massage achieves its reported effects.
Purpose:
To develop and validate the Client Expectations of Massage Scale (CEMS), a measure of clients’ clinical, educational, interpersonal, and outcome expectations.
Setting:
Offices of licensed massage therapists in Iowa.
Research Design:
A practice-based research methodology was used to collect data from two samples of massage therapy clients. For Sample 1, 21 volunteer massage therapists collected data from their clients before the massage. Factor analysis was conducted to test construct validity and coefficient alpha was used to assess reliability. Correlational analyses with the CEMS, previous measures of client expectations, and the Life Orientation Test–Revised were examined to test the convergent and discriminant validity of the CEMS. For Sample 2, 24 massage therapists distributed study materials for clients to complete before and after a massage therapy session. Structural equation modeling was used to assess the construct, discriminant, and predictive validity of the CEMS.
Participants:
Sample 1 involved 320 and Sample 2 involved 321 adult massage clients.
Intervention:
Standard care provided by licensed massage therapists.
Main Outcomes:
Numeric Rating Scale for pain and Positive and Negative Affect Schedule–Revised (including the Serenity subscale).
Results:
The CEMS demonstrated good construct, convergent, discriminant and predictive validity, and adequate reliability. Client expectations were generally positive toward massage and their massage therapists. Positive outcome expectations had a positive effect on clients’ changes in pain and serenity. High interpersonal expectations had a negative effect on clients’ changes in serenity.
Conclusions:
Client expectations contribute to the nonspecific effects of massage therapy.
PMCID: PMC3457721  PMID: 23087774
massage therapy; validity; practice-based research; pain; affect
12.  Striking in Vivo Phenotype of a Disease-Associated Human SCN5A Mutation Producing Minimal Changes in Vitro 
Circulation  2011;124(9):1001-1011.
Background
The D1275N SCN5A mutation has been associated with a range of unusual phenotypes including conduction disease and dilated cardiomyopathy (DCM) as well as atrial and ventricular tachyarrhythmias. However, when D1275N is studied in heterologous expression systems, most studies show near-normal sodium channel function. Thus, the relationship of the variant to the clinical phenotypes remains uncertain.
Methods and results
We identified D1275N in a patient with atrial flutter, atrial standstill, conduction disease, and sinus node dysfunction. There was no major difference in biophysical properties between wild-type and D1275N channels expressed in CHO or tsA201 cells in the absence or presence of β1 subunits. To determine D1275N function in vivo, the Scn5a locus was modified to knock out the mouse gene, and the full-length wild-type (H) or D1275N (DN) human SCN5A cDNAs were then inserted at the modified locus using recombinase mediated cassette exchange. Mice carrying the DN allele displayed slow conduction, heart block, atrial fibrillation, ventricular tachycardia, and a DCM phenotype, with no significant fibrosis or myocyte disarray on histological examination. The DN allele conferred gene-dose dependent increases in SCN5A mRNA abundance, but reduced sodium channel protein abundance and peak sodium current amplitudes (H/H, −41.0±2.9 pA/pF at −30mV; DN/H, 19.2±3.1 pA/pF, P<0.001 versus H/H; DN/DN, −9.3±1.1 pA/pF, P<0.001 versus H/H).
Conclusions
Although D1275N produces near normal currents in multiple heterologous expression experiments, our data establish this variant as a pathological mutation that generates conduction slowing, arrhythmias, and a DCM phenotype by reducing cardiac sodium current.
doi:10.1161/CIRCULATIONAHA.110.987248
PMCID: PMC3297976  PMID: 21824921
genetics; ion channels; cardiomyopathy; electrophysiology
13.  Neural stem cell transplantation benefits a monogenic neurometabolic disorder during the symptomatic phase of disease 
Stem cells (Dayton, Ohio)  2009;27(9):2362-2370.
Although we and others have demonstrated that neural stem cells (NSCs) may impact such neurogenetic conditions as lysosomal storage diseases when transplanted at birth, it has remained unclear whether such interventions can impact well-established mid-stage disease, a situation often encountered clinically. Here we report that when NSCs were injected intracranially into the brain of adult symptomatic Sandhoff (Hexb−/−) mice, cells migrated far from the injection site and integrated into the host cytoarchitecture, restoring β-hexosaminidase enzyme activity and promoting neuropathologic and behavioral improvement. Mouse lifespan increased, neurological function improved, and disease progression was slowed. These clinical benefits correlated with neuropathological correction at the cellular and molecular levels, reflecting the multiple potential beneficial actions of stem cells, including enzyme cross-correction, cell replacement, tropic support, and direct anti-inflammatory action. Pathotropism, i.e., migration and homing of NSCs to pathological sites, could be imaged in real time by magnetic resonance imaging. Differentially expressed chemokines might play a role in directing the migration of transplanted stem cells to sites of pathology. Because many of the beneficial actions of NSCs observed in newborn brains were recapitulated in adult brains to the benefit of Sandhoff recipients, NSC-based interventions may also be useful in symptomatic subjects with established disease, even in symptomatic patients.
doi:10.1002/stem.163
PMCID: PMC3411354  PMID: 19591217
neural stem cell therapy; lysosomal storage disorders; Sandhoff disease; neurodegenerative disease; metabolic cross-correction
14.  Informatic and functional approaches to identifying a regulatory region for the cardiac sodium channel 
Circulation research  2011;109(1):38-46.
Rationale
Although multiple lines of evidence suggest variable expression of the cardiac sodium channel gene SCN5A plays a role in susceptibility to arrhythmia, little is known about its transcriptional regulation.
Objective
We used in silico and in vitro experiments to identify possible non-coding sequences important for transcriptional regulation of SCN5A. The results were extended to mice in which a putative regulatory region was deleted.
Methods and Results
We identified 92 non-coding regions highly conserved (>70%) between human and mouse SCN5A orthologs. Three conserved non-coding sequences (CNS) showed significant (>5-fold) activity in luciferase assays. Further in vitro studies indicated one, CNS28 in intron 1, as potential regulatory region. Using Recombinase-Mediated Cassette Exchange (RMCE), we generated mice in which a 435 bp region encompassing CNS28 was removed. Animals homozygous for the deletion showed significant increases in SCN5A transcripts, NaV1.5 protein abundance, and sodium current measured in isolated ventricular myocytes. ECGs revealed a significantly shorter QRS (10.7±0.2ms in controls vs. 9.7±0.2ms in knockouts) indicating more rapid ventricular conduction. In vitro analysis of CNS28 identified a short 3′ segment within this region required for regulatory activity and including an E-box motif. Deletion of this segment reduced reporter activity to 3.6±0.3% of baseline in CHO cells and 16±3% in myocytes (both P<0.05), and mutation of individual sites in the E-box restored activity to 62±4% and 57±2% of baseline in CHO cells and myocytes, respectively (both P<0.05).
Conclusions
These findings establish that regulation of cardiac sodium channel expression modulates channel function in vivo, and identify a non-coding region underlying this regulation.
doi:10.1161/CIRCRESAHA.110.235630
PMCID: PMC3135383  PMID: 21566215
Gene Expression Regulation; Sodium Channels; Mice; Transgenic
15.  Reliability of tanning behaviors and sunburns history among sororities and fraternities students 
The need for both valid and reliable measurement is crucial for the assessment of sun behavior. We used test-retest reliability of a self-administered survey to examine the reliability of reporting on behavior relevant to artificial ultraviolet tanning, sunburns and sun sensitivity among sorority and fraternity-affiliated university students. Subjects were members of sororities and fraternities who participated in an initial survey and skin examination. Students were prompted by specific recall queries. The students completed a second survey 2-4 weeks later. High reliability on test-retest for questions evaluating the number of artificial UV tanning sessions and the number of sunburns during specific time periods was found. Moderate reliability for measures reporting the use of self-tanning creams, can be largely explained by only one third of the students reporting they had ever used self-tanning creams. Overall this study suggests that members of sororities and fraternities report lifetime artificial ultraviolet tanning consistently when required to recall time period specific exposures prior to estimating their lifetime exposure.
doi:10.1111/j.1600-0781.2008.00359.x
PMCID: PMC3374482  PMID: 18717960
reproducibility of results; skin neoplasms; ultraviolet rays
16.  Tanning behavior among young frequent tanners is related to attitudes and not lack of knowledge about the dangers 
Health education journal  2009;68(3):232-243.
Objective
To examine the importance of tanning among students in relation to attitudes and knowledge regarding skin cancer prevention.
Design
A cross-sectional survey.
Setting
College students at a major Midwestern university
Methods
Students were recruited to complete a self-administered questionnaire that included information on sun-sensitivity, knowledge and tanning attitudes and behaviors. Survey sampling statistical techniques that account for clustering among the 163 students recruited were used.
Results
We found a high level of skin cancer prevention knowledge; however knowledge was not related to a reduction in the importance of tanning. In many cases, higher levels of knowledge corresponded to a greater emphasis on the importance of tanning. Sunscreen use was low among this population. Those who placed an importance on tanning more often checked that they believed that “sunless tanning creams are safer than the sun”.
Conclusions
This population’s belief that they look healthier and feel better with a tan strongly influences the desire to tan. Therefore, future cancer information campaigns or other prevention efforts should directly address the desire to tan by encouraging the use of sunless tanning products as an alternative method of tanning.
doi:10.1177/0017896909345195
PMCID: PMC3374486  PMID: 22707763
Knowledge; Attitudes; Cancer Prevention; Tanning; Ultraviolet Rays
17.  Partial Agonists of the α3β4* Neuronal Nicotinic Acetylcholine Receptor Reduce Ethanol Consumption and Seeking in Rats 
Neuropsychopharmacology  2010;36(3):603-615.
Alcohol use disorders (AUDs) impact millions of individuals and there remain few effective treatment strategies. Despite evidence that neuronal nicotinic acetylcholine receptors (nAChRs) have a role in AUDs, it has not been established which subtypes of the nAChR are involved. Recent human genetic association studies have implicated the gene cluster CHRNA3–CHRNA5–CHRNB4 encoding the α3, α5, and β4 subunits of the nAChR in susceptibility to develop nicotine and alcohol dependence; however, their role in ethanol-mediated behaviors is unknown due to the lack of suitable and selective research tools. To determine the role of the α3, and β4 subunits of the nAChR in ethanol self-administration, we developed and characterized high-affinity partial agonists at α3β4 nAChRs, CP-601932, and PF-4575180. Both CP-601932 and PF-4575180 selectively decrease ethanol but not sucrose consumption and operant self-administration following long-term exposure. We show that the functional potencies of CP-601932 and PF-4575180 at α3β4 nAChRs correlate with their unbound rat brain concentrations, suggesting that the effects on ethanol self-administration are mediated via interaction with α3β4 nAChRs. Also varenicline, an approved smoking cessation aid previously shown to decrease ethanol consumption and seeking in rats and mice, reduces ethanol intake at unbound brain concentrations that allow functional interactions with α3β4 nAChRs. Furthermore, the selective α4β2* nAChR antagonist, DHβE, did not reduce ethanol intake. Together, these data provide further support for the human genetic association studies, implicating CHRNA3 and CHRNB4 genes in ethanol-mediated behaviors. CP-601932 has been shown to be safe in humans and may represent a potential novel treatment for AUDs.
doi:10.1038/npp.2010.191
PMCID: PMC3055681  PMID: 21048701
α3β4* nicotinic acetylcholine receptor; ethanol; drug abuse; addiction; rat; alcohol & alcoholism; animal models; drug discovery/development; neuropharmacology; alpha3beta4* nicotinic acetylcholine receptor; ethanol; drug abuse; addiction; rat
18.  Flying in a flock comes at a cost in pigeons 
Nature  2011;474(7352):494-497.
Flying birds often form flocks, with social1, navigational2 and anti-predator3 implications. Further, flying in a flock can result in aerodynamic benefits, thus reducing power requirements4, as demonstrated by a reduction in heart rate and wingbeat frequency in pelicans flying in a V-formation5. But how general is an aerodynamic power reduction due to group-flight? V-formation flocks are limited to moderately steady flight in relatively large birds, and may represent a special case. What are the aerodynamic consequences of flying in the more usual ‘cluster’ 6,7 flock? Here, we use data from innovative back-mounted GPS and 6 degree of freedom inertial sensors to show that pigeons 1) maintain powered, banked turns like aircraft, imposing dorsal accelerations of up to 2g, effectively doubling body weight and quadrupling induced power requirements; 2) increase flap frequency with increases in all conventional aerodynamic power requirements; and 3) increase flap frequency when flying near, particularly behind, other birds. Therefore, unlike V-formation pelicans, pigeons do not gain an aerodynamic advantage from flying in a flock; indeed, the increased flap frequency – whether due to direct aerodynamic interactions or requirements for increased stability or control – suggests a considerable energetic cost to flight in a tight cluster flock.
doi:10.1038/nature10164
PMCID: PMC3162477  PMID: 21697946
19.  Endothelial Heparan Sulfate Controls Chemokine Presentation in Recruitment of Lymphocytes and Dendritic Cells to Lymph Nodes 
Immunity  2010;33(5):817-829.
SUMMARY
Heparan sulfate can bind several adhesion molecules involved in lymphocyte trafficking. However, the in vivo function of endothelial heparan sulfate in lymphocyte homing and stimulation of the immune response has not been elucidated. Here, we generated mutant mice deficient in the enzyme Ext1, which is required for heparan sulfate synthesis, in a Tek-dependent and inducible manner. Chemokine presentation was diminished in the mutant mice, causing the lack of appropriate integrin-mediated adhesion, and resulted in a marked decrease in lymphocyte sticking to high endothelial venules and in recruitment of resident dendritic cells through lymphatic vessels to the lymph nodes. As a consequence, mutant mice displayed a severe impairment in lymphocyte homing and a compromised contact hypersensitivity response. By contrast, lymphocyte rolling was increased due to loss of electrostatic repulsion by heparan sulfate. These results demonstrate critical roles of endothelial heparan sulfate in immune surveillance and immune response generation.
doi:10.1016/j.immuni.2010.10.018
PMCID: PMC2996097  PMID: 21093315
20.  Partial Agonists of the α3β4* Neuronal Nicotinic Acetylcholine Receptor Reduce Ethanol Consumption and Seeking in Rats 
Alcohol use disorders (AUDs) impact millions of individuals and there remain few effective treatment strategies. Despite evidence that neuronal nicotinic acetylcholine receptors (nAChRs) play a role in AUDs, it has not been established which subtypes of the nAChR are involved. Recent human genetic association studies have implicated the gene cluster CHRNA3-CHRNA5-CHRNB4 encoding the α3, α5 and β4 subunits of the nAChR in susceptibility to develop nicotine and alcohol dependence; however, their role in ethanol-mediated behaviors is unknown due to the lack of suitable and selective research tools. To determine the role of the α3, and β4 subunits of the nAChR in ethanol self-administration, we developed and characterized high affinity partial agonists at α3β4 nAChRs, CP-601932 and PF-4575180. Both CP-601932 and PF-4575180 selectively decrease ethanol but not sucrose consumption and operant self-administration following long-term exposure. We show that the functional potencies of CP-601932 and PF-4575180 at α3β4 nAChRs correlate with their unbound rat brain concentrations suggesting that the effects on ethanol self-administration are mediated via interaction with α3β4 nAChRs. Also varenicline, an approved smoking cessation aid previously shown to decrease ethanol consumption and seeking in rats and mice, reduces ethanol intake at unbound brain concentrations that allow functional interactions with α3β4 nAChRs. Furthermore, the selective α4β2* nAChR antagonist, DHβE, did not reduce ethanol intake. Together, these data provide further support for the human genetic association studies implicating CHRNA3 and CHRNB4 genes in ethanol-mediated behaviors. CP-601932 has been shown to be safe in humans and may represent a potential novel treatment for AUDs.
doi:10.1038/npp.2010.191
PMCID: PMC3055681  PMID: 21048701
α3β4* nicotinic acetylcholine receptor; ethanol; drug abuse; addiction; rat
21.  Social Support Patterns of Collegiate Athletes Before and After Injury 
Journal of Athletic Training  2010;45(4):372-379.
Abstract
Context:
Social support has been identified as an important factor in facilitating recovery from injury. However, no previous authors have prospectively assessed the change in social support patterns before and after injury.
Objective:
To examine the preinjury and postinjury social support patterns among male and female collegiate athletes.
Design:
Prospective observational study.
Setting:
A Big Ten Conference university.
Patients or Other Participants:
A total of 256 National Collegiate Athletic Association Division I male and female collegiate athletes aged 18 or older from 13 sports teams.
Main Outcome Measure(s):
Injury incidence was identified using the Sports Injury Monitoring System. Social support was measured using the 6-item Social Support Questionnaire. Data on preinjury and postinjury social support patterns were compared.
Results:
Male athletes reported more sources of social support than female athletes, whereas female athletes had greater satisfaction with the support they received. Athletes' social support patterns changed after they became injured. Injured athletes reported relying more on coaches (P  =  .003), athletic trainers (P < .0001), and physicians (P  =  .003) for social support after they became injured. Athletes also reported greater postinjury satisfaction with social support received from friends (P  =  .019), coaches (P  =  .001), athletic trainers (P < .0001), and physicians (P  =  .003).
Conclusions:
Our findings identify an urgent need to better define the psychosocial needs of injured athletes and also strongly suggest that athletic trainers have a critical role in meeting these needs.
doi:10.4085/1062-6050-45.4.372
PMCID: PMC2902031  PMID: 20617912
psychology; athletic trainers
22.  Principles for Establishing Trust When Developing a Substance Abuse Intervention With a Native American Community 
Creative nursing  2011;17(2):68-73.
This article traces the development of a research project with a Native American community. Four principles were used to guide the development of the “Community Partnership to Affect Cherokee Adolescent Substance Abuse” project using a community-based participatory research approach. The principles suggest that establishing trust is key when developing and conducting research with a Native American community.
PMCID: PMC3103821  PMID: 21563633
23.  Variation in Prescription Use and Spending for Lipid-Lowering and Diabetes Medications in the VA Healthcare System 
Objectives
Very little is known about variation in prescription drug use and spending. We examine variation in outpatient prescription use and spending for diabetes and hyperlipidemia in the Veterans Affairs (VA) system and its association with quality measures for these conditions.
Study Design and Methods
We compared outpatient prescription use, spending and quality of care across 135 VA Medical Centers (VAMCs) in 2008, including 2.3 million patients dispensed lipid-lowering medications and 981,031 dispensed diabetes medications. We calculated VAMC-level cost/patient for these medications, proportion of patients on brand-name drugs, and Healthcare Effectiveness Data and Information Set (HEDIS) scores for hyperlipidemia (LDL<100mg/dl) and diabetes (HgA1c>9 or not measured) at each facility.
Results
The median cost/patient for lipid-lowering agents in 2008 was $49.60 and varied from $39.68 in the least expensive quartile of VAMCs to $69.57 in the most expensive (p<0.001). For diabetes agents, median cost/patient was $158.34 and varied from $123.34 in the least expensive quartile to $198.31 in the most expensive (p<0.001). The proportion of patients dispensed brand name oral drugs in these classes in VAMCs in the most expensive quartile was twice that in the lowest quartile (p<0.001). There was no correlation between VAMC-level prescription spending and performance on HEDIS measures for lipid-lowering drugs (r=.12 and r=.07) or diabetes agents (r=-.10).
Conclusions
Despite a closely managed formulary, significant variation in prescription spending and use of brand name drugs exists in the VA. Although we could not explicitly risk adjust, there appears to be no association between spending on medications and quality of care.
PMCID: PMC3096004  PMID: 20964470
24.  The Notch ligands Jagged2, Delta1, and Delta4 induce differentiation and expansion of functional human NK cells from CD34+ cord blood hematopoietic progenitor cells 
Notch receptor signaling is required for T cell development, but its role in NK cell development is poorly understood. We compared the ability of the five mammalian Notch ligands (Jagged1, Jagged2, Delta1, Delta3, or Delta4) to induce NK cell development from human hematopoietic progenitor cells (HPCs). CD34+ HPCs were cultured with OP9 stromal cell lines transduced with one of the Notch ligands or with OP9 stromal cells alone, in the presence of IL-7, Flt3L, and IL-15. Differentiation and expansion of CD56+CD3− cells was greatly accelerated in the presence of Jagged2, Delta-1, or Delta-4, versus culture in the absence of ligand or in the presence of Jagged1 or Delta3. At four weeks, cultures containing Jagged2, Delta1, or Delta4 contained 80–90% NK cells, with the remaining cells being CD33+ myeloid cells. Notch-induced NK (N-NK) cells resembled CD56bright NK cells in that they were CD16-, CD94−, CD117+, and KIR-. They also expressed NKp30, NKp44, NKp46, 2B4, and DNAM-1, with partial expression of NKG2D. The N-NK cells displayed cytotoxic activity against the K562 and RPMI-8226 cell lines, at levels similar to activated peripheral blood NK cells, although killing of Daudi cells was not present. N-NK cells were also capable of IFN-γ secretion. Thus, Notch ligands have differential ability to induce and expand immature but functional NK cells from CD34+ HPCs. The use of Notch ligands to generate functional NK cells in vitro may be significant for cellular therapy purposes.
doi:10.1016/j.bbmt.2009.06.002
PMCID: PMC2730186  PMID: 19660715
25.  Cherokee Self-Reliance and Word-Use in Stories of Stress 
This study examined the relationship between Cherokee self-reliance and related values expressed through word-use in stories of stress written by Cherokee adolescents. The overall aim of this pilot study was to test the feasibility of using cultural appropriate measurements for a larger intervention study of substance abuse prevention in Cherokee adolescents. A sample of 50 Cherokee adolescent senior high school students completed the Cherokee Self-Reliance Questionnaire and wrote their story of stress. The Linguistic Inquiry and Word Count (LIWC) program, a word-based computerized text analysis software, was used to report the percentage of words used in the selected word categories in relation to all the words used by a participant. Word-use from the stories of stress were found to correlate with Cherokee self-reliance.
PMCID: PMC2914319  PMID: 20669397
Cherokee Self-Reliance; Linguistic Inquiry and Word Count; Culture; Stress

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