An association between testosterone therapy (TT) and cardiovascular disease has been reported and TT use is increasing rapidly.We conducted a cohort study of the risk of acute non-fatal myocardial infarction (MI) following an initial TT prescription  in a large health-care database. We compared the incidence rate of MI in the 90 days following the initial prescription  with the rate in the one year prior to the initial prescription  (post/pre). We also compared post/pre rates in a cohort of men prescribed phosphodiesterase type 5 inhibitors , and compared TT prescription post/pre rates with the PDE5I post/pre rates, adjusting for potential confounders using doubly robust estimation.trend = 0.03), while no trend was seen for PDE5I (ptrend = 0.18). In men under age 65 years, excess risk was confined to those with a prior history of heart disease, with RRs of 2.90  for TT prescription and 1.40  for PDE5I, and a RRR of 2.07 .In all subjects, the post/pre-prescription rate ratio (RR) for TT prescription was 1.36 . In men aged 65 years and older, the RR was 2.19  for TT prescription and 1.15  for PDE5I, and the ratio of the rate ratios (RRR) for TT prescription relative to PDE5I was 1.90 . The RR for TT prescription increased with age from 0.95  for men under age 55 years to 3.43  for those aged ≥75 years (pIn older men, and in younger men with pre-existing diagnosed heart disease, the risk of MI following initiation of TT prescription is substantially increased. Testosterone therapy (TT) has been used in healthy older men to treat diminished extremity strength and physical function associated an age-related decline in serum testosterone. The Truven Health MarketScan® Commercial Claims and Encounters Database includes employees, dependents and retirees with commercial or Medicare insurance whose employers license healthcare data to Truven Health Analytics (Truven). The MarketScan data contributors include Fortune 500 employers (60 percent) and health plans covering numerous other companies and unions (40 percent). The data include diagnoses, procedures, and prescriptions for all enrollees. Enrollees in 2011 are distributed regionally within the U.S. as Northeast (24 percent), North Central (37 percent), South (20 percent), and West (19 percent). We used the data from 2006 to 2010, including patient-specific enrollment history, year of birth, gender, inpatient and outpatient diagnoses (ICD-9 codes) and treatments, and outpatient prescriptions. The data for the study were hosted in secure facilities of Consolidated Research, Inc. (CRI), as required by the Agreement between CRI  and Truven Health Analytics . We received IRB exemption for this study from the NIH Office of Human Subjects Research Protections (OHSRP) since the study was conducted in claims data that were anonymized or de-identified by Truven prior to release. The database is available from Truven under licensing agreements similar to ours.We formed cohorts from the database from men with a minimum of 22 months of continuous enrollment for analyses with post-prescription follow-up intervals of 90 days, and 25 months for analyses with post prescription follow-up intervals of 91 to 180 days. From this cohort, we selected men who filled a first prescription for any of several TT prescriptions not containing estrogen  and, for a comparison population, men who filled a first prescription for phosphodiesterase type 5 inhibitors , between January 1, 2008 and September 30, 2010. We did not have data on how much of the prescribed medication was consumed. The most common TT prescriptions were testosterone gel, testosterone micronized, testosterone cypionate, and testosterone transdermal system. We selected men receiving PDE5I prescriptions as a comparison group because some indications for prescription are similar to those for TT prescription. In addition, PDE5I is commonly prescribed to older men, does not have androgenic effects, and is not metabolized to other sex steroid hormones, such as dihydrotestosterone or estrogens. Also, while PDE5I is recommended for men healthy enough to engage in sexual activity, the drugs themselves, after extensive scrutiny, have not been associated with adverse cardiovascular events The covariates were those recorded in the 18 to 12 month interval prior to the initial prescription for TT prescription or PDE5I. The pre-prescription interval was the one year prior to the initial prescription, the post-prescription interval was 90 days following the initial prescription, thus the prescription cohorts were restricted to those men with a minimum of 18 months enrollment prior to and three months after their initial prescription. In the post-prescription interval, patients were followed until a diagnosis of acute non-fatal myocardial infarction, refilled first prescription, or 90 days following initial prescription, whichever occurred first.For those who did not refill their initial prescription, we analyzed an additional 90-day interval (91–180 days post-prescription), a time when this group likely had minimal use of these drugs. A refill and a subsequent prescription were treated equally in the analysis. Patients with first prescriptions for both TT prescription and PDE5I during follow-up were excluded from this analysis.The study outcome was a diagnosis of acute MI (ICD-9: 410). Men with a history of MI prior to the first prescription for TT or PDE5I were excluded from the post-prescription analyses.Age at the time of initial prescription was included as a covariate. Diagnostic covariates were identified by the ICD 9 codes recorded for inpatient or outpatient diagnoses, reported to be associated with MI, We examined risk of MI with TT prescription in all subjects. Since previous studies indicated that cardiovascular events appeared early in treatment, we focused on the 90 day interval following the filling of a first prescription. For those who did not refill their prescription, we also assessed the interval of 91–180 days. To examine potential effect modification by pre-existing disease, we estimated the effect of TT prescription by prior history of a heart disease diagnosis . We also examined the RR for PDE5I and compared the RRs in the TT prescription and PDE5I cohorts.We examined the effect of the medications by estimating the ratio of the MI incidence rate in the post-prescription interval to the MI incidence rate in the pre-prescription interval (post/pre RR). To estimate the effect of TT prescription relative to PDE5I, we weighted the PDE5I patients with weights derived from propensity scores, specifically their estimated odds of being prescribed TT prescription rather than PDE5I. We also estimated the ratio of rate ratios (RRR). trend = 0.03). Men aged 65 years and older, whose follow-up was not right-censored because they did not refill their prescription, were followed for an additional 90 day period (91–180 days post-prescription) during which the RR was 0.98 . In men under age 65 the corresponding RR was 1.15 .For all TT prescription subjects combined, the post/pre prescription rate ratio for MI (RR)was 1.36  . In men For the comparison group of PDE5I users, the baseline distributions of prior cardiovascular diagnoses, risk factors, and medication use were less common than in the TT prescription cohort, but after weighting, the distributions of covariates in each prescription cohort were nearly identical . This prThe data for both prescription groups were also divided into men with a previously recorded diagnosis of any heart disease, and those without . For TT As odds-of-treatment weighting is less familiar than unweighted regression methods, we also examined the effect of controlling for the covariates using an unweighted Poisson regression. In subjects aged 65 years and older, the RRR was 2.27 , about 20 percent higher than the weighted estimate of 1.90, and in those under age 65 years with a history of heart disease, the RRR was 2.33 , about 13 percent higher than the weighted estimate of 2.07. The lower estimates suggest that weighting in the primary analysis helped reduce possible upward confounding.Among men aged 65 years and older, we observed a two-fold increase in the risk of MI in the 90 days after filling an initial TT prescription, the risk declined to baseline in the 91 to 180 days after initial TT prescription, among those who did not refill their prescription. Since we censored follow-up at the first refill, and the supply for most prescriptions was 30 to 90 days, it is likely that there was little use of the medication in the 91 to 180 day post-prescription interval when the risk declined. Thus, the pattern of change in risk by supply of testosterone is consistent with an effect of the drug, and underscores the concerns raised by three recent studies in predominantly older men P = 0.41) Among younger men with a history of heart disease, we observed a two to three-fold increased risk of MI in the 90 days following an initial TT prescription and no excess risk in younger men without such a history. Among older men, the two-fold increased risk was associated with TT prescription regardless of cardiovascular disease history, although this analysis was based on relatively small numbers of MI cases in each subgroup. More relevant perhaps is the rapid increase with age in the prevalence of diagnosed and undiagnosed coronary artery disease reported from autopsy studies, both overall and among accident victims, serious adverse cardiovascular-related events  which included myocardial infarction along with other conditions. Our findings are consistent with a recent meta-analysis of placebo-controlled randomized trials of testosterone therapy lasting 12 or more weeks among mainly older men, which reported that testosterone therapy increased the risk of adverse cardiovascular-related events , as well as endogenous testosterone levels may also be positively associated with cardiovascular events Taken together, the evidence supports an association between testosterone therapy and risk of serious, adverse cardiovascular-related events–including non-fatal myocardial infarction–in men. However, there is some evidence that low Despite plausible biologic mechanisms linking TT prescription to an elevated risk of MI, our study has limitations related to use of a health-care database that did not include information on the serologic or diagnostic indications for treatment. It also identified only subjects with non-fatal MIs, typically representing about 75% of the total incidence, and was based on the diagnosis of an attending physician, rather than a structured evaluation as might occur in a randomized trial. However, the accuracy of an MI diagnosis is considered to be reliable in such databases, We addressed potential confounding from measured and unmeasured risk factors by using each treated group as its own control, comparing risk before versus after the start of medication use during the short time-frame of our study, and by controlling for differences in the prevalence of risk factors between the two treatment groups. In the prescription-odds weighted regressions, we found no association between PDE5I prescriptions and the risk of MI, suggesting that the TT prescription-related risk of MI is more likely a drug effect, rather than a result of behavioral or other factors associated with prescription.Furthermore, there is no reason to suspect that physicians excluded high-risk individuals from TT prescription or monitored them more closely, since the hypothesis relating TT prescription to adverse cardiovascular events was not widely known during the study period. Indeed, the initial prescription for over 90 percent of the patients in our study occurred prior to publication of this potential concern, Further study is needed to examine the risk of a variety of specific serious adverse cardiovascular events in relation to TT dose and duration, and to assess if the risks of TT vary by level of serum testosterone and presence or absence of hypogonadal disease. The observed excess MI risk in younger men with a history of heart disease is a particular public health concern, as about 10 percent of the men in our study under age 65 years with a TT prescription had a history of heart disease.Given the rapidly increasing use of TT, the current results, along with other recent findings emphasize the urgency of the previous call for clinical trials adequately powered to assess the range of benefits and risks suggested for such therapy. Until that time clinicians might be well advised to include serious cardiovascular events in their discussions with patients of potential risks, particularly for men with existing cardiovascular disease.Table S1Distribution of baseline covariates in men 65 and older in the TTand PDE5 inhibitor cohorts before and after weighting. The TT patients were unweighted and the PDE5 inhibitor patients were weighted to match the TT cohort based on odds of TT prescription.(DOC)Click here for additional data file.Table S2Distribution of baseline covariates in men under 65 in the TT and PDE5 inhibitor cohorts before and after weighting. The TT patients were unweighted and the PDE5 inhibitor patients were weighted to match the TT cohort based on inverse prescription probability.(DOC)Click here for additional data file.


Attrition from follow-up is a major methodological challenge in randomized trials. Incentives are known to improve response rates in cross-sectional postal and online surveys, yet few studies have investigated whether they can reduce attrition from follow-up in online trials, which are particularly vulnerable to low follow-up rates.Our objective was to determine the impact of incentives on follow-up rates in an online trial.Two randomized controlled trials were embedded in a large online trial of a Web-based intervention to reduce alcohol consumption . Participants were those in the DYD pilot trial eligible for 3-month follow-up (study 1) and those eligible for 12-month follow-up in the DYD main trial (study 2). Participants in both studies were randomly allocated to receive an offer of an incentive or to receive no offer of an incentive. In study 1, participants in the incentive arm were randomly offered a £5 Amazon.co.uk gift voucher, a £5 charity donation to Cancer Research UK, or entry in a prize draw for £250. In study 2, participants in the incentive arm were offered a £10 Amazon.co.uk gift voucher. The primary outcome was the proportion of participants who completed follow-up questionnaires in the incentive arm(s) compared with the no incentive arm.P < .001). The incremental cost per extra successful follow-up in the incentive arm was £110 in study 1 and £52 in study 2.In study 1 (n = 1226), there was no significant difference in response rates between those participants offered an incentive  and those with no offer  . There was no significant difference in response rates among the three different incentives offered. In study 2 (n = 2591), response rates were 9% higher in the group offered an incentive  than in the group not offered an incentive   Attrition from follow-up is a major methodological challenge in randomized trials, and the proportion of participants who provide follow-up data is a recognized quality marker ,2. Poor One approach to increasing response is the use of incentives, which has been effective at increasing response rates in surveys -9. IncenWith the paucity of empirical research in this area, there is a clear need to evaluate the impact of different incentive levels and types before using them to boost retention in online trials. Even relatively small incentives such as £5 can have an important impact on research budgets, particularly in online trials where large numbers of participants can be recruited reasonably easily . To deteThe primary hypothesis in both incentive studies was that offer of an incentive would increase the response rate compared with no offer of incentive. Secondary objectives were to determine the relative effectiveness of 3 different types of incentive (study 1 only), identify predictors of response to incentives, and calculate the cost of achieving an additional response.We conducted two randomized controlled trials. Ethical approval was obtained for both trials from University College London ethics committee.Both incentive studies were embedded in the Down Your Drink online trial of a Web-based intervention to reduce alcohol consumption ,13. The In both incentive studies, participants were already enrolled in the larger DYD study and, thus, were drinking above recommended levels of alcohol and were interested in reducing their drinking see . The firAim                                            To determine the effectiveness and cost-effectiveness of the Down Your Drink (DYD) website in reducing alcohol consumption.Design                                            A 2-arm randomized controlled trial. Participants were randomized to receive access to either an online behavior change program to help people reduce their alcohol consumption or an information-only website on the potential harms of alcohol.Methods                                            The trial was conducted entirely online through the DYD website . ParticiThe primary outcome was total past week alcohol consumption, measured by the TOT-AL . SecondaParticipant profile                                            The trial randomized 7935 people who had self-recruited to the trial. The trial recruited slightly more women than men (57%). The majority of participants were white British (84%), with a mean age of 38 years. Around half of the participants were educated to degree level and above (52%). Average alcohol consumption (geometric mean) was 46 (SD 31.2) units per week, where 1 UK unit = 8 g ethanol. Follow-up rates were 55% at one month and 42% at 3 months  and 46% at 3 months and 34% at 12 months .In view of the paucity of literature on incentives for this population, we undertook some preliminary research to identify a range of potentially effective incentives. This included identification of commonly used incentives in the survey literature, discussion with the DYD user representatives, and interviews with a convenience sample of hazardous drinkers demographically similar to the target audience. This preliminary work resulted in the choice of 3 incentives for initial study. Amazon is one of the most popular websites in the United Kingdom, with online shopping being a common use of the Internet . CharitaThe results of study 1 informed the decision on level and type of incentive in study 2, for which a £10 Amazon.co.uk voucher was chosen.In both studies, offer of an incentive was compared with no offer of incentive.In study 1, DYD pilot trial participants were emailed a request to provide follow-up data at 3 months . The email contained a hyperlink to the study questionnaires, stressed the importance of providing follow-up data, and conveyed our gratitude to participants for providing this information. Those participants who had not completed the outcome measures 1 week after the first email request were randomized to receiving an offer of an incentive or no offer of an incentive. Study 1 is thus restricted to those who did not respond to the initial request to provide follow-up data. Within the incentive arm, participants were also randomly allocated to receive either the £5 Amazon.co.uk voucher, £5 donation to Cancer Research UK, or entry in a £250 prize draw. Offer of an incentive was given in the second and third email prompts .Participants responding in each incentive arm were sent an email , which thanked them for their time and contained, as appropriate a unique Amazon.co.uk voucher code and instructions on how to claim; a hyperlink to the charity’s website, which detailed the amount donated to Cancer Research UK as a result of participants completing the questionnaires see ; and conThe results of study 1 informed the decision on level and type of incentive in study 2. In study 2, all DYD-RCT participants were randomized to receive either an offer of an incentive (£10 Amazon.co.uk voucher) or no incentive at the first request for data at the final (12-month) follow-up  . All parIn both studies, randomization was performed by a computer-generated randomization sequence that triggered automatic emails to participants. Hence, randomization could not be subverted by the study team, and allocation was thus fully concealed. Randomization was stratified by DYD experimental group (DYD intervention vs DYD comparator). The randomization function in Java was used to generate random assignment.In both studies, the outcome was the proportion of participants who responded, defined as completing the questionnaires within 40 days of the first email reminder after randomization. Additional data already obtained at entry into the DYD trial, including age, gender, baseline weekly alcohol consumption, and DYD experimental group (intervention or comparator) were used to explore possible variability in outcome.Data gathered for the economic analysis included the costs of developing the database for each study, researcher time in sending personalized emails, and costs of the incentives themselves.For both studies, the sample size was calculated to detect a 6% difference in response rates between incentive and no incentive arms with 90% power at 5% significance level. The response rate in the no incentive arm was assumed to be 11% for study 1 and (building on the results of study 1) 26% for study 2. This gave total sample size requirements of 1468 for study 1 and 2400 for study 2.The primary analysis compared response rates between the no incentive arm and incentive arm (3 incentive arms combined for the first study). For study 1, secondary analyses explored the differences between incentive types. Statistical significance was calculated using chi-square tests.Subgroup analyses were conducted for gender, age, and heavy drinking at baseline (> 35 units per week for women and > 50 units per week for men where 1 unit = 8 g ethanol). Interactions between these variables and allocation to incentive in affecting response rates were tested on a risk difference scale using the binreg command in STATA. The statistical analyses were undertaken by authors EK and IW in STATA version 10 .A simple economic analysis was conducted for both studies. It cost £822 to set up a database for the research for study 1 and £1180 for study 2. Identifying which participants completed the questionnaires and were, therefore, eligible to receive an incentive or not and sending emails to deliver the incentive took 10 minutes per 10 participants at a cost (including overheads) of £0.95 per minute. In practice, offering incentives would involve some but not all of these costs. For example, if all participants were offered an incentive, then some of the selection and computer programming time would be saved. As the purpose of the economic evaluation was to compare the additional costs of incentives compared with the control condition of no incentives, a reasonable estimate of the additional setup costs is 50% of the database costs plus an additional minute of researcher time per incentive offered. The final costs of the scheme are those of the incentive. The cost-effectiveness ratios were calculated as the additional cost per successful additional completed follow-up, that is, the total cost of offering incentives divided by the number of additional responses see . The ecoP = .37)  or offer of an incentive (n = 615) . The chaP = .37) . There wThe costs associated with offering incentives in study 1 are outlined in P < .001)  or offer of a £10 Amazon.co.uk voucher (n = 1296) . Charact < .001) . There wThe incremental cost per successful follow-up in the incentive arm was £52  .These trials provide a valuable contribution to the limited literature on the use of incentives for reducing attrition in online trials. Study 1 found that promising a low level incentive  had no significant impact on follow-up rates, whereas in study 2, a higher-level incentive (£10 Amazon.co.uk voucher) improved response rates by 9%. It should be borne in mind, however, that direct comparisons between the 2 studies are limited by differences in the study populations  and follow-up study time frames (3 and 12 months respectively). Notwithstanding these caveats, the higher incentive was also more cost-effective, in terms of costs per additional response. Researchers should, therefore, not assume that any level of incentive will necessarily improve follow-up rates.The types of incentives offered in study 1 were comparable to those shown to have a positive impact on improving response rates to postal and electronic surveys . HoweverAltruism is a commonly cited motive for trial participation -28, wherOur conclusions are strengthened by the large sample sizes employed, the randomized design, and the completeness of the data. We were also able to inform the design of the second study using the results from the first. The £5 Amazon voucher in study 1 resulted in the highest response rate of the three incentive types, although not higher to a statistically significant degree (test results not reported). So, in the second study, participants were randomized to a higher-level incentive (£10 Amazon voucher). Study 2 was undertaken in a population and setting that were similar to study 1, the main differences being that study 1 was conducted among pilot DYD trial participants at 3 months who had not responded 1 week after an email request for follow-up, whereas study 2 was conducted among all main DYD trial participants eligible for 12-month follow-up within a defined time period. Our novel context of online trials is important, since it is likely to be the vehicle for an increasing number of studies of delivering health care and health promotion in the future. A potential limitation of the first incentive study is that it failed to meet its planned sample size because the DYD pilot phase ended slightly earlier than anticipated (due to programming commitments necessary for the commencement of the main DYD-RCT). For this reason and because response rates in the control arm were higher than expected, the results of study 1 were somewhat inconclusive, with a confidence interval including both no difference and the 6% difference in response specified in the power calculation.This pair of studies has two important implications for researchers. Firstly, researchers should not assume that all levels of incentive would improve follow-up rates; instead, use of incentives for this purpose needs careful consideration and piloting of both level and type of incentive to be offered in a particular study population and setting. The second is that the costs of offering incentives can be substantial, and whether such costs are a good use of research funds needs to be considered. Further research that explores levels of different incentive types, offered and selected in different ways, and other means of reducing attrition in online trials should be prioritized if online health care delivery is to be well informed by strong research evidence.


Zimbabwe suffers from one of the greatest burdens of HIV/AIDS in the world that has been compounded by social and economic instability in the past decade. However, from 2001 to 2009 HIV prevalence among 15-49 year olds declined from 26% to approximately 14%. Behavior change and condom use may in part explain this decline.PSI-Zimbabwe socially markets the Protector Plus (P+) branded line of condoms. When Zimbabwe converted to a dollar-based economy in 2009, the price of condoms was greatly increased and new marketing efforts were undertaken. This paper evaluates the role of condom marketing, a multi-dimensional scale of brand peceptions (brand equity), and price in condom use behavior.We randomly sampled sexually active men age 15-49 from 3 groups - current P+ users, former users, and free condom users. We compared their brand equity and willingness to pay based on survey results. We estimated multivariable logistic regression models to compare the 3 groups.We found that the brand equity scale was positive correlated with willingness to pay and with condom use. Former users also indicated a high willingness to pay for condoms. We found differences in brand equity between the 3 groups, with current P+ users having the highest P+ brand equity. As observed in previous studies, higher brand equity was associated with more of the targeted health behavior, in this case and more consistent condom use.Zimbabwe men have highly positive brand perceptions of P+. There is an opportunity to grow the total condom market in Zimbabwe by increasing brand equity across user groups. Some former users may resume using condoms through more effective marketing. Some free users may be willing to pay for condoms. Achieving these objectives will expand the total condom market and reduce HIV risk behaviors. Zimbabwe suffers from one of the greatest burdens of HIV/AIDS in the world. Recent estimates from the Joint United Nations Program on HIV/AIDS (UNAIDS) indicate that approximately 1.3 million adults 15 years and older were living with HIV/AIDS in 2007 [Compounding the effects of HIV/AIDS, from 2001 to 2009, Zimbabwe's economy was characterized by hyperinflation and rapid currency devaluation, with associated deterioration in infrastructure, services, employment and purchasing power . ConcurrIn this context, social marketing of condoms as an important strategy to combat the spread of HIV/AIDS faces significant hurdles. Despite the severity of the epidemic, prevalence rates in Zimbabwe have shown signs of decline, from 26.0 percent prevalence among adults ages 15 to 49 in 2001 to 15.3 percent prevalence in the same age group in 2007 , and then to 14.2% in 2009 [http://www.psi.org is a non-governmental organization (NGO) that socially markets disease prevention and health promotion products and services. To promote safe sexual behavior, PSI-Zimbabwe markets the Protector Plus (P+) branded line of condoms. At the time of dollarization in late 2009, P+ was sold at the equivalent of less than $.01USD, representing a virtually free commodity. While such a low price makes the P+ condom highly affordable, it may lead to inefficient allocation of social marketing and public sector resources by excluding commercial products from the marketplace, offering little or no profit incentive for retailers, risking wastage in the distribution system, and smuggling of the product to other countries with higher prices [PSI r prices . Moreover prices .P+ are the following: 1) Distribution of an affordable brand of condoms in traditional and non-traditional outlets, and those located in areas where high risk behavior takes place ; 2) Promotion of condom use among groups at high risk and sexually active youth; 3) Developing behavior change communication programs that address the dynamics of high-risk behavior among target groups.PSI-Zimbabwe's objectives in marketing P+ brand is to grow the total market by increasing condom use among sexually active men and women. The brand therefore has a very important role in helping destigmatize condom use and to making it something "everybody is doing" and "everyone is talking about" because of its different presentations and characteristics. Brand equity represents the value that the brand holds for consumers. PSI-Zimbabwe's goal is to measure current brand equity in its P+ lines, actively manage brand equity, and increase equity through future brand repositioning, marketing, and management.The role of the P+ condoms to 10 cents in November 2009 (prices noted throughout are US currency). Immediately following this price increase, as with previous price increases, there was an observed steep decline in sales, from over 5 million units sold in October 2009 to just over 2 million units sold in November 2009. There was a rebound in sales to over 3 million in December 2009, but the question facing PSI-Zimbabwe faced was about the current brand equity of P+ and how the brand should be actively managed at the new price and in a changing overall marketplace.PSI-Zimbabwe increased the price of An in-depth audience profile was created to provide an illustrative description of the target audience to inform the intervention's communications strategy and activity planning. An 'archetype' of the targeted condom consumer was developed as a guide to the brand marketing. 'Mike' is a 25 year old single man living in with his extended family in Chitungwiza . He is an informal trader with secondary education and makes an average of $80 per month. He owns and listens to the radio and rides a kombi (shared taxi) to and from his trading place in the city centre. Mike dreams of having a steady girlfriend and providing for his extended family's material needs. He drinks opaque beer and occasionally clear beer whilst hanging out with his numerous friends at the shopping centre during weekends. He worries about HIV infection so he openly discusses condoms with his friends and they encourage each other to use condoms although Mike does not use condoms consistently with his partner.The P+ brand is positioned as follows to reach its audience: "For Mike, Protector Plus is the condom that gives him control of a demanding lifestyle by reducing the number of things he has to be concerned about." Marketing efforts seek to communicate two key brand attributes to the audience: 1) Protector Plus is easy to use, strong, reliable, and effective and gives me control to enjoy life - Always use Protector Plus (brand promise); 2) I am confident I can use Protector Plus correctly and consistently with my partner - Use Protector Plus every time (self-efficacy).P+ marketing efforts seek to: 1) Increase the percentage of men ages 20 -30 who believe Protector Plus condoms are reliable, strong and effective; and 2) Increase the percentage of men ages 20 -30 who are capable of using condoms correctly and consistently with all partners.Additionally, marketing efforts aim to increase positive brand perceptions and self-efficacy to use condoms among young men in the P+ target audience . The theassociations they represent, and the resulting behavior  they can engender. In this respect, brands are very much like reputations - they precede the individual or organization and shape how the world responds.Brands have been defined as "a set of associations linked to a name, mark, or symbol associated with a product or service" . Thus thBrand equity is what the brand stands for in the hearts and minds of consumers, and as a metric it captures their identification with and intentions to purchase, use, and engage with the brand ,11. It iBrand equity has been previously studied as a mediator, or mechanism by which marketing efforts promote product use and behavior change . Social P+ and other in-market brands? 2) What differences are there in P+ and other in-market brand equity between specific audience segments and brand attributes? 3) Is higher brand equity in P+ and other in-market brands associated with willingness to pay higher prices for condoms overall or for specific brands? The long-term study objective is to establish a baseline of brand equity for future P+ and condom use monitoring and evaluation studies. In this research, we seek to identify opportunities to improve P+ brand equity by addressing specific audience segments and brand attributes. We evaluate the effect of the price increase and examine the relationship between product price points and brand equity. In this way, social marketers can consider whether former P+ users who have switched or lapsed from condom use would be willing to use condoms or P+ again. Study results also inform decisions about how to grow the total condom market in Zimbabwe by establishing appropriate prices for socially marketed products that would permit commercial brands to enter the market in the future.We asked the following specific research questions: 1) What is the current brand equity in We conducted interviews with 890 Zimbabwean men in November and December 2010. Study participants were recruited by simple random selection and were a nationally representative sample of Zimbabwean men aged 18-49 residing in rural and urban areas. The sample was stratified to include men who reported having used P+ condoms within the past 3 months (defined as current P+ users), men who had used P+ condoms within the past year but not in the past 3 months (defined as lapsed users), and men who had used public sector condoms in the past 3 months but not the P+ brand.Additionally, the study included a qualitative study arm in which 30 Zimbabwean men meeting the same recruitment criteria and not included in the quantitative study arm were interviewed in an open-ended, in-depth interview (IDI) format. The IDI form included the same topics as the questionnaire. Results of the qualitative arm will be reported in a future publication.For the quantitative arm of the study a two stage cluster sampling strategy was utilized to select eligible participants. The first stage involved selection of Enumeration areas (EAs) from a list of EAs from the 2002 Zimbabwe national population census. Selection of EAs was dependent upon the size of the EA. A list of households for each of the selected EAs was compiled and became the basis for selecting households. A random selection procedure was employed to sample households for study recruitment. Trained interviewers screened participants following a study recruitment criteria. In the selected households, a Kish grid was used to select one participant in cases where the number of eligible respondents was more than one. After providing informed consent individually, participants completed a face-to-face interview following a structured, 45-item questionnaire. The questionnaire was programmed on a personal digital assistant (PDA) device and the interviewer entered responses directly into the PDA data form. The questionnaire took approximately 20 minutes to administer and a total of 890 individual interviews were completed for this study.P+ current users, public sector users, lapsed P+ users, and former users. The IDIs explored the same topics examined in the survey, but in the form of open-ended questions with probes and opportunities to explore the participants' reasons for their indicated level of brand equity and other measures. The sample was chosen based on previous experience in similar studies. A total of 30 in-depth interviews were conducted.The qualitative arm of the study comprised in-depth interviews (IDIs) with randomly selected 7 to 8 participants among We developed and fielded an interviewer-administered, 45-item questionnaire that included items on participant socio-demographics, media use, exposure to condom brand marketing, brand equity, willingness to pay for condoms, and use of specific condom brands. The following are the brand equity scales. Each item was asked on a 4-point scale from 'strongly agree' to 'strongly disagree' with a 'don't know' option. Each scale was asked for each of the brands evaluated in this study (P+ and public sector) for all respondents across the 3 sample strata.1) I will use this brand the next time I use a condom; 2) I would wear a baseball cap with this brand name and logo on it; 3) I would recommend my friends use this brand; 4) This is the best brand of condoms; 5) This brand has the best condom advertisements; 6) I would recommend my friends use this brand.1) This brand will not break during sex; 2) This brand gives a pleasant sensation during sex; 3) This brand does not smell like latex; 4) This brand has nice colors; 5) This brand feels good to the touch; 6) This brand is not too thick; 7) This brand is there when I need it; 8) This brand is available at an outlet near me; 9) This brand has sufficient lubricant.1) P+ is becoming more popular with people like me; 2) P+ is for people like me; 3) P+ is the leading brand of condoms.1) There are good reasons to buy this brand instead of other condom brands; 2) This brand is a good value for the price; 3) This brand is a better value than other condoms.1) People who use this brand are strong; 2) People who use this brand are confident; 3) People who use this brand have a lot of freedom; 4) People who use this brand have fun; 5) People who use this brand find it easy to have girlfriends; 6) People who use this brand are just like me; 7) People who use this brand are like the people that I hang out with.1) This brand costs more than I would expect; 2) I would be willing to pay more than the current price for this brand; 3) If the price were reduced by 3 cents, I would buy this brand; 4) If the price were reduced by 6 cents, I would buy this brand.Additionally, we asked participants how much they would be willing to pay for P+ brand condoms. We asked whether they would be willing to buy P+ at 6 levels of price including the actual price (10 cents) and ranging from free to 15 cents per pack of 3 condoms. We examined the relationship between brand equity and willingness to pay.We also asked about awareness of brands available for sale and free in Zimbabwe. The multivariable regression analyses reported below included 4 co-variates: age, education, marriage status, and socio-economic status. Age was self-reported in years. Education was self-reported by category with 'completed university' as highest. Marriage status included all potential (co-)habitation status. Socio-economic status (SES) was measured by a list of household amenities which in combination would be indicative of wealth relative to the local economy.Stata version 11  was used for all statistical analyses. Survey Participants were grouped into three categories - Current P+ User, Lapsed P+ User, and Public Sector User, based on recent and historic condom brand use. Descriptive analyses on socio-demographic and brand pricing points were performed to assess overall brand dynamics. Additionally, opinions on statements related to brand equity, structured by commonly-used categories, were then dichotomized (agree/disagree). Outcomes of brand awareness, satisfaction/loyalty, perceived quality, leadership/popularity, perceived value, brand personality, and market barrier to acceptance were described and compared between the three groups.Using confirmatory factor analysis procedures, factor loadings for variable in each brand equity construct were assessed, assuring adequate (>.5) individual factor loading and high overall (Chronbach's Alpha >0.6) inclusion between both questions asked concerning Protector Plus and public sector condoms. These thresholds for factor inclusion were chosen following the widely used Comrey and Lee (1992) criteria . After aUsing brand pricing point data, additional logistical regression analyses were performed to assess brand equity perceptions among those willing to pay more (or less) for either Protector Plus or public sector condoms.Table We conducted confirmatory factor analysis (CFA) for the brand equity scales. As found in previous studies the 5 brand equity scales - satisfaction/loyalty, quality, leadership/popularity, value and personality - each represented a single factor. Because we expected different reactions to the P+ and public sector brands, we ran separate CFA for the 2 sample strata. In each analysis, all factor loadings and Chronbach alpha statistics were high and above threshold for acceptable scales . The lowTable p < .10). These users have lower perceptions of popularity of public sector brand and higher associations with personality of the public sector. Thus they represent a group of individuals who might be convinced to switch back to using P+ given the right marketing and incentives.Table Table Overall, P+ currently has a strong brand equity and market position in the Zimbabwe condom market, with market share of 48% at the time of our study Declines in HIV rates reported in recent years may in part be due to changes in behavior including increased use of condoms ,2. HowevOne important implication of this research is that there are opportunities to grow the total market by further building up equity in the P+ brand ,12. We oOne long-term goal of the Total Market Approach is to create opportunities for private sector products to replace socially marketed and freely available condoms over time as economic conditions and consumer demand allow . It may While brand equity in P+ was much lower among public sector users than among current P+ users and lapsed users, levels of brand equity in P+ were still substantial among this group. Over 60% of public sector users self-reported awareness of P+, and the brand leadership/popularity factor differed by only 3 percentage points (45.3% v. 42%) between P+ and the public sector brand among these users  that some users who are currently not paying for condoms (lapsed P+ and public sector) are willing to pay, and 2) some current P+ users are willing to pay more for the brand than its current price of 10 cents for a pack of 3. Higher brand equity, especially the loyalty, quality, and value factors, is associated with higher willingness to pay for condoms among public sector and P+ users.Thus price does not appear to be a major barrier to condom use in Zimbabwe. Rather it is perceptions of the condom product, as measured by brand equity, that appear to be a greater driver of preferences for condom use. As noted in Table P+ as a brand, and condom use as a category of health behavior, appears positioned to grow in Zimbabwe. Clearly future economic conditions, resolution of outstanding political turmoil, and public perceptions of condoms as a protective health behavior and of the P+ brand of condoms will influence future growth in the market. Continued brand research is needed to evaluate changes in brand equity. The question remains how NGOs such as PSI-Zimbabwe should actively manage their condoms brands to grow the total market for condoms.The authors declare that they have no competing interests.WDE conceptualized the study, developed the analysis plan, supervised analysis, and wrote sections of the paper. NT contributed to the study design and analysis plan and edited the entire paper. KL edited the entire paper. JS conducted statistical analysis and wrote sections of the paper. All authors read and approved the final manuscript.


Annexins are a family of calcium- and phospholipid-binding proteins found in nearly all eukaryotes. They are structurally highly conserved and have been implicated in a wide range of cellular activities. In this paper, we focus on Annexin A2 (AnxA2). Altered expression of this protein has been identified in a wide variety of cancers, has also been found on the HIV particle, and has been implicated in the maturation of the virus. Recently, it has also been shown to have an important role in the establishment of normal apical polarity in epithelial cells. We synthesize here the known biochemical properties of this protein and the extensive literature concerning its involvement in the endocytic pathway. We stress the importance of AnxA2 as a platform for actin remodeling in the vicinity of dynamic cellular membranes, in the hope that this may shed light on the normal functions of the protein and its contribution to disease. A number of reviews have already been written on this large family of proteins. For the sake of brevity, we can only cursorily describe these roles. The annexins are found in all multicellular and some single-cell eukaryotes. They are a highly conserved family of calcium and phospholipid-binding proteins usually comprising four repeats of the characteristic alpha-helical-rich endonexin fold and an N-terminal domain that is unique to each annexin –5, FigurIn different phyla, they have evolved to perform a variety of cellular functions but all retain a set of core properties –7. They  In the most general terms, they have been implicated in calcium sensing and homeostasis –10, pH s Changes in their expression levels have been implicated in a number of human diseases, loosely termed “annexinopathies” (reviewed in –24). In  In this paper, we shall concentrate on Annexin A2 (AnxA2), which, whilst being an archetypal annexin, has a number of distinct properties rendering it particularly apposite to regulate actin-associated cellular processes at dynamic membranes.Importantly, of all the annexins, altered expression of AnxA2 is most correlated with disease progression. The protein has been found on the HIV particle and has been implicated in the maturation of the virus , 30. FurμM calcium to drive membrane association) [AnxA2 differs from other annexins in that its association to membranes is most sensitive to increasing calcium levels (only requiring 0.05 ciation)  and struciation) –36, has ciation)  and is fciation) . Ionophociation) , 39.in vitro, AnxA2 seems to have affinity for most anionic phospholipids and displays a calcium-enhanced affinity for phosphatidylinositol 4,5-bisphosphate P2) [AnxA2 has been shown to favour cholesterol-rich subdomains of membranes , suggestP2) –44, a liAccordingly, in cells lacking the inositol polyphosphate 5′-phosphatase OCRL1 P2, but also acts upon PIP3), AnxA2 is recruited to vesicular structures that contain presumed PIP2 accumulations , 45, 46.This observation demonstrates the properties we believe AnxA2 is critical for, that is, the remodeling of actin driving membrane rearrangements.μM calcium) and gives rise to a 1 : 1.9 stoichiometry of annexin bound to actin. Interestingly, the bundling activity of the tetrameric complex was found to be decreased by phosphorylation of the protein on Tyr23, the site favoured by the tyrosine kinase Src, suggesting a possible role for Src in the association of AnxA2 with actin [The ability of the AnxA2 and the AnxA2-S100A10 tetrameric complex to bind and bundle filamentous actin (F-actin) was first identified using proteins purified from the brush-borders of porcine epithelial cells  and fromth actin .in vitro, the AnxA2 monomer alone (without associated S100A10) is able to “cap” the fast-growing “barbed” end of the growing actin filament and possibly also interacts directly with G-actin monomers. Thus, AnxA2 monomer not only has the capacity to bundle preformed filaments, but may regulate the growth of newly forming ones [At micromolar calcium concentrations ing ones .in vivo, AnxA2 localises to regions of free-barbed ends (identified with labeled monomeric actin units) [Whilst the protein may perform this bundling and bridging function we suspect it is not its primary function. Accordingly, AnxA2 is not usually seen “coating” large actin bundles, such as stress-fibres and filopodia, as would be expected for a conventional actin bundling protein. Instead, AnxA2 is likely to have a more precise role in regulating actin cytoskeletal rearrangements. Consistently, n units) .Taken together, we propose that AnxA2 functions at sites of actin association with membranes enriched in cholesterol and/or phosphoinositides. Supporting this proposal, AnxA2 has been shown to have roles in many steps of membrane trafficking and cell polarity. This is reviewed below also see .A number of annexins have been implicated in vesicular trafficking events . A partide novo actin polymerization). It may partially restrict the spread and growth of the actin on the comet, to optimize its motility (capping adventitious filaments as they spread from the sides of the actin tail). Alternatively, it may act as a bridge, physically linking the actin to the membrane  or as aAnxA2 has also been implicated in the phagocytic process. It accumulates on actin-rich phagosomes produced when outer segments (the effete part of the photoreceptor) are internalized by retinal pigmented epithelial cells, and are lost from the phagosome at the same time as the actin which drives internalization. During phagocytosis, AnxA2 is tyrosine phosphorylated, possibly by Src that is also recruited to the phagocytic cup, suggesting a role for Src-mediated AnxA2 phosphorylation in regulation of the process. Accordingly, depletion of AnxA2 from these cells resulted in slower kinetics of phagocytosis in tissue culture, and AnxA2 knock-out mice show similarly altered kinetics of phagosome degradation . Recentlμ2 subunit of the AP-2 clathrin adaptor complex P2 [Annexins have also been shown to be involved in the first step of endocytosis, that is, the formation and pinching off of clathrin-coated vesicles (CCVs). AnxA2 and AnxA6 were first reported on CCVs in the adrenal gland . Associacreening ). AP2 isIP2 , we propIP2 , suggestThe first evidence that AnxA2 had a broader role in the endocytic pathway comes from observations by Harder and Gerke and Jost et al., , 64 idenAnxA2 association with early endosomes is suggested to be predominantly mediated by its association with cholesterol-rich domains . Indeed, Binding of AnxA2 to endosomes influences their localization. When a chimeric AnxA2/S100A10 fusion protein is introduced into cells (which causes aggregation of AnxA2), early endosomes are also aggregated. Depleting Anx2A also leads to a dramatic change in the general distribution and morphology of early and recycling endosomes that collapse into a perinuclear domain. Furthermore, the tubules of these endosomes become tortuous and bent , 64.Both early and late endosomes have been shown to be actin positive, and AnxA2 has been implicated in regulating this actin . It is tThe first indication that AnxA2 was involved in endosomal function came from studies using endosome fusion assays. Indeed, a subtle reduction in endosome fusion mediated by AnxA2-free cytoplasm was noted that was rescued by addition of recombinant protein. Furthermore, HeLa cells depleted of AnxA2 by RNA interference show reduced transfer of a fluid-phase marker of endocytosis and of EGF, to the lysosome, but not internalized LDL , suggestConversely, recycling of transferrin receptor is unaffected by AnxA2 loss, suggesting that the protein is involved specifically in maturation of late endosomes .Failure of late endosome maturation could be rescued by a pseudotyrosine-phosphorylated AnxA2 construct but not by a mutant in which the phosphorylation site was mutated . This stIn this respect, Morel et al., , 69 recein vivo. Depletion of AnxA2 resulted in reduced formation of APPL-positive endosomes and solubilisation of APPL2 protein. This suggests that AnxA2 plays a key role in the identity and/or formation of APPL-endosomes and warrants further investigation.On a speculative note, Anx2 could also have a role in early endosomal conversion. By this, we refer to the existence of a pool of APPL/Rab5-positive but EEA1/PI3P-negative early endosomes that acquire PI3P and EEA1. APPL endosomes are derived from both macropinosomes and a subset of vesicles formed by clathrin-mediated endocytosis and represent a very early stage in the endocytic cycle, lying upstream of the canonical PI(3)P-positive endosomes –72. The Interestingly, APPL proteins physically interact with OCRL1, which is recruited to the CCV after the scission event . This suThe second critical function for AnxA2 that engages both its biochemical properties is exemplified by its role in the establishment of epithelial polarity. In response to signals from the external environment, epithelial cells polarize their membrane domains into distinct apical and basolateral domains, separated by intercellular contacts such as adherens and tight junctions. AnxA2 is able to recruit and potentially activate Rho GTPases as well as key polarity complexes which act in concert to orchestrate the actin cytoskeletal rearrangements required for epithelial polarity. Although some links are yet to be clarified, it is apparent that AnxA2 is a central player in the genesis of epithelial cell polarity.Cell-cell adhesion is strongly regulated by the activities of Rho GTPases and their effects in regulation of the actin cytoskeleton , 75. ThiOn one hand, AnxA2 recruits Rho to the plasma membrane, for instance in wound closure assays in Caco-2 epithelial cells which facilitates cell motility/migration, a process antagonistic to cell junction formation. Correspondingly, in cells in which AnxA2 is depleted, Rho recruitment to lamellipods is decreased and cells fail to migrate .On the other hand, during the initial stages of cell-cell adhesion, another Rho GTPase, Rac1, becomes localized to nascent junctions, driven by an interaction with E-cadherin . InteresAnxA2 has also been shown to regulate the actin cytoskeleton via its interaction with the scaffold protein, AHNAK  , 80 whicA second critical role for AnxA2 in cell polarity is in the formation of E-cadherin-based adhesions downstream of Nectin-1. In a so-called “calcium switch”-experiment in which cell-cell contacts are disassembled and then can be observed as they are rebuilt over time, rerecruitment of E-cadherin to junctions fails in cells depleted of AnxA2. Despite the lack of E-cadherin targeting, the tight junction still is formed in these cells . This clThe role of AnxA2 is not limited to epithelial, but also endothelial cells. AnxA2 has been shown to play a key role in the structure of endothelial adherens junctions and associates with VE-cadherin , 83. DepAs described above, AnxA2 functions as an initiator of actin remodeling events and does so through the integration of spatiotemporal modulations of phosphoinositides and the recruitment and/or activation of Rho GTPases. In addition, use of 3D MDCK cysts reveal key findings that depletion of AnxA2 or overexpression of dominant negative AnxA2 prevents the formation of the central lumen . AnxA2 iRab11a recycling endosomes are now recognized as a key intermediate in the trafficking of junctional proteins, such as E-cadherin, to the plasma membrane –87. AnxAIn addition to recycling of apical markers, AnxA2 plays a key role at polarized plasma membrane domains. During the formation of 3D MDCK cysts, prior to lumenogenesis, PIP3 is enriched at the plasma membrane, relative to PIP2 , 89. At Vital to the formation of a lumen is the activity of the inositol lipid 3′-phosphatase PTEN, which is recruited to the presumptive apical surface where it generates PIP2 which AnxA2 binds to with a higher affinity. Upon generation of PIP2 at apical membranes by PTEN, AnxA2 shifts its localization to sites at which its preferred substrate is concentrated .AnxA2 binding to PIP2 at the apical surface initiates a cascade of events vital to epithelial morphogenesis. For instance, AnxA2 is proposed to recruit another Rho-subfamily member, Cdc42, and the key polarity proteins aPKC and Par6 to the nascent apical surface , 88. Although it seems most likely that AnxA2 functions to scaffold active Cdc42 and nucleate actin remodeling, it is also highly possible that the formation of the apical surface requires the fusion of small vesicles enriched with AnxA2. Interestingly, Cdc42 is required for TGN exit of basolateral proteins  and AnxANot only AnxA2 is expressed at particularly high levels in epithelia and endothelia intracellularly, but it also appears as an extracellular, cell-surface-associated pool in complex with S100A10. How the tetrameric complex exits cells to appear on the outer leaflet has also been a subject of much discussion. As a protein lacking a signal peptide, AnxA2 is one of a growing list of unconventionally secreted proteins –98, whicWhichever the means of cell egress, extracellular AnxA2 has been implicated in in membrane bridging , 103 andTetrameric AnxA2 complex colocalises with tight junction markers along the lateral plasma membrane and a pool of this protein was shown to be extracellular. Addition of an inhibitory peptide (aimed at interrupting the AnxA2/S100A10 complex formation) across a calcium switch experiment blocked tight junction reformation, consistent with the idea that extracellular AnxA2 plays an important role in remodeling of tight junctions .Phosphorylation of AnxA2 on Tyr23 leads to its increased recruitment to the plasma membrane and subsequently to sites of actin remodeling . Given tin vitro and in vivo at physiologically significant calcium concentrations to an affinity for PIP2-, PIP3- and/or cholesterol-containing membranes and the ability to cap the fast-growing ends of actin filaments. These are subtle but important refinements because they change the role of the protein from being that of a structural “contaminant” of membrane and cytoskeletal preparations to being critical in establishing a specific subset of membrane-actin interfaces. It may be the case that the annexin itself is sufficient to form this structure but it is likely that in vivo a number of annexin/actin/PIP2-associated proteins ultimately bulwark the membrane/actin interface.The principal biochemical properties of AnxA2 are calcium-dependent association with phospholipids and actin. These properties have now been refined both  In this paper, we have outlined the predominant role of AnxA2 in events that occur in the vicinity of the plasma membrane including clathrin-mediated budding, formation of macropinosomes and phagosomes, conversion of early endosomes and maturation of early-to-late endosomes . The potential role for AnxA2 at recycling endosome events and during endosome-maturation is less well established and may be a cholesterol-mediated function or occur under specific conditions, such as when the protein is hyper-phosphorylated, for example during Src-mediated transformation.AnxA2 may mediate these events P2 and calcium) either as a result of its inherent biological activity or indirectly through its Almost all these processes occur concomitantly with significant actin remodeling. This may be necessary to drive vesicle motility or to ease endosome movement through dense cortical actin filaments. However, the importance of short bursts of actin polymerization during vesicle fusion and fission is controversial and is less well understood with regard to the maintenance of endosomal structure. In the formation and motility of actin rockets and macropinosomes and in 3D epithelial polarity, both lipid and actin binding properties of AnxA2 have been implicated. In other events linked to AnxA2, only one of its biochemical properties has so far been involved. For instance, in our proposed model of early endosome transition, processes of CCV formation, and granule secretion, there is clearly a role for PIP2 dynamics; but a role for actin is less well established. Conversely, the maturation of early-to-late endosomes requires AnxA2-mediated actin polymerization, but a role for PIP2 regulation of AnxA2 in this process is still to be demonstrated. There is clearly room for further investigations of the link between the two biochemical properties of AnxA2 in these processes. AnxA2 is highly abundant in those cells in which it is expressed; but it is not ubiquitous. For this reason, it cannot be critical in all cell types for all the processes we have described in this paper. This may be partly explained by the potential redundancy between annexin family members that due to space limitation, we have not been able to discuss. The Annexin family is highly conserved and many of the other members, in spite of having unique sequences  have shared biochemical activities with AnxA2. Individual lipid/calcium/actin-binding affinities and specificities may vary, but the potential for overlap is clear. Indeed, in many cases, they have been implicated in related processes and may form oligomeric complexes; though as yet overexpression of one family member has not been shown to compensate for loss of another. This may explain the viability and fertility of the AnxA2 knockout mouse. Notwithstanding homeostatic alterations in annexin or other gene expression which may compensate for genetic lesion, the mouse does seem surprisingly healthy given the number of processes in which AnxA2 has been implicated. It appears that the major defects in the mouse are not in normal development; but rather in subsequent plastic changes: the mouse seems to be incapable of sustaining neovascularisation, for example, which may make AnxA2 a promising target for drug intervention in cancer treatment. Inherent redundancies in endocytosis, phagocytosis, and junction formation may mean that AnxA2 phenotypes are not as clear in the developing animal as they seem to be in culture. The AnxA2 mouse is also effectively an S100A10 knockout animal as AnxA2 serves to prevent S100A10 degradation in the cytoplasm. Overexpression of AnxA2 (as is seen in some cancers) or nonstoichiometric changes in the relative concentrations of AnxA2 and S100A10 may have more severe phenotypes than loss of both.There are still a number of important gaps in the AnxA2 story, such as a lack of AnxA2 crystal structure in complex with either actin or PIP2 or how phosphorylation of the protein  or PIP2 binding precisely affects its actin remodeling activity and membrane dynamics. We can hope that further research will throw more light on the role of this vital cellular component.As a final note, alterations in AnxA2 expression or activity have been demonstrated in many different cancers, particularly those of the prostate, the breast, the liver, the lung (there are too many references to cite meaningfully here), and in the formation of infective HIV particles , 30. Exa


Cigarette smoking remains a significant health threat for smokers and nonsmokers alike. Secondhand smoke (SHS) is intrinsically more toxic than directly inhaled smoke. Recently, a new threat has been discovered – Thirdhand smoke (THS) – the accumulation of SHS on surfaces that ages with time, becoming progressively more toxic. THS is a potential health threat to children, spouses of smokers and workers in environments where smoking is or has been allowed. The goal of this study is to investigate the effects of THS on liver, lung, skin healing, and behavior, using an animal model exposed to THS under conditions that mimic exposure of humans. THS-exposed mice show alterations in multiple organ systems and excrete levels of NNAL (a tobacco-specific carcinogen biomarker) similar to those found in children exposed to SHS (and consequently to THS). In liver, THS leads to increased lipid levels and non-alcoholic fatty liver disease, a precursor to cirrhosis and cancer and a potential contributor to cardiovascular disease. In lung, THS stimulates excess collagen production and high levels of inflammatory cytokines, suggesting propensity for fibrosis with implications for inflammation-induced diseases such as chronic obstructive pulmonary disease and asthma. In wounded skin, healing in THS-exposed mice has many characteristics of the poor healing of surgical incisions observed in human smokers. Lastly, behavioral tests show that THS-exposed mice become hyperactive. The latter data, combined with emerging associated behavioral problems in children exposed to SHS/THS, suggest that, with prolonged exposure, they may be at significant risk for developing more severe neurological disorders. These results provide a basis for studies on the toxic effects of THS in humans and inform potential regulatory policies to prevent involuntary exposure to THS. Despite efforts by governments and health organizations worldwide, cigarette smoking remains a serious health threat for smokers and nonsmokers alike The first complete ban in the world on indoor smoking in all public spaces (including bars and restaurants) occurred in 1990 in San Luis Obispo, CA. That legislation, and its expansion to many countries, was achieved only because of clear scientific evidence that SHS is dangerous to non-smokers. Now, more than 20 years later, evidence is emerging that THS exposure potentially poses similar health risks, especially for children. Several studies have affirmed THS as an underappreciated public health hazard 2 include: a) smoking; b) combustion sources such as improperly ventilated gas stoves and heaters and c) infiltration of outdoor air pollution generated by vehicle exhaust or biomass burning Contamination of the homes of smokers by SHS residues (THS) is high, both on surfaces and in dust, including in children's bedrooms The exposure to tobacco smoke toxicants in THS can occur via ingestion, skin adsorption/absorption and/or inhalation Although the potential risks attributed to THS exposure are increasing, virtually nothing is known about the specific health implications of acute or cumulative exposure. Therefore, there is a critical need for animal experiments to evaluate biological effects of THS-exposure that will inform subsequent human epidemiological and clinical trials. Such studies can determine potential human health risks, design of clinical trials and potentially can contribute to policies that lead to reduction in both exposure and disease.To address this need, we have conducted the first animal study on the effects of THS on several organ systems under conditions that simulate THS exposure of humans. We show that significant damage occurs in liver, lung and during healing of wounds. In addition, the mice display hyperactivity. The results of our study delineate the early stages of potentially serious THS-induced damage in each of these organs and in behavior, problems that are known to be associated in humans with THS and SHS but have not previously been identified in humans exposed to THS.3, a value that falls within the range detected by the Environmental Protection Agency (EPA) in the homes of smokers (15–35 µg/m3) We have developed a mouse model for THS exposure that approximates that of children and others in environments contaminated by THS . Materiain vitro showed that THS extracts and NNA itself are genotoxic to human cell lines Direct comparison between biomarkers in our mice and in humans is difficult because population studies concerning THS-exposure in humans are just beginning. Moreover, these comparisons are difficult to make because humans do not always comply with the needed experimental constraints. Some of the components of SHS undergo chemical reaction with the indoor air to produce additional toxicants, at least some of which can be highly carcinogenic To obtain a measure of the exposure to THS, we have measured NNAL (4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol), the principal metabolite of NNK. We chose to use nitrosamines to compare between humans and mice because the half-life of the nitrosamines (days) is much longer than that of cotinine (hours) and therefore makes the comparison between our well-controlled experiments and not-as-well-controlled human data collection more reliable not genetically altered) are placed in standard, well-ventilated, mouse cages containing SHS-exposed materials. The animals are not constrained; they can move about the cage normally and the cages are kept in a large room with normal ventilation. Therefore, the environmental conditions also closely mimic those of THS exposure of children in the homes of smokers. These mice live in this environment for 6 months; materials exposed to THS are refreshed periodically as explained in the At age 3 weeks, just-weaned wild-type mice , giving the liver a pale red color compared to the deep red in normal liver  at doses that normally would not be damaging. This is of particular concern for children because they are frequently treated with acetaminophen for fever and pain.A related important function of liver is regulation of insulin metabolism. We find that THS-exposed animals have fasting glucose levels that indicate they are pre-diabetic Fig. 2A In lung, we observe that in the region of the alveolar sacs in THS-exposed mice the walls of the alveoli are disrupted significantly more often than in the controls . Moreover, the expression of genes that are important in the inflammatory response and response to wounding is decreased . In the healing tissue, the level of fibrillar collagen (blue) is greatly decreased in THS-exposed animals . Time spent in the closed arms represents anxious behavior; rodents generally spend much more time in the closed arms. THS-exposed mice and controls display the same level of anxious behavior, as illustrated by their similar preference for closed over open arms of the maze . Individual mice were placed in the Open Field; walking, stationary and rearing behaviors were assessed, as well as the frequency of transition from one of these behaviors to another. THS-exposed mice spent significantly more time walking, much less time standing still and more time rearing than control mice (Video S1) and the other of a THS-exposed mouse during the first 10 minutes of the videotaped hour (Video S2). These movies are sped up by a factor of two to better illustrate the differences in activity during this period  and the last 10 minutes of the hour in which the control mice showed on average little activity whereas the THS-exposed mice remained very active  harm nonsmokers but that there is such an effect is now clear. Our studies in mice that are never exposed to smoke itself but are exposed to residues of the smoke, strongly implicate tobacco smoke residues in these pathologies. It follows that children in environments where smoking is, or has been allowed, are at significant risk for suffering from multiple short-term and longer health problems, many of which may not manifest fully until later in life.C57BL/6 mice were divided into control and experimental groups. The experimental group was exposed to THS from right after weaning to 24 weeks; the control group was never exposed to THS. All mice were fed a standard chow diet .Animal experimental protocols were approved by the University of California, Riverside, Institutional Animal Care and Use Committee (IACUC). The animal use protocol is A-2008024.2 pieces of carpet (fiber) to maintain equal exposure levels across experimental groups. Two packs of 3R4F research cigarettes were smoked each day, 5days/week and smoke was routed to a mixing compartment and distributed between two exposure chambers, each containing 8 cages with the materials. We use the gravimetric method to determine the particulate concentrations. Whatman grade 40 quantitative cellulose filter papers are first weighed, then introduced into the filtering device and, after running the test for 15 mins, the filter is weighed again to determine the particulate mass that has accumulated during this time. This procedure is repeated with 2 more filters and the average of the 3 masses calculated gives the TPM values for each chamber. All cigarettes were smoked and stored in accordance with the Federal Trade Commission (FTC) smoking regimen Using the Teague smoking apparatus Control animals were tested for behavior and wound healing in a pilot study to look for possible changes related to living in the (non-exposed) materials of this project. No significant differences were observed in relation to animals living in normal conditions in the vivarium. During these experiments, in all cases the cages for THS-exposed and control mice were the same, food was the same, temperature the same, etc. Hair was not removed in this particular set of experiments but we have removed the hair in control mice in many other studies of dermal exposure to tobacco chemicals and found no differences between controls with and without removed hair.2O) to visualize lipid or stained with Diff-Quick to visualize the structure of the tissue. Lung and skin sections were stained with Diff-quick for structure, Masson-trichrome for fibrillar collagen and examined by second harmonic generation imaging (SHIM) to evaluate the structure of the fibrillar collagen.Tissues were fixed in 4% paraformaldehyde overnight. Following fixation, tissues were rinsed in PBS, incubated in 0.1 M glycine solution, cryoprotected by two successive incubations with 15% and 30% sucrose solution and embedded in optimal cutting temperature (O.C.T.) embedding medium. Liver sections were stained for 15 min in freshly prepared Oil-Red-O solution  were utilized. Plasma samples were prepared according to the manufacturers guidelines and measured at 570 nm.Fasting blood glucose (FBG) levels were measured using a commercially available kit (TRUEresult) and gold sensor-laser accuracy strips. Mice were fasted for 12 hrs for obtaining FBG levels. A nick was made on the tail vein and the FBG level was measured using the strip brought into contact with a drop of blood from the nicked tail vein. Intraperitoneal insulin tolerance test (IPITT) was performed on mice fasted for 12 hrs. Blood glucose reading was taken at time 0 followed by humulin (recombinant human insulin) injection at a dose of 10 ml/kg body weight. Humulin solution was prepared from stock humulin at a 1∶2000 dilution in saline. Glucose levels were taken every 15 min for the following 60 min. Intraperitoneal glucose tolerance test (IPGTT) was done following a 12 hr fast. Blood glucose was taken at time 0 followed by glucose administration. Mice received 20% glucose solution at a dose of 10 ml/kg body weight.Lung tissue was homogenized in RIPA at 4°C for 5–10 minutes. Protein concentration in the tissue was determined, the assays conducted according to the manufacturers' protocols  and read using a Luminex™ 100 instrument , which quantifies cytokine levels by monitoring R-Phycoerythrin RPE fluorescence associated with bead sets.ex = 800 nm). Each image presented in this work is 12 bit, 512×512 pixels representing 225 µm×225 µm field of view.An inverted Zeiss LSM 510 non-linear optics (NLO) META laser scanning microscope equipped with standard illumination for transmitted light and epi-fluorescence detection was used. The microscope was also equipped with an NLO interface for a femtosecond Titanium:Sapphire laser excitation source  for multi-photon excitation. The Chameleon laser provided femtosecond pulses at a repetition rate of about 80 MHz, with the center frequency tunable from 690 to 1040 nm. A long working distance objective  was used to acquire the images. The two-photon signals from the sample were epicollected and discriminated by the short pass 650 nm dichroic beamsplitter. The SHG images were collected using a META detection module with signals sampled in a 394–405 nm detection range . Tissue samples were homogenized in water and hydrolyzed with HCl in pressure-tight Teflon capped vials at 120°C for 3 hrs. Hydrolyzed samples were dried in a 96 well plate in a hot air oven at 70°C followed by addition of chloramine T and dimethylaminoborane and incubated for 90 mins at 60°C. The hydroxyproline forms a chromogen with an absorbance maximum at 560 nm.Experiments were approved by the Institutional Animal Care and Use Committee of the University of California, Riverside. After removal of dorsal hair, excision wounds were created on the dorsum of both control and THS-exposed mice using a 7 mm biopsy punch . Wound tissues were collected using a 10 mm diameter Acupunch at various time points following injury. Half of each sample was homogenized for the Affimetrix array assays and the remainder prepared for frozen sections.2 expression measure for each probe set was calculated from image files (CEL format) using the robust multi-array analysis (RMA) procedure using Agilent GeneSpring GX software. The changes of expression level between non-exposed and exposed wound tissues were compared. Only genes that were over- or under-expressed by >2-fold were considered.We used Affymetrix Mouse Genome 430 2.0 Arrays, which covers over 39,000 transcripts on a single array. Wound tissues were collected at 14 days post-healing and total RNA extracted and evaluated for quality using the Agilent Bioanalyzer 2100 using the Agilent RNA 6000 Nano Assay kit  and the concentration determined using the NanoDrop ND-1000 spectrophotometer . After the arrays were performed, a single logFig. S1A). The two arms that were closed had 8.5 inch black plastic walls. The maze was used in a quiet room and was enclosed by a black curtain with low light. All trials were video recorded. At the beginning of each trial, mice were placed in the middle of the maze facing a closed arm. The duration of time spent in the open or closed arms and the frequency of entries into open and closed arms were scored using the event recorder JWatcher .Singly-housed mice were transferred from the vivarium to the test room the morning of the trials. The elevated plus maze was manufactured in house as described previously Fig. S1B). Six control and six THS mice were used. Each mouse was placed in the center of the apparatus and videotaped for one hour. Analysis was performed during the first 10 min using the event–recorder JWatcher program. Rearing, Stationary and Walking behaviors were assessed, as well as the transition from one of these behaviors to the next and frequency of transition. A mouse was considered hyperactive if it spent more time walking than stationary and had more frequency of behavior transitions from one behavior to the next. To confirm these findings, the experiment was repeated; another set of 6 control mice and 6 THS-exposed mice were tested for 1 hr. This time the analysis was performed using Ethovision 7.1 video tracking software and the first 10 min and last 10 min of the tape were analyzed for distance and velocity of movement of each mouse. The software also produced raster plots that showed tracings of the movements of the mice superimposed on the image of the maze. The tracings shown in To test for hyperactivity we used the Open Field test Determination of the NNK metabolite NNAL was carried out using liquid chromatography – tandem mass spectrometry (LM-MS/MS) Significance was determined using Student's t-test for comparison between 2 means and ANOVA for comparison between more than 2 means. All data were examined to assure homogeneity of variance. Means were considered significantly different when p<0.05.Figure S1Schematic representation of the apparatus used to compare the behavior of normal vs THS-exposed mice. (A) Elevated Plus maze, used to assess anxiety. (B) Open Field Test Used to test levels of hyperactivity.(TIF)Click here for additional data file.Figure S2Quantification of data in : Graphs of the distances traveled (A) and (B) velocities of the mice whose tracks are shown in . Yellow is the THS exposed mouse and the blue is the control normal mouse. Note that the THS-exposed mouse was running at a great speed initially but quickly slowed down to a velocity only slightly greater than the control. However, with time, the control slowed down significantly whereas the THS-exposed mouse remained approximately constant in activity.(TIF)Click here for additional data file.Video S1Control mouse 1 (2× speed)-Control non-exposed mouse in the open field chamber to test for hyperactivity. The speed of this video has been increased by two times for faster viewing purposes. The control mouse moves more slowly and less frantically than the THS-exposed mouse. It also has longer rest periods and stays and travels more in the center of the chamber, as is expected of a mouse that is not hyperactive. This mouse is representative from an n = 12.(ZIP)Click here for additional data file.Video S2THS mouse#1 (2× speed)THS-exposed mouse in the open field chamber to test for hyperactivity. The speed of this video has been increased by two times for faster viewing purposes. The THS-exposed mouse moves faster, with fewer rest periods and appears to be frantic. Furthermore, the THS-exposed mouse stays along the periphery of the chamber and rarely travels or goes through the center of the chamber, a characteristic found in hyperactive behavior. This mouse also puts its front paws up throughout the test as if it is trying to find a way to escape out of the chamber. This mouse is representative from an n = 12.(ZIP)Click here for additional data file.


Activation of dopamine receptors in forebrain regions, for minutes or longer, is known to be sufficient for positive reinforcement of stimuli and actions. However, the firing rate of dopamine neurons is increased for only about 200 milliseconds following natural reward events that are better than expected, a response which has been described as a “reward prediction error” (RPE). Although RPE drives reinforcement learning (RL) in computational models, it has not been possible to directly test whether the transient dopamine signal actually drives RL. Here we have performed optical stimulation of genetically targeted ventral tegmental area (VTA) dopamine neurons expressing Channelrhodopsin-2 (ChR2) in mice. We mimicked the transient activation of dopamine neurons that occurs in response to natural reward by applying a light pulse of 200 ms in VTA. When a single light pulse followed each self-initiated nose poke, it was sufficient in itself to cause operant reinforcement. Furthermore, when optical stimulation was delivered in separate sessions according to a predetermined pattern, it increased locomotion and contralateral rotations, behaviors that are known to result from activation of dopamine neurons. All three of the optically induced operant and locomotor behaviors were tightly correlated with the number of VTA dopamine neurons that expressed ChR2, providing additional evidence that the behavioral responses were caused by activation of dopamine neurons. These results provide strong evidence that the transient activation of dopamine neurons provides a functional reward signal that drives learning, in support of RL theories of dopamine function. There is extensive evidence, particularly from pharmacological studies, that dopamine can cause positive reinforcement of stimuli and actions RPE drives reinforcement in computational models of RL First, although the transient response of dopamine neurons has the appropriate properties to drive RL from a computational perspective, there is little direct evidence that it actually does. Virtually all the direct evidence that dopamine causes reinforcement has come from manipulations that modulate dopamine for periods of at least minutes or longer It has not previously been possible to directly test for a causal role of the transient dopamine signal in reinforcement. Pharmacological and other manipulations act on a much slower timescale, whereas electrical stimulation is not able to selectively activate dopamine neurons. However, it should now be possible to test the RL theory of dopamine function in a direct and definitive manner by using optogenetic techniques, which allow temporal control of neurons with a precision in the range of milliseconds, together with the ability to target genetically defined neuronal populations Tsai and colleagues first demonstrated the feasibility of optogenetic stimulation of dopamine neurons in mice It is critical to note that the study of Tsai and colleagues Channelrhodopsin-2 (ChR2) was introduced into the right VTA of dopamine transporter (DAT) IRES-Cre mutant mice Behavioral experiments commenced 14 days or more after injection of vector. A nose poke to the “active” port caused delivery (after a 100 ms delay) of a single, 200 ms square-wave pulse of blue light to the right VTA via an optic fiber . The precise temporal pattern of depolarization and action potentials that was induced in dopamine neurons is not known and is not critical to our hypothesis. The only critical point is that this excitation will increase firing rate within a temporal window of a few hundred milliseconds Each mouse was conditioned in an operant chamber over 9–18 days. After several days of conditioning, each of the four ChR2-H mice showed significant responding for optical stimulation  Movie S. On daysIn order to better quantify the reward value of the optical stimulation, we used a progressive ratio schedule (PRS) of reinforcement in which animals had to perform a larger number of responses for each additional pulse of light. The PRS was not administered in separate sessions, but rather in the same sessions described above. Each day (excluding extinction training on days 9–18), each of the first 20 nose pokes was followed by a pulse of light (a FR-1 schedule). Additional responses resulted in light pulses according to the PRS. Whereas In contrast to the ChR2-H mice, none of the ChR2-L or AAV- mice showed clear evidence of an increase in responding on the active port over 9 days of conditioning, nor did they show any difference in responding between active and inactive ports  2-way A.In addition to its reinforcing effects, dopamine from VTA is also known to increase locomotion The responding observed in ChR2-H mice  & Fig. 32 = 0.99, p = 0.01) and ChR2-L  mice (without correction for multiple comparisons) . There was also evidence of correlations across mice between the three behavioral responses in both ChR2-H and ChR2-L mice, as can be seen by comparing the responses of individual mice between the panels of 2 = 0.98, p = 0.01), and the same trend was found in ChR2-L mice .The level of ChR2 expression across mice correlated strongly with each of the 3 behavioral responses (p<0.01 in each case) . This isarisons) . Operantarisons)  and contarisons)  showed tThese correlations suggests that despite the very small numbers of ChR2+ neurons in ChR2-L mice, optical stimulation may have elicited a small but behaviorally relevant activation of dopamine neurons. The more general inference that we make based on these correlations is that the number of ChR2 positive neurons is tightly linked to the magnitude of the optically-driven dopamine response, and that the magnitude of the dopamine response is tightly linked to behavior.The three types of behaviors measured here  are all known to result from increases in dopamine transmission that are sustained over periods of minutes or longer e.g. . Given tThe optically driven reinforcement of operant responding observed here is comparable to the electrically driven “brain stimulation reward” (BSR) described first by Olds and Milner Adamantidis and colleagues have recently tested the effect of optical activation of dopamine neurons on operant responding in mice, using an optogenetic technique virtually identical to that used here Our results are complemented by those of Zweifel and colleagues Although the present results demonstrate that the transient activation of dopamine neurons is sufficient for positive reinforcement, it does not appear to be necessary for reinforcement It has long been thought that that some drugs are addictive because they mimic the natural reward signal of dopamine neurons Male dopamine transporter (DAT) IRES-Cre mice http://www.addgene.org/pgvec1?f=c&cmd=findpl&identifier=18917 fromthe University of North Carolina Gene Therapy Vector Core). AAV-Flex-ChR2-tdTomato virus  was injected through a cannula into the right VTA  over a 10 minute period. Behavioral experiments began 14 or more days following injection of virus.We used an adeno-associated virus (AAV-FLEX-ChR2-tdTomato) to drive Cre-dependent expression of Channelrhodopsin-2 (ChR2) A 100 mW, 473 nm blue laser  was coupled to a 200 µm-diameter optical fiber  through a rotary joint (Doric Lenses). Light irradiance, as measured with an 818-SL photodetector (Newport Co.), was 477 mW/mm2 at the fiber tip, within the typical range of light intensities used for brief ChR2 activation. The optic fiber was inserted into the brain through the same cannula that was used for injection of virus. We found no evidence of any behavioral effects of light in the absence of ChR2 .The duration of 200 ms for the pulse of light was chosen to mimic the duration of the increase in firing rate of dopamine neurons that is observed following the sudden onset of a natural reward event e.g. . The parThe short delay of 100 ms between detection of a nose poke and onset of optical reward was chosen to maximize the speed and efficacy of reinforcement. Longer delays would be expected to result in slower acquisition of operant responding, and after acquisition, a lower asymptotic reward value due to temporal discounting. It is noteworthy that 100 ms is also the approximate delay between onset of a reward-predicting sensory stimulus and onset of the increase in firing rate of dopamine neurons e.g. .Operant responding was measured in separate sessions in a chamber that was dark except for light emanating from each of two nose poke ports . Inside If a mouse completed 20 nose pokes within one session, for the remainder of the session a progressive ratio schedule (PRS) was applied in which a larger number of nose pokes were required for each additional pulse of optical stimulation. The schedule was 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40 and 50 nose pokes, with 50 being the maximum break point that we observed across all sessions in all mice. Our use of the PRS may have quantitively altered responding on the FR-1 schedule (on the subsequent day following the PRS), but any such alteration is not a primary concern given that our interest is simply in determining whether or not the dopamine RPE is sufficient to drive reinforcement.Locomotor responses to optical stimulation were measured in separate behavioral sessions. Both head speed and rotations were tracked with a Noldus EthoVision automated tracking system. Mice were habituated to the behavior chamber for 20 minutes. In the following 20 minutes, there were alternating periods of 2 minutes of optical stimulation followed by 2 minutes without stimulation, for a total of 5 periods and 10 minutes of each condition . During The counts of TH-positive and ChR2-positive neurons  were perWe did not find evidence of any ChR2-tdTomato expression in SN, but we do not exclude the possibility that some SN dopamine neurons may have expressed ChR2 and may have contributed to behavioral responses. Although we targeted dopamine neurons of the VTA, it was not the purpose of this study to distinguish VTA from SN.2) was calculated to measure correlations.Statistical significance was defined as p<0.05 in all tests. Operant responses in individual mice  were anaMovie S1A mouse working for optical stimulation on a FR1 schedule. Light can be seen emanating from both the active and inactive ports.(MOV)Click here for additional data file.Information S1We reviewed the literature to investigate the possibility that our AAV vector might confer light sensitivity in the absence of ChR2 expression. We cited 29 studies that have used AAV vector in combination with optical stimulation of neural tissue, and we categorized these using a Venn diagram. The text reads “In the present work, we controlled for nonspecific effects of light, both in our AAV- group and by demonstrating a strong correlation of behavioral responses with ChR2 expression. We did not control for the possibility that AAV might confer light sensitivity in the absence of ChR2 expression. While this is certainly possible, we consider it extremely unlikely given basic knowledge of photochemistry and membrane excitability. In addition, the technique of optical stimulation in combination with AAV vector is no longer so new. To investigate the available data on this issue, we have reviewed a total of 29 papers that have used AAV for optogenetic experiments, including 6 papers on dopamine neurons. 13 of these 29 papers included AAV control experiments (3 of 6 papers on dopamine neurons). None of the 13 papers found any effect of light in AAV controls. 16 of the 29 papers did not include any AAV controls. We note that most of the papers without AAV controls were published in well respected journals, suggesting that many reviewers were not particularly concerned about the lack of AAV controls.”(PDF)Click here for additional data file.