This study examined trends in premarital sex, multiple partnership and condom use among young people (15–24 years) in Zambia from 2000 to 2009, and assessed the effects of individual and neighbourhood variables on these sexual behaviour indicators in 2000 and 2009.We analysed data from the Zambia Sexual Behaviour Survey, conducted in 2000, 2003, 2005 and 2009. Multi-stage cluster sampling was used to select 385 neighbourhoods, giving a population sample of 6,500 young people. Using linear-by-linear trend test, trends in the three indicators were examined. Multilevel logistic regression was used to assess the effects of individual and neighbourhood variables on the indicators.Premarital sex among young people decreased significantly from 51 to 42% between 2000 and 2009. Multiple partnerships of men also decreased from 26 to 14% during the same period. The use of condoms by young people remained stable during this period. Full multilevel regression models explained 29 and 34% of the neighbourhood variance of premarital sex in 2000 and 2009. For multiple partnerships and condom use, the explained variance was 29 and 18% in 2000; whereas in 2009 it was extremely low. Urban residence and living in neighbourhood with higher average duration of residence were associated with low premarital sex and higher condom use. Living in a neighbourhood with higher average level of comprehensive knowledge of HIV was associated with less risky sexual behaviour.Declining trends in premarital sex and multiple partnerships are among the factors that might explain the decrease in HIV incidence in Zambia among young people. However, condom use among young people has remained low and stable over the years. The results also suggest that behaviour change interventions should take stock of the social context when introducing individual-level programmes because neighbourhood factors play a considerable role in influencing sexual behaviour. The Joint United Nations Programme on HIV/AIDS (UNAIDS) finally acknowledged in its 2010 report that the HIV incidence had declined by >25% between 2001 and 2009 in 22 sub-Saharan African countries According to the UNAIDS 2010 report, young people (15–24 years) accounted for 46% of all new infections in Zambia, with an estimated 120,000 young men and women currently being HIV positive Most previous studies on factors affecting sexual risk behaviours have been restricted to individual-level factors In this study, we used data from the 4 latest rounds of the Zambia Sexual Behaviour Surveys (ZSBS) to examine trends in premarital sex, multiple partnerships and condom use at last premarital sex in different subgroups of young people. The effects of individual and neighbourhood-level factors on the 3 sexual risk behaviour indicators among young people were also assessed.Zambia is a landlocked country located in Southern Africa and has an estimated population of 13 million people The Sexual Behaviour Survey (ZSBS) is a nationally representative population based cross-sectional survey that has been conducted in Zambia in 1998, 2000, 2003, 2005 and 2009, and has collected data on HIV/AIDS/STIs knowledge, attitudes, sexual behaviour, and health-care seeking behaviours Ethical approval for the ZSBS was granted by the University of Zambia Ethics Committee. All eligible participants were informed of the purpose of the survey, and both written and oral consent to participate in the survey was sought. Selected participants were given the option of accepting or declining the interview. For participants <18 years, a parent or guardian was also asked for permission to interview them. This study was anonymous, and confidentiality was assured in all the 4-rounds of the surveys A 2-stage probability random sampling procedure was used in all 4-rounds of the ZSBS. The first stage of the sampling involved the selection of urban and rural clusters in every province of Zambia. These clusters, which we used as proxies for neighbourhoods, are census tracts or standard enumeration areas (SEAs), with an average size of 130 households or 600 people The second stage of the sampling process involved the selection of households in the sampled clusters. About 16 households (20 households in 2000) were sampled per urban cluster and 34 households (30 households in 2000) were sampled per rural cluster. A higher number of households were sampled in rural clusters because rural households on average have fewer adult members. This resulted in a total number of sampled households to 1,851 in 2000, 2,497 in 2003, 2,465 in 2005, and 2,500 in 2009. All females aged 15–49 years and males aged 15–59 years in the selected households were eligible to participate in the survey.Since the design of the ZSBS was not based on a theoretical model, the selection of variables to include in the analyses of this paper was based on an extensive review of published articles that have examined sexual behaviour Three dependent variables considered for this study were: premarital sex, multiple partnership and condom use at last premarital sex. Premarital sex was defined as the number of young people who reported sexual intercourse in the last 12 months among all single young people what kind of work they mainly did” or “their occupation”. Those who mentioned specific work or occupation were categorised as “employed”, whereas students, family workers without pay, retirees, housewives and respondents who reported that they were not working were categorised as “unemployed”. In the 2005 and 2009 surveys, respondents were asked whether they were employed or not, and those responding positively were categorised as “employed” and those not employed as “unemployed”.The following independent individual variables were analyzed: sex, age, marital status, educational attainment, employment status, religion and urban/rural residence . Descriptive statistics for the population distribution and variables are shown in (xtmelogit) on the 3 sexual risk behaviour indicators were analysed using STATA 11.1 . All neighbourhood variables were analysed as continuous variables, but were standardised before running the multilevel analysis. The multilevel models tested included the following:Multilevel mixed effects logistic regression a random intercept-only model (model 1)a random intercept model including individual-level variables only (model 2)a random intercept model including neighbourhood-level variables only (model 3)a random intercept model including both individual and neighbourhood level variables (model 4).2), the unexplained variance at the neighbourhood-level (or intraclass correlation (ICC)) and log-likelihood tests. To estimate the explained variance of the models, we initially considered the formula suggested by Raudenbush and Bryk (2002) These model tests were run for the 3 dependent variables using 2000 and 2009 data. Interaction tests were conducted for individual and neighbourhood variables, but none was found to be statistically significant. Other multilevel statistics estimated included the explained variance (RWhere: 2/3 = 3.29).2/3 = 3.29) In the logistic distribution for the level-one residual, the variance is assumed to be a constant  than young men who were interviewed. The majority had never married (61–73%) and lived in urban areas (60–64%). The relative distribution of participants by gender, age, religion, urban/rural residence was relatively stable across the surveys. However, the proportion with secondary or higher education increased with time from 38 to 51%, as did the mean neighbourhood residential stability (from 11.9 to 12.6 years) and comprehensive knowledge of HIV (from 12% to 48%). The “proportion employed” in 2000 and 2003 was substantially higher than the “proportion employed” according to the definition applied in 2005 and 2009. Similarly, neighbourhood labour force participation estimates in 2000 and 2003 were much higher than the estimates in 2005 and 2009 .There was a 17% decrease in premarital sex among young women (from 47 to 39%) and men (from 54 to 45%) from 2000 to 2009 . The decIn For 2009, the intercept-only model showed that premarital sex varied across neighbourhoods, with a neighbourhood difference of 11%, which was statistically significant at p <0.05. Model-2 indicates that individual variables were much stronger predictors of premarital sex, such that their addition increased the unexplained neighbourhood-level variance from a proportion of 0.40 to 0.78. The inclusion of these variables explained 33% of the variance in premarital sex. Model-3, with neighbourhood-level variables, had a very low neighbourhood variance of premarital sex among the neighbourhoods. In contrast, model-4, which included both neighbourhood and individual-level variables, explained ∼34% of the variance in premarital sex, but the difference between neighbourhoods remained at 11%. The log-likelihood test revealed that despite these results, the inclusion of both individual and neighbourhood variables significantly improved the fit. Model-4 also showed that 2 individual-level variables (age and being in employment) were significantly associated with higher odds of premarital sex among young people. Urban residence and residing in a neighbourhood with high labour force participation and high comprehensive knowledge of HIV gave significantly lower odds of reporting premarital sex .There was no clear overall trend in multiple partnerships among young women between 2000 and 2009. However, a statistically significant decrease of ∼45% was generally observed among young men, and almost all subgroups showed statistically significant declining trends. The only subgroup of men where a decline could not be seen was among those living in neighbourhoods with high comprehensive knowledge of HIV, but the change was not statistically significant .The intercept-only model in 2000 showed that multiple partnerships varied across neighbourhoods with a neighbourhood difference of 14%. The addition of individual variables in model-2 reduced the neighbourhood difference in multiple partnerships to 10%, whereas these variables explained ∼29% of the variance in multiple partnerships. Model-3 showed that the inclusion of neighbourhood variables explained ∼2% of the variance in multiple partnerships. The addition of both individual and neighbourhood variables in model-4 explained ∼29% of the variance in multiple partnerships, while the difference between neighbourhoods was reduced to 9%. Model-4 showed that the inclusion of both individual and neighbourhood variables significantly improved the fit. Being female was the covariate significantly associated with lower odds of multiple partnerships in model-4. However, having secondary/higher educational attainment and being in employment were significantly associated with higher odds of multiple partnerships among young people .For 2009, the intercept-only model showed an extremely low variation in multiple partnerships across neighbourhoods. Model-4, however, showed that being females and residing in neighbourhoods with high comprehensive knowledge gave lower odds of having multiple partners .Condom use at last premarital sex generally remained stable among men between 2000 and 2009, whereas it decreased non-significantly among women. Significant falling trends occurred among women in neighbourhoods with medium residential stability and low comprehensive knowledge of HIV (women) .Model-1 for 2000 showed that condom use varied across neighbourhoods, with a neighbourhood difference of 18%. The addition of individual variables (model-2) explained ∼10% of the variance in condom use among young people. The difference across neighbourhoods was reduced to 9%. Model-3, with neighbourhood variables only, explained 15% of the variance and the difference across neighbourhoods was reduced to 5%. Model-4 explained ∼18% of the variance, whereas the difference across neighbourhoods was reduced to 3%. The background variables that were significantly associated with lower odds of condom use in model-4 were being a protestant Christian and residing in neighbourhoods with lower labour force participation. Young people residing in neighbourhoods with high educational attainment had higher odds of condom use .The intercept-only model in 2009 showed that condom use varied across neighbourhoods, with a neighbourhood difference of 12%. The inclusion of individual variables in model-2 and neighbourhood variables in model-3 reduced the differences across neighbourhoods to 2 and 3%, respectively. However, individual variables explained ∼7% of the variance in condom use, while neighbourhood variables explained ∼6% of the variance. The addition of both individual and neighbourhood variables in model-4 reduced the difference between neighbourhoods to an extremely low level. Young people residing in neighbourhoods with high residential stability and high comprehensive knowledge of HIV had higher odds of condom use .Sexual behaviour surveys based on the nationally representative samples of young adults in Zambia showed a decline in premarital sex for both females and males between 2000 and 2009. A substantially downward trend of multiple partnerships occurred among young men, whereas the proportion with multiple partnerships among young women during this period was relatively low and stable. However, condom use at last premarital sex remained relatively stable among males over the years, but decreased  among females. These findings have also been reported in the 2009 Zambia Sexual Behaviour Survey report It is clear from our findings that the downward trends of premarital sex provide a good indication that young people are postponing their sexual debut in Zambia. The decline does not seem to be due to an increased rate of early marriage, since the proportion of young people who had never been married decreased. Combined with decreasing trends of multiple partnerships among young men, this suggests positive sexual behaviour changes among young people in Zambia. The behavioural changes could have contributed to the decline in HIV incidence in Zambia since 2001. It is possible that the decline in premarital sex and multiple partnerships is a sign that HIV prevention campaigns promoting abstinence and faithfulness to one partner could have had an impact. It is also likely however that young people's personal experience of the effects of the HIV epidemic have led to these behavioural changes One of the targets set in the millennium development goal-6 was to increase condom use during high-risk sex among young people to > 90% by 2015 Findings from the multilevel analyses offer insights into possible neighbourhood contextual effects on premarital sex, multiple partnerships and condom use among young people in Zambia. We have shown that the addition of individual factors to the intercept-only model substantially reduced, or in some cases increased, the difference between neighbourhoods. Increase in the total unexplained neighbourhood variance of premarital sex and multiple partnerships compared to the intercept-only model's variance when the individual-level factors were added to the models may be explained by the fact that the formula used assumes simple random sampling, so when grouped data are entered into the model, the lower level variance tends to increase This study also found that urban residence was significantly associated with lower likelihood of premarital sex and a higher likelihood of condom use in both 2000 and 2009, suggesting reduction in sexual risk behaviour among young urban males and females. These findings support the observation that HIV prevention efforts have been more intensive in urban settings. An example of this can be drawn from the condom distribution campaigns in Zambia, where an estimated 14 million condoms were distributed in 2008, with the majority of the distribution outlets being located in urban settings 2) is a proportion of the total variance, because first level variables in principle, can explain all variation, including that at the second level To check for independent neighbourhood effects, we entered neighbourhood factors into the intercept-only models. This resulted in a substantial decline in the unexplained variance of premarital sex (in 2000) and condom use at last premarital sex (in 2000 and 2009) at the neighbourhood-level. This suggests that neighbourhood variables have a considerable influence on the sexual risk behaviour indicators. To assess the neighbourhood-level variance explained by these variables, we initially considered the formula suggested by Raudenbush and Bryk Neighbourhood residential stability was a statistically significant predictor of both premarital sex and condom use . We argue that this predictor can be used as a proxy for social capital Another striking neighbourhood predictor of lower risk of premarital sex and multiple partnerships, and higher condom use in our study, is comprehensive knowledge of HIV. The 2007 Zambia Demographic and Health Survey indicated that >90% of Zambia's population were aware of HIV, but only 37% of young men and 34% of women had comprehensive knowledge of HIV transmission Neighbourhood educational attainment may be viewed as a proxy for neighbourhood socio-economic position. High neighbourhood socio-economic position has been found to reduce the risk of premarital sex The different aspects of average neighbourhood employment, for example labour force participation, the prevalence of full time employment, and employment opportunities, have been used as proxies of the ability of adults to supervise young people's behaviour To measure the mediating effects of individual and neighbourhood factors, they were entered into the same model. For example, the full multivariate model for premarital sex in 2000 showed that neighbourhood labour force participation became statistically insignificance. It is possible that neighbourhood labour force participation was a confounder in model-3 and that the important association was between the individual-level employment status and premarital sex. Furthermore, neighbourhood comprehensive knowledge in 2000 lost its statistical significance in the full multivariate models for multiple partnerships, which might have been a result of partial confounding of the effect of rural-urban residence variable. Urban-rural residence seemed to be a confounder in relation to condom use at last premarital sex in 2000, since the significant association seen in model-2 disappeared when adjusted for neighbourhood educational attainment and neighbourhood labour force participation. In contrast, after the urban-rural residence variable was added to the full multivariate models in 2009, neighbourhood comprehensive knowledge became a significant predictor of multiple partnerships, neighbourhood residential stability became significantly associated with condom use at last premarital sex, and neighbourhood labour force participation became a significant predictor of premarital sex. In these cases urban/rural residence seems to have functioned as a negative confounder that distorted the true association toward the null.The results of this study should be considered with some caution. For instance, it was based on self-reported sexual behaviour data, i.e. subject to bias. Some studies have found considerable errors in self-reporting of sexual activity, for example, males over-reporting and females under-reporting their sexual activities Furthermore, it is possible that using face-to-face interviews to gather this type of information may have contributed to reporting errors as people might not reveal all their sex life details to a stranger. If the magnitude of the bias has changed over time, this would have affected our trend estimates. The cross-sectional nature of the data limits our ability to draw causal inferences about the associations we have found. Moreover, the use of repeated cross-sectional studies conducted in different clusters, with different households, to estimate trends would affect estimates if there had been a major population shift over the years being analysed. However, such major shifts do not seem to have occurred. The number of neighbourhood and individual-level variables used in this study was restricted by the changes in the questionnaires over the years. Large proportions of the total variance of the three indicators were not explained by the individual or the neighbourhood factors, and the explanation for this could be that we did not include all the important factors. We have only assessed trends in 3 sexual behaviour indicators, and there may be other important aspects of changes in sexual behaviour among young people that might have been missed whether neighbourhood factors influence sexual behaviour but begin to tackle critical questions of how these factors affect sexual risk behaviour. Furthermore, there is need for these future studies to be based on theory at design stage, for example, the Social Capital theory or the Ecological model of human development, so that the relationships found can be properly explained.To be useful to policy makers in Zambia, future neighbourhood research on the effect of neighbourhood on sexual risk behaviour should not only be focused on This study showed that premarital sex and multiple partnerships have been decreasing over the years but condom use has remained almost stable since 2000. In addition, the present study showed that both individual and neighbourhood contextual factors played a considerable role in influencing the sexual risk behaviours of young people.Table S1Operational Definitions of the Variables.(DOC)Click here for additional data file.Table S2Descriptive statistics of young people aged 15–24 years stratified by survey year, in ZSBS 2000–2009 (percentage).(DOC)Click here for additional data file.Table S3Premarital sex trends among young people (15–24 years) from 2000 to 2009 (in percentage).(DOC)Click here for additional data file.Table S4Multiple Partnerships trends among young people (15–24 years) from 2000 to 2009 (in percentage).(DOC)Click here for additional data file.Table S5Condom use at last sex trends among young people (15–24 years) from 2000 to 2009 (in percentage).(DOC)Click here for additional data file.


Sexual behaviour and relationships are key components of wellbeing and are affected by social norms, attitudes, and health. We present data on sexual behaviours and attitudes in Britain  from the three National Surveys of Sexual Attitudes and Lifestyles .We used a multistage, clustered, and stratified probability sample design. Within each of the 1727 sampled postcode sectors for Natsal-3, 30 or 36 addresses were randomly selected and then assigned to interviewers. To oversample individuals aged 16–34 years, we randomly allocated addresses to either the core sample  or the boost sample . Interviewers visited all sampled addresses between Sept 6, 2010, and Aug 31, 2012, and randomly selected one eligible individual from each household to be invited to participate. Participants completed the survey in their own homes through computer-assisted face-to-face interviews and self-interview. We analysed data from this survey, weighted to account for unequal selection probabilities and non-response to correct for differences in sex, age group, and region according to 2011 Census figures. We then compared data from participants aged 16–44 years from Natsal-1 (1990–91), Natsal-2 (1999–2001), and Natsal-3.Interviews were completed with 15 162 participants  from 26 274 eligible addresses (57·7%). 82·1% (95% CI 81·0–83·1%) of men and 77·7% (76·7–78·7%) of women reported at least one sexual partner of the opposite sex in the past year. The proportion generally decreased with age, as did the range of sexual practices with partners of the opposite sex, especially in women. The increased sexual activity and diversity reported in Natsal-2 in individuals aged 16–44 years when compared with Natsal-1 has generally been sustained in Natsal-3, but in men has generally not risen further. However, in women, the number of male sexual partners over the lifetime , proportion reporting ever having had a sexual experience with genital contact with another woman , and proportion reporting at least one female sexual partner in the past 5 years  increased in Natsal-3 compared with Natsal-2. While reported number of occasions of heterosexual intercourse in the past 4 weeks had reduced since Natsal-2, we recorded an expansion of heterosexual repertoires—particularly in oral and anal sex—over time. Acceptance of same-sex partnerships and intolerance of non-exclusivity in marriage increased in men and women in Natsal-3.Sexual lifestyles in Britain have changed substantially in the past 60 years, with changes in behaviour seeming greater in women than men. The continuation of sexual activity into later life—albeit reduced in range and frequency—emphasises that attention to sexual health and wellbeing is needed throughout the life course.Grants from the UK Medical Research Council and the Wellcome Trust, with support from the Economic and Social Research Council and the Department of Health. Improving sexual and reproductive health remains a public health priority in Britain ,8The first National Survey of Sexual Attitudes and Lifestyles  was done in a probability sample of 18 876 adults aged 16–59 years in Britain in 1990–91.19Here, we report data on sexual behaviours and attitudes in Britain from the latest survey, Natsal-3, and the two previous surveys. The combination of data from all three Natsal surveys enables both period and birth cohort analyses; together, the surveys sampled people born between the 1930s and the 1990s. We examine changes in sexual lifestyles throughout the life course and trends over time.Full details of the methods used in Natsal-3 have been reported elsewhere.Within each primary sampling unit, 30 or 36 addresses were randomly selected and then assigned to interviewers from NatCen Social Research. To allow detailed exploration of behaviours in the age group at highest risk of some sexual health outcomes , we oversampled individuals aged 16–34 years. We randomly allocated addresses to either the core sample  or the boost sample . Letters and leaflets giving background information about Natsal-3 were sent to sampled addresses before visits began.questionnaire and technical report will be available on the Natsal website on the day of publication.Interviewers visited all sampled addresses, identified residents in the eligible age range, and randomly selected one individual to be invited to participate in the survey using a Kish grid technique. Participants then completed the survey in their own homes through a combination of face-to-face interviews with computer-assisted personal interview and a self-completion format with computer-assisted self-interview. Interviewers were present in the room while participants completed the computer-assisted self-interview and could provide assistance as necessary, but did not view responses. On completion of computer-assisted self-interviews, answers could not be accessed by interviewers. No names or other potentially identifying information was attached to the interviews. Interviews lasted about 1 h on average. Participants received a £15 gift voucher as a token of appreciation. An anonymised dataset will be deposited with the UK Data Archive, and the complete These methods are broadly the same as those used in Natsal-1 and Natsal-2. However, in Natsal-1, paper was used (rather than computers) during face-to-face interviews and self-interview. Descriptions of the development stages of previous surveys are available elsewhere.As in Natsal-1 and Natsal-2, we weighted Natsal-3 data to adjust for the unequal probabilities of selection in terms of age and the number of adults in the eligible age range at an address. After application of these selection weights, the Natsal-3 sample was broadly representative of the British population compared with 2011 Census figures,27We compared data for participants aged 16–44 years in each survey. This age group was common to all three surveys. Information about variables that were compared was derived from identically worded questions. All three surveys had been weighted for differential selection probabilities. Natsal-1 was post-stratified to 1991 Census figures and Natsal-2 to 2001 Census figures, with procedures described for Natsal-3,23The Natsal-3 study was approved by the Oxfordshire Research Ethics Committee A (reference: 09/H0604/27). Participants provided oral informed consent for interviews.We did all analyses with the complex survey functions of Stata (version 12.1) to incorporate weighting, clustering, and stratification of data. We present descriptive statistics by sex and age group. We used binary logistic regression to calculate age-adjusted odds ratios (aAORs) to investigate how reporting of key sexual behaviours and attitudes varied by three measures of social status: the National Statistics Socio-Economic ClassificationThe sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.A resident within the eligible age range (16–74 years for the core sample or 16–34 years for the boost sample) was identified at 23 360 of 59 412 addresses selected for sampling. Eligibility could not be established at 4143 addresses (7·0%), because contact was attempted on several occasions but could not be made or all information was refused. By assuming the percentage of residents who would be eligible at the addresses for which this percentage was unknown was the same as for the addresses for which eligibility was known (separately for core and boost addresses), we estimated that 2914 of these addresses would have housed a resident in the eligible age range, giving an estimated total of 26 274 eligible addresses.The response rate was 57·7%; interviews were completed with 15 162 participants  from the 26 274 eligible addresses. Individuals from 6343 eligible addresses refused to participate, contact was not made at 327 addresses, and interviews were not completed for 1528 for other reasons. Therefore, the co-operation rate (how many interviews were completed from eligible addresses for which contact was made) was 65·8%.Median age at first heterosexual intercourse was 17 years in both sexes , Table 3The reported number of lifetime sexual partners of the opposite sex by time of interview varied substantially by sex and age group , Table 382·1% of men and 77·7% of women reported at least one sexual partner of the opposite sex in the year before interview. The proportion peaked in men aged 35–44 years (92·5%) and women aged 25–34 years (91·8%). The proportions of men and women reporting at least one sexual partner of the opposite sex in the past year were largely similar in the three youngest age groups, but after the age of 55 years, men were more likely to have had a sexual partner of the opposite sex in the past year than were women , Table 3We recorded substantial variability by age in the number of occasions of sexual intercourse with a partner of the opposite sex in the 4 weeks before interview in both men and women who reported at least one sexual partner of the opposite sex in the past year , Table 360·7% of participants reported having had vaginal sex in the past 4 weeks. This proportion decreased substantially after the age of 55 years in both sexes, as did the proportion reporting heterosexual oral sex in the past year , Table 3The proportion of participants of all ages reporting ever having a sexual experience or contact with an individual of the same sex was slightly higher in women (11·5%) than in men 8·0%; , Table 3We generally recorded less variation in sexual behaviours and attitudes between groups of participants divided by area-level social-status measures than between those in groups divided by the two individual-level measures . Of the Odds of almost all sexual behaviours in participants aged 16–44 years were significantly lower in Natsal-1 than in Natsal-2 , which rIn men, we noted little change between Natsal-2 and Natsal-3 in number of female sexual partners over the lifetime, number of female sexual partners in the past year, or numbers of individuals who had had a sexual experience with another man . The proIn women, we recorded increases between Natsal-2 and Natsal-3 in the reported number of male sexual partners over the lifetime, reporting of anal sex with a male partner in the past year, reporting of ever having had sexual experience or contact with another woman, reporting of ever having had a sexual experience with genital contact with another woman, and reporting of at least one female sexual partner in the past 5 years .We noted increased acceptance of same-sex partnerships in men and women in Natsal-3 compared with Natsal-2, although women continued to be more accepting than men . By contVaginal sex was almost universal in the group aged 25–34 years in all three birth cohorts for whom data about this age group were available . By contWe have presented findings from Natsal-3 on sexual attitudes and lifestyles in Britain in 2010–12. By also including data from the two previous Natsal surveysWe have shown wide variability in sexual lifestyles by sex, age, and birth cohort, and, for the first time, have recorded behaviour patterns and attitudes in individuals aged up to 74 years. Most adults at all ages are sexually active, but sexual frequency and the range of practices reported reduces with age, especially in women. Although many aspects of health behaviour have strong social determinants,Sexual frequency, and particularly reporting of recent vaginal sex, has decreased in the past decade. Increases in the reporting of oral sex with a partner of the opposite sex between Natsal-1 and Natsal-2 have not continued; prevalence was the same in Natsal-3 as in Natsal-2. By contrast, the reporting of anal sex with a partner of the opposite sex—albeit much lower than that of oral sex—increased in men and women between Natsal-1 and Natsal-2, and between Natsal-2 and Natsal-3. This finding suggests that heterosexual repertoires—or at least experimentation—have expanded in successive birth cohorts and over time.More substantial changes in behaviour occurred between Natsal-1 and Natsal-2 than between Natsal-2 and Natsal-3, possibly because Natsal-1 coincided with public concern around the beginning of the HIV/AIDS epidemic. The start of the 1990s might have represented a low point in terms of sexual activity and risk behaviour, a hypothesis that is corroborated by the large decrease in diagnoses of sexually transmitted infections that occurred during the 1980s and early 1990s.As has been reported elsewhere,Although response in the previous Natsal surveys was higher than for Natsal-3 , we recorded little change in partner acquisition since the increase between Natsal-1 and Natsal-2.48The large changes in sexual lifestyles that have occurred in the past 60 years that have been captured by the three Natsal studiesExamination of the sexual lifestyles of older as well as younger people in Britain emphasises the importance of sexual health and wellbeing across the life course, and underscores the limitations of restricting sexual health policy and services to younger age groups. Although the sexual health and wellbeing needs of the British population might have diversified, they remain as important as when the national strategies for sexual health were launched earlier in the new millennium.17


Presence of circulating tumor cells (CTC) in patients with metastatic breast, colorectal and prostate cancer is indicative for poor prognosis. An automated CTC (aCTC) algorithm developed previously to eliminate the variability in manual counting of CTC (mCTC) was used to extract morphological features. Here we validated the aCTC algorithm on CTC images from prostate, breast and colorectal cancer patients and investigated the role of quantitative morphological parameters.Stored images of samples from patients with prostate, breast and colorectal cancer, healthy controls, benign breast and colorectal tumors were obtained using the CellSearch system. Images were analyzed for the presence of aCTC and their morphological parameters measured and correlated with survival.Overall survival hazard ratio was not significantly different for aCTC and mCTC. The number of CTC correlated strongest with survival, whereas CTC size, roundness and apoptosis features reached significance in univariate analysis, but not in multivariate analysis. One aCTC/7.5 ml of blood was found in 7 of 204 healthy controls and 9 of 694 benign tumors. In one patient with benign tumor 2 and another 9 aCTC were detected.CTC can be identified and morphological features extracted by an algorithm on images stored by the CellSearch system and strongly correlate with clinical outcome in metastatic breast, colorectal and prostate cancer. Case studies showing the presence of tumor cells in blood of cancer patients have been reported for more than a century 5 and in case studies in breast and colorectal cancer it was found that breast cancer CTC are somewhat rounder than cells from colorectal samples The recent emergence of different technologies to identify CTC has been accompanied with a large range of CTC definitions urging the need for standardization Blood samples enriched for CTC of patients before they received treatment (baseline) and at first follow-up after initiation of a new line of therapy from prospective multicenter CTC studies were used in this study. For metastatic breast cancer, fluorescence images of 283 blood samples processed on the CellSearch system from 179 patients could be imported from the IMMC-01 study and 482 image sets from 248 patients from the IC2006-04 study 2 sized pixels. For each cartridge, 144–180 4-layer tiff images of DAPI, FITC, PE, APC are saved per patient. After imaging, the system preselects objects that are positive for DNA and CK and shows the images of all fluorescence channels together with a DNA/CK overlay of all selected objects in a gallery. Finally, a trained operator selects objects from the gallery as manual CTC (mCTC) if they are DNA+CK+, CD45-, have a cell-like morphology, and are larger than 4×4 µm2.The CellSearch system  was used to enumerate CTC. The system consists of a CellTracks Autoprep® for sample preparation and a CellTracks Analyzer II® for sample analysis 2). However, the upper aCTC size limit was increased to 2000 pixels (∼900 µm2) after the observation that aCTC sizes in metastatic breast cancer are larger The recorded image sets from the patients were copied to central hard drives and analyzed by an algorithm developed in Matlab 2009a, using the DipImage toolbox . The algorithm was optimized using survival of 185 castration resistant prostate cancer patients as a training parameter and is described in more detail elsewhere 2 . If multiple DNA-DAPI+ objects were found close to the CK signal, the center-of-mass distances between the CK and DNA signals were determined. The object with  overlap between CK and DNA and the smallest distance  was selected to determine the NCK-ratio. Clusters of cells were defined as the number of DNA-DAPI objects larger than 10 µm2 and located within each CK-PE signal. Finally, speckles within the CK-PE signals are indicative of damaged or apoptotic CTC We performed a number of measurements on the aCTC, including cell roundness, nuclear to cytokeratin (NCK) ratio, the presence of cell clusters, and the number of speckles within the cytoplasm. Cell roundness was defined as the ratio of the signal perimeter squared divided by 4π times the signal area (termed P2A in the literature). Thus, roundness equals one for a perfect circle. The NCK-ratio was defined as the area of the DNA-DAPI signal, divided by the area of the CK-PE signal. For this parameter, an extra segmentation step for DNA-DAPI was performed to find DNA-DAPI+ objects larger than 10 µm2). Morphological parameters were extracted as described for aCTC.Due to a-specific binding of EpCAM-ferrofluid particles during the CTC enrichment, leukocytes are carried over and are present at the analysis surface of the sample cartridge. Leukocytes are identified by their DAPI staining, expression of CD45 and lack of cytokeratin expression. We applied the triangle threshold method to determine the outline of leukocytes in the CD45-APC channel SPSS 16  was used for survival analysis. Least-squares linear regression analysis was performed to compare aCTC and mCTC, baseline and follow-up samples, and to compare different studies of the same type of cancer. Kaplan-Meier analysis was performed by dichotomizing the patient population on the clinically used cut-off value of five CTC for breast and prostate cancer, and three CTC for colorectal cancer. Beyond these cut-offs, other cut-off values of 1, 5, 10, and 100 for aCTC or mCTC were tested to dichotomize the patient population per study for univariate Cox hazard regression. Log-rank tests were used to compare survival between groups. Cox hazard ratios (HRs) with 95% confidence intervals (CI) were determined and collected in a table. Survival was defined as the time between baseline sample and death from any cause.inp 0.10 and outp 0.20), controlling for cancer type , with all morphological parameters and the aCTC count as continuous variables. The number of aCTC was 10log transformed before inclusion in the regression To determine whether the morphological parameters determined on aCTC provide additional information that is relevant for prognosis, multivariate Cox proportional hazards regression was performed  distribution for colorectal, breast and prostate cancer is shown in panels A, B and C respectively. The NCK-ratio (range 0.5 to 5) for breast, colorectal, and prostate cancer CTC is shown in panels E, F and G respectively. aCTC appear in a large range of shapes. To illustrate this diversity in CTC shape, a measure of roundness (range 1 to 5) for colorectal, breast and prostate cancer CTC is shown in panels H, I, and J respectively. For comparison the size and roundness properties of leukocytes are shown in 2 correlation between measurements before and after initiation of therapy is provided in each panel. aCTC morphological parameters were quantified by mean and standard deviation and are provided in p = 0.18. For median roundness of aCTC p-values were 0.27, 0.008, and 0.15 for comparison between breast-colorectal, breast-prostate, and colorectal-prostate, respectively. For all other morphological parameters from p was less than 0.001 between all types of cancer.Recording of the outline of aCTC permits the extraction and quantification of morphological parameters from the identified aCTC. The distribution of the size, NCK ratio and roundness of aCTC in blood of patients with metastatic breast, colorectal, and prostate cancer before initiation of therapy and at first follow up after initiation of therapy is shown in . None of the patients had a significant rise or drop in the number of speckled aCTC between baseline and first follow-up measurement.The size of the objects can be influenced by the presence of multiple CTC representing CTC clusters. These clusters are counted as one CTC in both the manual identification as well as the automated identification of CTC. The presence of aCTC clusters was estimated by the number of nuclei that could be detected within the CK-PE area. Percentages of clustered cells per patient are shown in To illustrate the relationship between survival and CTC, Kaplan Meier plots were generated dividing the patients in favorable and unfavorable groups based on a threshold of 3 CTC for metastatic colorectal cancer and 5 CTC for metastatic prostate and breast cancer. The Kaplan-Meier plots shown in Next, all variables of aCTC were entered in a multivariate Cox regression analysis. mCTC were left out of this analysis, as their quantitative parameters are unknown. Hazard models were calculated using all first follow-up image sets for all cancer types together and for each type separately. When all image sets were combined, we controlled for differences in overall survival due to cancer type. Results are shown in 2 and 224 µm2.Recently, we showed that counting CTC by an automated algorithm is preferable to manual counting by a trained reviewer 2 to 900 µm2, concordant with a recent study showing a larger size of CTC in breast cancer Here we applied the aCTC algorithm to image sets of patients with benign and malignant breast and colorectal cancer, as well as to image sets of healthy controls. The upper size limit was increased from 224 µmComparison of aCTC morphological parameters between cancer types shows a larger size, a relatively large nucleus to cytokeratin ratio and rounder CTC in breast cancer as compared to prostate and colorectal cancer. The distribution of the size of aCTC is exponential in CTC of both prostate and colorectal cancer, which is cut off at the lower end by the CTC algorithm, in contrast with leukocytes that display a more bell-shaped size distribution. These distributions shows that a wide range of CTC and CTC fragments are present in the blood of patients, as was reported earlier p<0.05) when we included all cancer types in the multivariate analysis, with rounder cells predicting poorer survival. When we performed multivariate analysis by cancer type, small cells with small nuclei compared to the cytoplasm were significantly (p<0.05) associated with poor survival only for breast cancer patients. Currently, we have no biological explanation for this phenomenon. However, research focused on separating normal blood cells from CTC on the basis of size should consider this observation. The association of rounder and/or smaller cells with poorer survival suggest that these maybe features of cancer stem cells, similar to the observations made for hematopoietic stem cells, which are clearly smaller and rounder as compared with the other hematopoietic progenitor cells We performed continuous univariate and multivariate survival analysis to investigate the influence of aCTC and its morphological parameters on survival. CTC measurements at first-follow up after treatment were used for this analysis as the CTC “surviving” this treatment more strongly related to survival and might contain typical morphological features. Median roundness was significant Click here for additional data file.Table S2.(DOCX)Click here for additional data file.


FOXP2, is the first gene implicated in a speech and language disorder. Since its discovery, many studies have been carried out in an attempt to explain the mechanism by which it influences these characteristically human traits. This review presents the story of the discovery of the FOXP2 gene, including early studies of the phenotypic implications of a disruption in the gene. We then discuss recent investigations into the molecular function of the FOXP2 gene, including functional and gene expression studies. We conclude this review by presenting the fascinating results of recent studies of the FOXP2 ortholog in other species that are capable of vocal communication. WIREs Cogn Sci 2013, 4:547–560. doi: 10.1002/wcs.1247The forkhead box P2 gene, designated  FOXP2 (forkhead box P2) gene (OMIM#605317) and the elucidation of its involvement in a speech and language disorder begins with the study of a multigenerational British family known as the KE family.The story of the discovery of the et al.Studies of the KE family appeared in the scientific literature in the early 1990s. A first examination of the pedigree was carried out by Hurst et al.A 1998 study found that the affected KE family members were individually impaired on three tests: word repetition, nonword repetition, and simultaneous and sequential orofacial movements.Ten affected and seven unaffected KE family members were subsequently tested using MRI in order to try to find any structural differences in their brains that could account for the functional abnormalities in the affected members who were tested using PET. Several regions where affected members had significantly more or significantly less gray matter than unaffected members were identified: affected members had more gray matter in the lentiform nucleus and the angular gyrus , and less gray matter in preSMA/cingulate cortex, Broca's area and the caudate nucleus . The caudate nucleus was therefore implicated in both the PET and MRI analyses, and further examination showed that the affected family members had significantly smaller left and right caudate volumes than the unaffected family members.7A subsequent functional MRI (fMRI) studyA behavioral analysis by Watkins et al.ATP2C2  and CMIP (c-Maf inducing protein),CNTNAP2 .ATP2C2 and CMIP were significantly associated with nonword repetition ability in children affected by SLI, but not in the general population, which suggests that these genes are involved in phonological short-term memory in language-impaired children. Each of the two genes had an independent effect on nonword repetition ability. The variants in CNTNAP2, which were identified in another study, were also significantly associated with nonword repetition in children with SLI, as described in the text.Specific language impairment (SLI) is diagnosed when a child has major problems in the acquisition of language, despite showing normal development in all other areas.FOXP2, due to structural similarities shared with other FOX proteins (transcription factors that have a characteristic forkhead DNA-binding domain).FOXP2 gene in the KE family resulted in the detection of a G to A transition in exon 14 which co-segregated perfectly with the disorder.FOXP2 haplo-insufficiency in the brain at a key stage of embryogenesis leads to abnormal development of neural structures that are important for speech and language.24While the behavioral and neuroimaging studies were taking place, researchers began looking for the gene that was the cause of the disorder in the KE family. The first analysis done by Hurst et al. suggested that the disorder from which half of the KE family members suffered was autosomal-dominant.FOXP2 gene.A mutation screening of children with verbal dyspraxia found three previously unreported exonic variants in the FOXP2 was reported for a child with oromotor and verbal dyspraxia and developmental delay.FOXP2 have also been reported in the context of developmental verbal dyspraxia, in which the absence of paternally inherited FOXP2 was associated with the condition.FOXP2 still resulted in verbal dyspraxia,FOXP2 is not imprinted.FOXP2 gene, not all cases of developmental verbal dyspraxia are the result of such disruptions in FOXP2. TableA deletion in chromosome 7q31-q32 which included FOXP2 may be involved in the etiology of neurological disorders. An association analysis and mutation screen of the FOXP2 gene was done on families with autism or specific language impairment, but no evidence for the direct involvement of FOXP2 in either of the disorders was found.FOXP2 showed association with some language and reading abilities and sequential motor activities in a dyslexia cohort.FOXP2 gene in healthy subjects found some associations with variations in activation during reading (detected with fMRI) in the left inferior frontal gyrus and the left precentral gyrus.32Several studies have investigated the possibility that normal genetic variation, as opposed to genetic mutation, across FOXP2 was found to be associated with poverty of speech in schizophrenia patients,FOXP2 and FOXP1 (discussed below) disruptions.Interestingly, a genetic variant in FOXP2 gene cause both linguistic and non-linguistic deficits. What mechanism might explain this fact? The FOXP2 protein is a transcription factor.FOXP2 expression was detected in fetal brains.Foxp2 expression was detected in the lungs and in neural, intestinal and cardiovascular tissues during development.FOXP2 expression was also detected in the caudate nucleus of adult human brains,FOXP2 expression can be detected from as early as the 44th day of gestation.FOXP2 expression is first detected in the midline of the hindbrain, and subsequently becomes more complex as the embryonic development progressed.FOXP2 expression was detected only in some brain regions, including the cerebellum, thalamus, caudate nucleus, and the putamen, and was sometimes limited to specific structures in those regions, e.g., Purkinje cells in the cerebellum.As has been shown, disruptions in the FOXP2 can potentially affect a variety of genes that could be relevant to speech, language, and/or motor control. In theory, it could also be influencing each of these phenotypes independently. Researchers thus set out to find the genes that FOXP2 regulates, i.e., its targets. One studyFOXP2 shows high expression in the lung.It is thus clear that, as a transcription factor expressed in the brain, Another study looked for FOXP2 targets in human neuron-like cells.40FOXP2 in neural development have also been carried out. One investigation showed that the knockdown of Foxp2 expression in the developing mouse cortex prevented the transition of radial precursors suggesting that murine Foxp2 regulates neurite (axon or dendrite) outgrowth during brain development.Foxp2 in the embryonic mouse neocortex delays neurite outgrowth and impairs radial migration, a process which is normally repressed through microRNA mediation.FOXP2 and mouse Fopx2 have been shown to regulate the genesis of intermediate progenitors and neurons from multipotent radial precursors, only the human FOXP2, when overexpressed, enhances the genesis of intermediate progenitors and neurons.FOXP2 is involved in neural development, and, particularly, in neurite outgrowth.Studies into the specific involvement of FOXP2 may be influencing language is that of CNTNAP2 . CNTNAP2 encodes a transmembrane protein, CASPR2, which is found at the nodes of Ranvier (gaps in the myelin sheaths insulating the axons of neurons).CNTNAP2 shows enriched expression in brain regions that are important for language, and its expression pattern in the human brain is remarkably different from its expression pattern in the rodent brain.46A good example of a gene in a pathway through which CNTNAP2 have been implicated in epilepsy, language regression and mental retardation,48Mutations in CNTNAP2 was a FOXP2 target, and that it was down-regulated by it. Moreover, this study also found an association between variants in CNTNAP2 and nonword repetition in probands with specific language impairment: nine intronic SNPs located between exons 13 and 15 of CNTNAP2 were significantly associated with nonword repetition.CNTNAP2 implicated in studies of specific language impairment49A study by Vernes et al.CNTNAP2 in a proband who also had a de novo mutation in FOXP1 (forkhead box P1).FOXP2: CNTNAP2 is regulated by FOXP2, and FOXP1 interacts with it. Findings, such as this, assist in the further characterization of pathways involving FOXP2 and assist in the identification of major players thereof. See Box 2 for more information on the possible interaction between FOXP2 and FOXP1.More recently, an exome-sequencing study of sporadic autism spectrum disorders identified a potentially deleterious missense mutation in CNTNAP2, a FOXP2 target, appears to be involved in several neurodevelopmental disorders in which language is impaired. This example shows how FOXP2 can be influencing various overlapping pathways in various disorders. Considering the fact that FOXP2 has many neural targets,FOXP2 can manifest itself as a very complex phenotype.Thus, FOXP2 is not found only in humans; FOXP2 orthologs are found in other vertebrates, and the FOXP2 protein is conserved across different species.FOXP2 protein is highly conserved across different species.FOXP2 and FOXP1 (OMIM#605515) both belong to the same gene family, the forkhead/winged-helix family of transcription factors, and their proteins share a high degree of similarity in their DNA-binding domain.FOXP2, FOXP1 is also expressed in neural tissue during development.FOXP2 and FOXP1 in developing and adult mice brains and in human fetal brain tissue found that while both genes were expressed in the brain, they each had their own distinct expression pattern with regards to different brain regions, with the exception of the basal ganglia.FOXP2 and FOXP1 expression in human and zebra finch (a songbird) brains found that both genes were expressed in brain circuits related to song learning and production.FOXP1, in contrast to that of FOXP2, consistently showed a difference in expression levels between males and females in sexually dimorphic brain regions, in that males had high levels of FOXP1 expression in the song-related brain regions, whereas the corresponding regions in the female brain did not.FOXP1 have been implicated in speech delay, intellectual disability, and autism, OMIM#6055 both beFOXP2 orthologs differ at only three amino acids, whereas the human and chimpanzee orthologs differ at two of those three amino acids.FOXP2. The asparagine-to-serine substitution occurred independently in some carnivore and bat species,FOXP2. In addition to that, these two amino acid substitutions were not found in whales, which are also vocal-learning mammals.57The human and mouse FOXP2 is involved in speech and language in humans, and it is found in other species, the question was raised of whether any changes in FOXP2 could be found in animal species with more complex forms of vocal communication  compared with species with simpler forms of vocal communication. Webb et al.FOXP2 gene of song-learning birds with that of non-song-learning birds, and examined whether other vocal-learning mammals such as whales and dolphins shared any of the amino acid substitutions that were thought to be human-specific. No difference in the amino acid sequence encoded by exon 7 of FoxP2 between song-learning birds and non-song-learning birds was found,FOXP2 orthologs in echolocating bats were quite diverse in their sequences, and some variation in exons 7 and 17 of the bat FoxP2 seemed to be correlated with echolocation type and phylogenetic boundaries, suggesting that FoxP2 may have played a role in the development of these echolocation systems.58As FOXP2 and the selective sweep originated in the common ancestor of Neanderthals and modern humans, suggesting that they took place sometime between 300,000 and 400,000 years ago.FOXP2 that is not present in Neanderthals.FOXP2 and chimpanzee FoxP2 in human neuronal cells found 116 genes that were significantly differentially regulated by FOXP2 and FoxP2.64A 2007 study by Krause et al.FOXP2 in neural tissue in a variety of species. Lai et al.FOXP2 in the developing human and mouse brains. Their results indicated high conservation of expression patterns at onset in the brains of both species, with regards to both the timing and tissue distribution. Even as brain development progressed, both expression patterns continued to resemble each other (it should be noted that the studying of human embryonic expression was confined to early gestation). Teramitsu et al. found highly similar expression patterns in human and zebra finch brains (discussed in more detail in Box 2).53Several studies investigated the expression patterns of FoxP2 expression in avian vocal learners and non-learners found that striatal expression was similar between birds and mammals, whereas pallial expression was not.FoxP2 expression levels in Area X  increased in young zebra finches and in adult canaries when the former learned to imitate song and when the latter remodeled their songs.65A study of FoxP2 expression in zebrafish found that the expression pattern in the brain of the zebrafish was similar to that of humans and included regions that correspond to brain regions which showed abnormalities in the affected KE family members.66A study of FoxP2 expression across various species. These include a study of the rat brain, which found high levels of expression in the striatum of the basal ganglia during brain development,Foxp2 expression in four species of mice, which also found a high and heterogeneous Foxp2 expression pattern in the caudate nucleus and putamen across species.69Other studies have investigated FOXP2 orthologs across various species suggest that FOXP2 may have a more general role in the development of the nervous system.The high degree of conservation in the expression patterns in neural tissue and amino acid sequence of FOXP2 has been the subject of several functional studies using animal models. Shu et al.Foxp2 gene in mice, thus producing heterozygous mice or knockout mice, respectively. The knockout mice showed severe motor impairment, and died prematurely.Foxp2 disruption.Foxp2 corresponding to the mutation found in the affected KE family members. Mice homozygous for this mutation showed severe motor abnormalities.Foxp2 mutation also prevented the normal development of the cerebellum in homozygous mice and the normal maturation of dendrites in the Purkinje neurons in both homozygous and heterozygous mice.73A recent study75FoxP2 expression levels in Area X increased during song learning.FoxP2 regulation in Area X of adult zebra finches, in a social context, during directed and undirected singing, i.e., when males sing during social interactions and when they are alone or do not sing toward a conspecific, respectively.FoxP2 regulation in Area X during adult song production and not song learning. Interestingly, FoxP2 in Area X was down-regulated in the context of undirected singing, compared to that of directed singing.FoxP2 is down-regulated during undirected singing in adult zebra finches in a brain region usually associated with song learning in young zebra finches, may suggest a role for FoxP2 beyond the developmental stages of vocal control circuits in the brain.78Haesler et al.FoxP2 regulation in Area X in both hearing and deafened zebra finches.FoxP2 was down-regulated in both hearing and deafened young zebra finches, similar to the case of adult zebra finches, after they were allowed to sing for 2 h, suggesting that the FoxP2 regulation did not solely depend on auditory feedback, although only in the hearing birds did the down-regulation depend on the amount of singing, suggesting a more complex regulatory mechanism.79A subsequent study examined FOXP2 gene was studied in mice. The two previously discussed amino acid substitutions that are found in humans (but not in chimpanzees) (T303N and N325S) were introduced into the endogenous murine Foxp2.80In a recent study a humanized version of the FOXP2 might have on the mouse brain found the following: a reduction in dopamine levels in humanized FOXP2 mice compared to the wild-type and heterozygous mice; longer dendritic trees of medium spiny neurons in the striatum of humanized FOXP2 mice compared with the wild-type and heterozygous mice; long-term synaptic depression was higher in medium spiny neurons of humanized FOXP2 mice than in those of wild-type mice; altered frequencies of ultrasonic vocalizations in humanized FOXP2 mice compared with wild-type mice.FOXP2 was not found to be influencing physiological measurements unless they were related to the central nervous system, it is likely that the two amino acid substitutions in question affect the brain circuits in the mouse, of which the corresponding regions in the human brain could arguably be relevant to speech and language.FOXP2 mice and wild-type mice.80Further investigations of the effect the humanized FOXP2 increased dendritic length only in neurons that form part of the cortico-basal ganglia circuits, and that it influences synaptic plasticity in medium spiny neurons, but not in Purkinje neurons.81A subsequent study examined whether the changes in long-term synaptic depression and dendritic length occur in other brain regions, and concluded that the humanized FOXP2 gene is the first gene found to be involved in a speech and language disorder. The disorder was first observed and studied in a British family known as the KE family, but similar, independent cases have since been reported. Various disruptions in the FOXP2 gene have been reported, and the conditions caused by said disruptions tend to include a broad spectrum of deficits, including: speech and language problems, verbal dyspraxia, low performance IQ, developmental delay, and brain abnormalities. The FOXP2 protein acts as a transcription factor, and has been shown, for the main part, to repress the expression of neural targets. A thoroughly studied example of a gene repressed by FOXP2, CNTNAP2, has been implicated in disorders such as specific language impairment and autism, among others, which sheds some light on the mechanism through which FOXP2 may be influencing speech and language. FOXP2 is a highly conserved protein and exists in many other vertebrates. Studies of FOXP2 function in species that communicate vocally and/or are vocal learners show that there exists a high degree of conservation in expression patterns across different species, and that FOXP2 is involved in brain circuits that are important for vocal learning and communication. The human FOXP2 ortholog contains amino acid substitutions which, when introduced into the mouse, have been shown to affect the function of the gene in the brain. Given the evidence from the wide variety of studies that have been performed at various levels, from the gene and up to the brain, it is clear why a disruption in FOXP2 may have a cascade of different effects: it can cause changes in the regulation of FOXP2 target genes, many of which are expressed in the brain. Functional studies of FOXP2 have shown that the gene plays a crucial role in the development of several brain regions that are known to be part of speech and language-related circuits. Molecular studies of the effects of FOXP2 disruptions in individuals with a speech and language disorder show that its function is considerably affected, which may result in the abnormal development of important brain regions. The exact mechanisms and pathways of FOXP2 and its neuronal targets and the way in which they affect speech and language are not yet completely clear, but continuing work is being carried out by scientists from various disciplines, from genetics to neuroscience, in order to shed some light thereon. Future studies may focus on the genes that regulate FOXP2 as well as continue to study the genes that FOXP2 regulates. The genetic and biochemical study of these complex genetic pathways, as well as functional studies in vocal learning species will allow us to better understand the biological foundations of human language.The


Answering questions such as "Which genes are related to breast cancer?" usually requires retrieving relevant publications through the PubMed search engine, reading these publications, and creating gene lists. This process is not only time-consuming, but also prone to errors.We report GLAD4U , a new, free web-based gene retrieval and prioritization tool. GLAD4U takes advantage of existing resources of the NCBI to ensure computational efficiency. The quality of gene lists created by GLAD4U for three Gene Ontology (GO) terms and three disease terms was assessed using corresponding "gold standard" lists curated in public databases. For all queries, GLAD4U gene lists showed very high recall but low precision, leading to low F-measure. As a comparison, EBIMed's recall was consistently lower than GLAD4U, but its precision was higher. To present the most relevant genes at the top of a list, we studied two prioritization methods based on publication count and the hypergeometric test, and compared the ranked lists and those generated by EBIMed to the gold standards. Both GLAD4U methods outperformed EBIMed for all queries based on a variety of quality metrics. Moreover, the hypergeometric method allowed for a better performance by thresholding genes with low scores. In addition, manual examination suggests that many false-positives could be explained by the incompleteness of the gold standards. The GLAD4U user interface accepts any valid queries for PubMed, and its output page displays the ranked gene list and information associated with each gene, chronologically-ordered supporting publications, along with a summary of the run and links for file export and functional enrichment and protein interaction network analysis.http://bioinfo.vanderbilt.edu/glad4u.GLAD4U has a high overall recall. Although precision is generally low, the prioritization methods successfully rank truly relevant genes at the top of the lists to facilitate efficient browsing. GLAD4U is simple to use, and its interface can be found at:  The physical development and phenotype of organisms can be thought of as a product of genes interacting with each other and with the environment. Therefore, it is common for a scientist to ask questions like "Which genes are related to breast cancer?", "Which genes are involved in embryonic development?", and "Which genes are functionally related to TP53?"The current answers to these questions are primarily contained in the articles indexed in the MEDLINE database. Traditionally, answering these questions requires individuals to retrieve relevant publications through the PubMed search engine and then to create gene lists by manually extracting gene-centered information from retrieved literature. This process is not only time-consuming, but also prone to errors. First, it is difficult to ascertain that all relevant literature is processed. Second, it is unlikely that all relationships in a publication will be detected. Third, individual researchers tend to extrapolate based on domain knowledge.Over the past decade, bioinformatics approaches have been developed to address this issue. One of the most successful projects in this area is the Gene Ontology (GO) project . GO prodA useful resource specifically designed for disease studies is the Online Mendelian Inheritance in Man (OMIM ) projectIn addition to manual curation, text mining tools have been developed to assist gene list creation . As an eHere, we report GLAD4U , a new web-based gene retrieval and prioritization tool. GLAD4U takes advantage of existing resources at the National Center for Biotechnology Information (NCBI) to ensure computational efficiency. It provides a simple user interface that facilitates intuitive usage and interpretation of results. The quality of gene lists created by GLAD4U is assessed using corresponding "gold standard" lists curated in GO, GAD (Genetic Association Database ), and OMGLAD4U relies on the NCBI eSearch API to find publications related to a user query and on the gene-to-publication link table to identify genes from the retrieved publications. We used three GO biological process terms  and three disease terms  as queries to evaluate the overall quality of the retrieved gene lists. For each query, using a corresponding gene list curated by GO or GAD/OMIM as a gold standard, we calculated the precision, recall and F-measure of the retrieved gene list. As shown in Table The low precision of GLAD4U may be partially attributed to the incompleteness of the annotation in GO and GAD/OMIM. However, it is likely that the original gene lists include many irrelevant genes. In this case, a prioritization step that ranks truly relevant genes at the top of a list would certainly facilitate efficient browsing.We studied the performance of two methods to prioritize the gene lists. The first, "GLAD4U Counts", is based solely on the number of supporting publications as commonly implemented in other software ,11. The Figure k" is usually used to measure precision at a fixed low level of retrieved results, e.g., the top k results. To this end, we calculated the precisions for the top 50 (k = 50) and top 100 (k = 100) genes for all three methods, for each query  is part of the renin-angiotensin system (RAS). Proteins in this system are thought as important regulators of blood pressure and are involved in the onset of hypertension -32. OverFrom these publications, we believe that MDM2 and IGN1 should be part of the apoptosis list, as well as REN and ADIPOQ should be part of the hypertension list. These results accentuate the incompleteness of the gold standards and suggest that GLAD4U can help in the completion of the gold standard lists.http://compbio.charite.de/genewanderer). We extracted 32 "disease-gene families" to use as standards for evaluating GLAD4U performance before and after thresholding. On average, GLAD4U performs 2.90-time better when genes with low prioritization scores  are removed, as illustrated by comparing the F-measures , the number of genes to present per result page, the maximum number of publications supporting each gene returned in the result page and the number of pages to build for each of the algorithm runs.The output page displays the ranked gene list and information associated with each gene Figure . As eachhttp://cytoscapeweb.cytoscape.org/).At the top of the output page, a summary of the run is also given: query term and options chosen, number of genes and publications processed, as well as a hyperlink to download the complete results in the comma-separated values (CSV) format. Although this file may be difficult to interpret by humans, it can be used as input for other computational analysis tools. For example, we have implemented a "send data to Functional Enrichment Analysis" link in the result page Figure  of GLAD4Reading through all relevant literature to generate a gene list is time consuming ,51-53, aOne important feature of GLAD4U is its information processing. Based on our survey among experimentalists, GLAD4U follows the exact same steps that an experimentalist would follow: gather literature, extract gene information and create an expert list . WhetherAnother important feature of GLAD4U is its simplicity. Researchers will be at ease using GLAD4U because its searching engine is powered by PubMed's API ,52, and Several tools rely on PubMed to build disease candidate genes lists ,52,56,57p value--the higher the score will be for the gene. Thus, even if GLAD4U is solely retrieving its data from the biomedical literature, it prioritizes following a statistical analysis of the retrieved data.Although using the biomedical literature as a knowledge source seems intuitive ,58,59, cThe most obvious usage of GLAD4U is to generate a gene list for an input concept, which has been demonstrated in this paper. This can be extremely useful for the design of targeted high-throughput experiments. If one needs to create a custom array or selected proteins for targeted quantitative proteomic analysis using the selected reaction monitoring (SRM) assay, one can use GLAD4U and review the ranked list of genes that likely should be included in the experimental design. Besides generating gene lists for individual concepts, GLAD4U is very flexible and allows production of gene lists related to multiple concepts, which cannot be done by searching GO or OMIM databases. For example, a query of "smoking AND cancer" can generate a gene list that could potentially help exploring gene-environment interactions in cancer. GLAD4U also holds the potential to assist in improvement of the functional annotation of genes. Although GO contains more than 17,000 terms  and is rFinally, because GLAD4U prioritization algorithm assigns scores to genes, removing the genes with a low score consistantly improves the quality of the results. This result justifies thresholding GLAD4U results by default.GLAD4U is a freely available web-application for creating expert candidate gene lists tailored to a user's query. It follows the same steps that the experimentalist would follow: gather literature, extract gene information and create an expert list. The simple interface of GLAD4U ensures easy usage and interpretation. Because GLAD4U relies on existing biomedical literature, it has an immediate credibility with experimentalists, who use this resource as a primary means for enhancing their knowledge and expertise. Although the gene list directly returned from a PubMed query is usually lengthy and noisy, the prioritization method implemented in GLAD4U successfully ranks truly relevant genes at the top of the list and facilitates efficient browsing of the list.GLAD4U relies on the eSearch application programming interface (API) developed by the NCBI for retrieving publications from the MEDLINE database . For a uGenes associated with PMIDs are retrieved based on the gene-to-publication link table provided by Entrez-Gene . Links bn be the number of publications retrieved for the query and present in the gene-to-publication link table (query-relevant publications) and k be the number of query-relevant publications that involves the gene G. Let us further assume that there are m publications in the gene-to-publication link table, j of which involve the gene G (gene-relevant publications). This method calculates the probability of observing k or more query-relevant publications for the gene by chance, based on the hypergeometric test and scores the gene using the following formula:We studied two methods to prioritize the retrieved genes based on publication counts or the hypergeometric test. To prioritize using counts ("GLAD4U Counts"), each gene receives a score equal to the number of publications describing it in the link table. The other method ("GLAD4U Hypergeometric") uses the hypergeometric test to prioritize all retrieved genes. Specifically, for a given query Q and a gene G, let pr/(p+r), where p is the precision defined as r is the recall defined as k retrieved genes (k = 50 and k = 100) to evaluate the performance of our gene prioritization methods, and compared it to the performance of the ranked lists generated by EBIMed [We used GO and disease terms as queries to evaluate the performance of the GLAD4U algorithms. Gene lists curated in GO, OMIM and GAD  were usey EBIMed . All perhttp://bioinfo.vanderbilt.edu/glad4u) is platform-independent and under a GNU GPL license [The GLAD4U user interface was developed in HTML and PHP languages. The scripts to deploy and update the algorithm on web servers were written in Perl, while the generation of hypergeometric test scores is using C. JQuery was used to implement user-features such as the ability to hide/ show options and functions. An email notification module was implemented to allow users to retrieve their results at a later time. GLAD4U ( license . It was ADIPOQ: adiponectin; API: application programming interface; CSV: comma-separated values; GAD: genetic association database; GLAD4U Counts: GLAD4U prioritization algorithm using counts; GLAD4U Hypergeometric: GLAD4U prioritization algorithm using the hypergeometric test; GLAD4U: gene list automatically derived for you; GO: gene ontology; GOTM: GOTree Machine; ING1: inhibitor of growth family: member 1; AP: average precision; NCBI: national center for biotechnology information; OMIM: online mendelian inheritance in man; PMIDs: publication identification IDs; REN: renin; SRM: selected reaction monitoring.The authors declare that they have no competing interests.BZ and JJ conceived of the study, which was coordinated by BZ. JJ carried out the work with PHP and Perl, DD implemented the C version of the algorithm, ZS implemented the cytoscape web plugin for network visualization. JJ, DD, ZS and BZ participated in testing. JJ and BZ participated in the analysis of the results and in writing of the manuscript.False-positive genes retrieved by querying "apoptosis" with GLAD4U. This table shows all genes retrieved by GLAD4U with the query "apoptosis" that were not among the gold standards. The table presents the rank and score of these genes and all the retrieved supporting publications.Click here for fileFalse-positive genes retrieved by querying "hypertension" with GLAD4U. This table shows all genes retrieved by GLAD4U with the query "hypertension" that were not among the gold standards. The table presents the rank and score of these genes and all the retrieved supporting publications.Click here for fileGLAD4U prioritization of disease candidate genes. This table shows the number of genes associated with each GeneWanderer hereditaty disease, retrieved by GLAD4U and overlapping between the two lists before and after thresholding. F-measure fold change between the GLAD4U prioritized list before and after thresholding, as well as the actual F-measures are also displayed in the table.Click here for file


Physical activity patterns of a population remain mostly assessed by the questionnaires. However, few physical activity questionnaires have been validated in Asian populations. We previously utilized a combination of different questionnaires to assess leisure time, transportation, occupational and household physical activity in the Singapore Prospective Study Program (SP2). The International Physical Activity Questionnaire (IPAQ) has been developed for a similar purpose. In this study, we compared estimates from these two questionnaires with an objective measure of physical activity in a multi-ethnic Asian population.Physical activity was measured in 152 Chinese, Malay and Asian Indian adults using an accelerometer over five consecutive days, including a weekend. Participants completed both the physical activity questionnaire in SP2 (SP2PAQ) and IPAQ long form. 43subjects underwent a second set of measurements on average 6 months later to assess reproducibility of the questionnaires and the accelerometer measurements. Spearman correlations were used to evaluate validity and reproducibility and correlations for validity were corrected for within-person variation of accelerometer measurements. Agreement between the questionnaires and the accelerometer measurements was also evaluated using Bland Altman plots.The corrected correlation with accelerometer estimates of energy expenditure from physical activity was better for the SP2PAQ  than for the IPAQ . For moderate activity, the corrected correlation between SP2PAQ and the accelerometer was higher for Chinese (r = 0.38) and Malays (r = 0.57) than for Indians (r = -0.09). Both questionnaires overestimated energy expenditure from physical activity to a greater extent at higher levels of physical activity than at lower levels of physical activity. The reproducibility for moderate activity  and vigorous activity  was moderate to high for all instruments.The agreement between IPAQ and accelerometer measurements of energy expenditure from physical activity was poor in our Asian study population. The SP2PAQ showed good validity and reproducibility for vigorous activity, but performed less well for moderate activity particularly in Indians. Further effort is needed to develop questionnaires that better capture moderate activity in Asian populations. Globally, non communicable diseases (NCDs), consisting mainly of cardiovascular diseases, cancers, chronic respiratory diseases and diabetes make up to 60% of all deaths . WHO proThere are different methods for assessing physical activity. These include criterion methods such as doubly labeled water, indirect calorimetry, and direct observation; objective methods such as heart rate monitor, pedometer, accelerometer; and subjective methods including questionnaires and activity diaries -6. HowevThe International Physical Activity Questionnaire (IPAQ) was designed to provide a set of well-developed instruments that can be used internationally to obtain estimates of physical activity that can be compared across different populations . In ordeBetween 2003 and 2007 we conducted a population-based study, the Singapore Prospective Study Program (SP2) , which cThe aim of this study was to assess the validity of IPAQ long form in a multi-ethnic population of Chinese, Malays, and Indians living in Singapore and compares it against the SP2PAQ using accelerometer measurements as the reference instrument.We studied 164 participants, aged above 21 years. These were mainly students and staff from local university and hospital. Ethics approval was obtained from the National University of Singapore Institutional Review Board (NUS IRB). Written informed consent was obtained from all participants. One participant withdrew from the study after 2 days. Hence, 163 participants completed the study.Anthropometric measurements were taken (height to the nearest 0.1 cm and weight to the nearest kg). Participants then completed one of the two evaluated questionnaires (SP2PAQ or IPAQ) before the physical activity monitoring period. Physical activity was monitored using an Actical accelerometer for five consecutive days including 3 weekdays and 2 weekend days. Participants were instructed to wear the accelerometer for all waking hours except during water-based activities. After completion of the five- day monitoring period, participants returned the accelerometer and completed the remaining questionnaire. The first 120 participants answered IPAQ before the monitoring period and SP2PAQ immediately after the monitoring period. The order of the questionnaires was reversed for the last 43 participants to evaluate whether the order of questionnaire assessment may have affected the results.Of the 163 participants, 52 participants were re-recruited to test reproducibility of the questionnaires and the accelerometer. The reproducibility of the accelerometer was evaluated at the same time as the questionnaires were administered using the same device in both periods. The mean interval between the two assessments was 175 days (SD = 64 days) with minimum 63 days and maximum 308 days.The 8 versions of International physical activity questionnaire (IPAQ) were developed by an International Consensus Group between 1997 and 1998. These were developed as an instrument for cross-national monitoring of physical activity with a recall period of 7 days. In 2000, the reliability and validity of the questionnaires were evaluated in 12 countries and the result was published in 2003, which showed acceptable reliability and validity . Since iThe physical activity questionnaire used in Singapore Prospective Study Program (SP2PAQ) is an interviewer-administered questionnaire with a recall period of the previous 3 months. As mentioned before it was adapted from several established questionnaires validated in other populations -14 and eThe questions on transportation activity were adapted from National Health Survey 2004 questionnaire ,21. Part® physical activity monitor  which is a water resistant, lightweight (17 g) and small (28 × 27 × 10 mm) device. The monitors are initialized and downloaded through the ActiReader PC serial port interface. According to manufacturer, the Actical is an omnidirectional, piezoelectric accelerometer, which is able to detect movements in all directions. It is sensitive to movements in the range of 0.5-3 Hz and its sensitivity allows for detection of sedentary movement as well as high-energy movements. Its reduced frequency range also minimizes the effect of undesirable noise impulses, which tend to skew energy expenditure [Objective measurement of physical activity was obtained by using the Acticalall 28 ×  × 10 mm enditure  and has enditure . The phyenditure .Compared to ankle and wrist, hip was the best location for monitor placement to predict the energy expenditure from physical activity when validated against with VmaxST portable metabolic system (R = 0.90). For thiThe quality of the devices was monitored by checking the coefficient of variation (CV) of the devices monthly . All the-1 × min-1 or 1 kcal × kg body weight-1 × h-1 [For IPAQ, we used the IPAQ data processing rules  for our -1 × h-1 . For bot-1 × h-1 ,37. Then-1 × h-1 .The resulting measures from the two questionnaires expressed in Kcal per week were divided by 7 and the total Kcal for each moderate and vigorous intensity category from accelerometer for 5-day wearing period was divided by 5, to derive average Kcal per day for all methods.The Actical accelerometer recorded physical activity in a series of activity counts which were proportional to the magnitude and duration of the sensed accelerations. The raw minute-by-minute activity counts were then transformed into energy expenditure by the computer program using MET prediction algorithms of Klippel et al . The outThe accelerometer data was considered valid only when 10 or more hours of data per day were collected for five days. Thus, 8 participants were excluded because they did not meet this criterion. In addition, 3 participants who reported the sum total of all walking, moderate and vigorous time more than 16 hours per day in IPAQ were treated as outliers and excluded from analysis according to IPAQ data processing rule . As a rewheret = "true" correlation coefficientr0 = observed correlation coefficientraccelerometer = interclass correlation coefficient of accelerometerwhere ICCaccelerometer = number of repeated accelerometer measurements within-subjectnThe 95% and 99.99% confidence intervals for correction correlations were calculated using the formulas suggested by Willett et al . In addi2. The majority of participants had a job, but there were also students, homemakers, retired and unemployed participants. Nearly 70% had a higher education while 21% achieved secondary education and less than 10% had no education or primary level. There was a large variation in household income among participants (Table The study population N = 152) had mean age of 38.3 years and mean BMI of 24.54 kg/m52 had meAdditional file The validity of the questionnaires was further assessed by stratifying the study population according to age, gender, and ethnic group. Compared with the younger age group, the correlation between the energy expenditure from physical activity assessed by questionnaire and accelerometer in the older group tended to be higher for moderate activity, but lower for vigorous activity. This was observed for both questionnaires. The correlation was higher in men for both moderate and vigorous activity when the IPAQ was used, whereas the correlation was higher in women than men for moderate activity when the SP2PAQ was used.The performance of the SP2PAQ was similar in all three ethnic groups for vigorous activity, but for moderate activity, Malays showed a higher correlation with accelerometer measurements than Chinese and particularly Indians. For the IPAQ, reasonable correlations were only observed with the accelerometer in Chinese for moderate activity and Indians for vigorous activity.The agreement between the questionnaires and the accelerometer was also evaluated using Bland-Altman plots. Both IPAQ and SP2PAQ underestimated average energy expenditure from moderate activity, but overestimated average energy expenditure from vigorous activity as compared with the accelerometer. The mean difference of daily energy expenditure between the measurements of IPAQ and accelerometer for moderate activity was -169 Kcal/day (95%CI: -236 to -90) and that of between SP2PAQ and accelerometer was -196 Kcal/day (95% CI: -295 to -97). SP2PAQ showed good agreement with the accelerometer for moderate activity when the energy expenditure was below approximately 1200 Kcal per day. However, it tended to overestimate energy expenditure when energy expenditure increased above that level  physical activity questionnaire used for monitoring physical activity across the U.S.A. compared with the accelerometer was 0.31 for moderate activity and 0.17-0.26 for vigorous activity , whereasWhen we compared questionnaire and accelerometer estimates of energy expenditure from physical activity using Bland-Altman plots, greater differences between the two methods were observed with increasing means of measurements for both moderate and vigorous activity. This may be due to either the questionnaire increasingly over-estimating activity with increasing activity or the accelerometer increasingly under-estimating activity with increasing activity. In a study done by Klesges et al reported that participants overestimated the duration of their physical activities, especially for aerobic activities . In addiSeveral methodological differences exist between SP2PAQ and IPAQ. It should be noted that the IPAQ assesses physical activity in the past week, whereas the SP2PAQ assesses habitual physical activity of at least the past 3 months. For the first 120 participants, the week recorded by IPAQ was different from that of the week measured by the accelerometer as IPAQ was administered before the accelerometer wearing period. However, we reversed the order of questionnaire administration for the subsequent 43 participants so that the IPAQ questionnaire applied to the same week of accelerometer measurement and found that the order of questionnaire administration did not affect the agreement with accelerometer measurements. The mode of administration of the questionnaires was also different as SP2PAQ is administered by an interviewer whereas IPAQ is self-administered. In a comprehensive review of physical activity instruments, it was concluded that the accuracy of interviewer-administered questionnaires tends to be greater than for self-administered questionnaires . FinallyTo our knowledge, this is one of a few studies that validated physical activity questionnaires in an Asian population. In addition, the correlations were corrected for within-person variation in the accelerometer measurements. Within-person correlation in the reference instrument will reduce the correlation with the evaluated questionnaires and should be corrected for in validation studies . The droOur study showed that the IPAQ had limited accuracy for distinguishing physical activity levels of individuals and performed poorly in our study population as compared with Western populations. However, its performance was comparable to that observed in a Chinese population in Hong Kong. The SP2PAQ had acceptable validity and reproducibility and can be used in large epidemiological studies particularly for the assessment of vigorous physical activity. For moderate activity, however, adaptation of the questionnaire for the Indian population may be needed. For both the IPAQ and SP2PAQ questionnaire, considerable measurement error observed for the estimation of absolute physical activity levels particularly at higher levels of activity, which should be taken into account when the adequacy of activity levels of population groups are assessed. When we compare our results for IPAQ with results from studies in other populations, it is evident that the validity of the IPAQ differs substantially between populations. Thus, validation sub-studies using objective measures of physical activity within large epidemiological studies are desirable to quantify measurement error and later correct estimates of physical activity and health outcomes for measurement error.The authors declare that they have no competing interests.EEKN was responsible for data collection, statistical analysis and drafting the manuscript. SAGN and YW involved in data collection and helped with the data analysis. AS helped in data analysis. EST and JL directed the study and helped in revising the manuscript. RMVD helped in data analysis, interpretation of the results and led writing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/11/141/prepub"Singapore Prospective Study Program Physical Activity Questionnaire (SP2PAQ)" for the questionnaire used to assess physical activity in Singapore Prospective Study Program.Click here for file"Correlation between IPAQ and SP2PAQ measurements of energy expenditure from physical activity" for spearman correlations between IPAQ and SP2PAQ measurements of energy expenditure from moderate and vigorous activity.Click here for file